Microorganisms and Metabolic Pathways Involved in Anaerobic Benzene Biodegradation under Nitrate-Reducing Conditions
By
Roya Gitiafroz
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto
!Copyright by Roya Gitiafroz (2012) Microorganisms and Metabolic Pathways Involved in Anaerobic Benzene Biodegradation under Nitrate-Reducing Conditions Doctor of Philosophy, 2012, Roya Gitiafroz Chemical Engineering and Applied Chemistry, University of Toronto
Abstract
This thesis describes the characterization of benzene-degrading denitrifying cultures. Four objectives were pursued. The first objective was to identify conditions that promote or inhibit benzene decomposition and thus, to improve the biodegradation capacity of the cultures. FeS, resazurin, and nitrite had a detrimental impact on benzene degradation, whereas addition of supernatant from an active culture improved the benzene degradation activity by reducing the lag times.
The second objective was to determine the microbial community composition in enrichment cultures and to identify the bacterial species that mediate benzene mineralization. Five dominant bacterial Operational Taxonomic Units (OTUs) were identified. The most abundant phylotype was related to the gram-positive Peptococcaceae family. Other bacteria present were closely affiliated with Dechloromonas, Azoarcus, Chlorobi and Anammox species. To correlate the growth of these specific microbes with benzene degradation, the abundance of specific 16S rRNA genes was monitored during mineralization process using quantitative polymerase chain reaction (qPCR). Based on the result of qPCR experiments and information about the metabolisms of the above bacteria, a syntrophic mode of benzene degradation was hypothesized to occur under denitrifying conditions. In this process, Peptococcaceae initiate attack on benzene, and ferment benzene to hydrogen and low molecular weight products such as acetate.
ii These products are then consumed by nitrate-respiring Azoarcus and Dechloromonas or phototrophic Chlorobi. Anammox bacteria recycle and detoxify nitrite, and stabilize the culture.
The third objective was to isolate and characterize pure cultures with the ability to mineralize benzene anaerobically. Dechloromonas- and Dechlorosoma-like microorganisms were isolated from several benzene-degrading microcosms. Theses bacteria, however, were not able to metabolize benzene anaerobically.
The fourth objective was to investigate the key metabolic steps in the anaerobic benzene degradation pathway and to identify enzymes that are involved in this process. Differential transcription during growth of the culture on benzene versus growth on a metabolite of benzene degradation, i.e. benzoate was examined. Carboxylase-related genes were specifically transcribed in the presence of benzene. Furthermore, mRNA sequences corresponding to the genes that encode different enzymes of the benzoyl-CoA degradation pathway were present in the culture. These findings suggest that mineralization of benzene starts by its activation to benzoate through a carboxylation reaction catalyzed by benzene carboxylase. Benzoate is further metabolized through benzoyl-CoA pathway.
iii Acknowledgements
I like to thank my supervisor Prof. E. A. Edwards and express my most sincere gratitude for her enthusiasm, patience, and support during this research. Her passion for research and life provided me with motivation and inspiration throughout this work.
I like to thank Prof L. Raskin for accepting me as a member of her research group and providing me with the guidance throughout this study. I also would like to thank her for always being there for me.
I like to thank my other thesis committee members: Prof. G. Allen and Dr. D. Major for their advice and guidance in all steps of this study.
I like to thank Prof. P. Adriaens for allowing me to use his lab facility.
I like to thank my colleagues in Edwards’ and Raskin’s Labs.
This study was made possible by the financial support of Natural Sciences and Research Council of Canada, Ontario Graduate Scholarship, and the Graduate School of Chemical Engineering and
Applied Chemistry at University of Toronto.
I especially like to thank my Husband Dr. Hossein Tavana and my parents Zahra Ghaffari and
Mohammad Hossein Gitiafroz for their love, support, and patience. This thesis is dedicated to my family.
iv Table of Contents Abstract...... ii Acknowledgements ...... iv Table of Contents ...... v List of Tables...... x List of Figures ...... xii Nomenclature ...... xiv Chapter 1: Introduction and Objectives...... 1 1.1. Introduction ...... 2 1.1.1. Benzene: a groundwater contaminant ...... 2 1.1.2. Benzene bioremediation...... 2 1.2. Research objectives...... 4 1.3. Thesis outline...... 6 1.4. References Chapter 1 ...... 7 Chapter 2. Background and Literature Review ...... 12 2.1. Background...... 13 2.1.1. Bacterial energetics and growth...... 13 2.2. Literature review...... 16 2.2.1. Aerobic benzene degradation...... 16 2.2.2. Anaerobic benzene degradation...... 19 2.3. Syntrophic mineralization of benzene ...... 33 2.4. Conclusions...... 35 2.5. References Chapter 2 ...... 36 Chapter 3: Maintenance and Optimization of Growth of Benzene-Degrading Nitrate-Reducing Enrichment Cultures ...... 45 3.1. Introduction...... 46 3.2. Materials and Methods...... 47 3.2.1. Maintenance of benzene-degrading nitrate-reducing enrichment cultures..47 3.2.2. Nitrite removal...... 48 3.2.3. Medium omission and addition experiments ...... 48
v 3.2.4. Analytical methods ...... 49 3.3. Results and discussion ...... 49 3.3.1. Monitoring benzene degradation and nitrate reduction in enrichment cultures...... 49 3.3.2. Preventing nitrite accumulation and inhibition...... 51 3.3.3. The effect of different components of medium on the lag time ...... 53 3.4. Conclusions...... 64 3.5. Recommendations for future work ...... 64 3.6. References Chapter 3 ...... 67 Chapter 4: Multiple Syntrophic Associations in Nitrate-Reducing Benzene- Degrading Cultures ...... 70 4.1. Introduction...... 71 4.2. Materials and Methods...... 74 4.2.1. Benzene-degrading nitrate-reducing cultures and subcultures ...... 74 4.2.2. DNA extraction, 16S rRNA gene cloning and sequencing...... 75 4.2.3. Quantification of specific species in enrichment cultures ...... 76 4.2.4. Time course experiments ...... 77 4.2.5. Analytical methods ...... 78 4.3. Results...... 79 4.3.1. Bacterial community structure of the denitrifying benzene-degrading cultures...... 79 4.3.2. Quantitative survey of enrichment Cultures ...... 81 4.3.3. Time course qPCR ...... 82 4.4. Discussion...... 88 4.4.1. Phylogenetic analysis and physiological roles of different bacteria in the culture...... 88 4.4.2. Stoichiometric and energetic considerations ...... 91 4.5. Conclusions...... 94 4.6. Recommendations for future work ...... 94 4.7. References Chapter 4 ...... 96 Chapter 5. Attempts at Isolating Pure Cultures of Benzene-Degrading Nitrate- reducing Microbes...... 103 5.1. Introduction...... 104 5.2. Materials and Methods...... 104
vi 5.2.1. Isolation procedure...... 104 5.2.2. Identification of isolated colonies...... 105 5.2.3. Analysis of 16S rRNA gene sequences ...... 106 5.2.4. Fluorescence microscopy...... 106 5.3. Results...... 106 5.3.1. Characterization of dilution cultures...... 106 5.3.2. Phylogenetic relationship between isolated bacteria and known microbes ...... 109 5.3.3. Catabolic study...... 109 5.4. Discussion...... 111 5.4.1. Phylogenetic and physiological characterization of isolated bacteria ...... 111 5.4.2. Metabolic characteristics ...... 112 5.5. Conclusions...... 113 5.6. Recommendations for future isolation trials ...... 113 5.7. References Chapter 5 ...... 115 Chapter 6: Metatranscriptome Analysis of a Benzene-Degrading Nitrate-Reducing Culture...... 118 6.1. Introduction...... 119 6.2. Materials and Methods...... 121 6.2.1. Nitrate-reducing enrichment cultures ...... 121 6.2.2. Substrate utilization experiments...... 121 6.2.3. Differential transcription experiments ...... 122 6.2.4. RNA extraction and Pyrosequencing...... 122 6.2.5. Community profiling and functional analysis of mRNA...... 123 6.2.6. Analytical methods ...... 124 6.3. Results and discussion ...... 126 6.3.1. Metabolism of benzoate and benzene by Cartwright Consolidated enrichment culture ...... 126 6.3.2. Analysis of RNA sequences...... 128 6.3.3. Comparison between community profiles of Cartwright Consolidated culture during growth on benzoate and benzene...... 129 6.3.4. Genes transcribed in the presence of benzoate ...... 125 6.3.5. Genes transcribed during growth of culture on benzene ...... 137
vii 6.3.6. A complex interaction between different bacteria in Cartwright Consolidated culture facilitates benzene degradation ...... 141 6.4. Conclusions...... 144 6.5. Recommendations for future work ...... 144 6.6. References Chapter 6 ...... 145 Chapter 7: General Discussion and Synthesis ...... 151 7.1. Optimizing growth of benzene-degrading nitrate-reducing cultures...... 152 7.1.1. Inhibitory impact of nitrite on mineralization of benzene ...... 152 7.1.2. Effect of omission of FeS and resazurin and addition of autoclaved culture on the lag time ...... 154 7.2. Exploring microbial interactions in benzene-degrading nitrate-reducing cultures...155 7.3. Carboxylation as the initial step in degradation of benzene ...... 156 7.4. References Chapter 7 ...... 158 Chapter 8: Conclusions and Engineering significance ...... 160 8.1. Summary...... 161 8.2. Conclusions...... 162 8.3. Engineering significance...... 163 8.4. Recommendations for future work ...... 165 8.5. References Chapter 8 ...... 167 Appendix A: Bacterial energetics and stoichiometric calculations...... 168 Calculation of free energy and overall energy reactions ...... 169
Calculation of fe, fs and overall stoichiometric equations...... 171
Calculation of fe, fs based on observed yields...... 174 Calculation of yield from the change in number of 16S rRNA gene copies per mole of benzene ...... 175 Calculation of yield based on ratio of nitrate to benzene...... 176 Calculation of reaction free energy for nonstandard conditions...... 177 Appendix B: Anaerobic medium composition ...... 179 Anaerobic medium used for maintaining our cultures ...... 180 Appendix C: Effect of addition of autoclaved culture on the observed lag times. 181 Benzene degradation curves for Cartwright pw1 10-5 and Cartwright Consolidated 10-3 and for their transfers into an autoclaved culture...... 182
viii Appendix D: Partial 16S rRNA gene sequences obtained from Swamp Consolidated enriched culture...... 183 Appendix E: Quantitative PCR (qPCR) calculations...... 186 qPCR experiments calculations ...... 187 Appendix F: 16S rRNA gene sequences of isolated colonies from benzene degrading nitrate-reducing enrichment cultures...... 190 Appendix G: Microscopy images of dilution cultures...... 193 Appendix H: Analysis of rRNA and mRNA sequences obtained from Cartwright Consolidated culture during growth on benzene and benzoate ...... 195 RNA extraction from cells grown on benzoate and benzene...... 196 Analysis of large subunit ribosomal RNA sequences...... 199 Assembly of mRNA sequences ...... 200 Analysis of mRNA sequences of benzoate- and benzene- grown cells...... 202 Nucleotide sequences of the genes that were transcribed in Cartwright Consolidated grown on benzoate ...... 238 Nucleotide sequences of the genes that were transcribed in Cartwright Consolidated grown on benzene ...... 252 References...... 255
ix List of Tables Table 2.1. Energy-yielding equations and standard free energy changes for oxidation of various electron donors coupled to reduction of nitrate to nitrogen gas or nitrate to nitrite...... 14 Table 2.2. Theoretical stoichiometric equations for oxidation of various electron donors coupled to either reduction of nitrate to nitrite or reduction of nitrate to nitrogen gas considering cell growth...... 15
Table 2.3.!Experimentally derived stoichiometric equations for oxidation of benzene coupled to either reduction of nitrate to nitrite or reduction of nitrate to nitrogen gas...... 16 Table 3.1. Benzene mineralization rates and nitrite accumulation in benzene-degrading nitrate- reducing cultures...... 51 Table 4.1. Microorganisms in enriched or pure cultures that are involved in anaerobic degradation of benzene under different electron accepting conditions...... 73 Table 4.2. List of qPCR primer sets used in this study with their sequences, annealing temperatures, and specificity...... 78 Table 5.1. List of dilution cultures showed benzene degradation activity...... 107 Table 5.2 Close Genbank matches to colonies isolated from serially diluted cultures...... 109 Table 6.1. Comparison between the lag times observed prior to degradation of benzene for culture bottles amended first with benzoate and after its consumption with the benzene, supplied simultaneously with benzoate and benzene, and positive controls that were fed only with benzene...... 128 Table 6.2. Total number of RNA, rRNA, and mRNA sequences for the RNA samples extracted from Cartwright Consolidated culture during its growth on benzoate and benzene...... 129 Table 6.3. List of the genes encoding the enzymes necessary for degradation of benzoate to 3- hydroxypimelyl-CoA/Pimelyl-CoA that were expressed in Cartwright Consolidated culture and those identified in other benzoate-degrading anaerobes...... 135 Table 6.4. List of several other genes that were expressed in Cartwright Consolidated culture during growth on benzoate and their possible functions in benzoate mineralization...... 136 Table 6.5. Aromatic hydrocarbon degradation genes that were specifically expressed in Cartwright Consolidated culture in the presence of benzene as the sole electron donor and carbon source ...... 139 Table 6.6. Peptococcaceae number of small and large subunit ribosomal RNA sequences in the cells amended with benzene and the ones supplied with benzoate...... 143 Table A.1. Half-reactions and their Gibb's standard free energy at pH=7.0...... 169 Table A.2. Free energy of formation of chemical species at 25°C...... 170 Table B.1. Components of our anaerobic medium and their corresponding concentrations...... 180 Table H.1. Quantity of RNA that is present in each individual RNA sample based on Nanodrop readings...... 197
x Table H.2. Denitrification pathway genes transcribed in Cartwright Consolidated grown on benzoate and benzene...... 201 Table H.3. Benzoate-related genes that were transcribed in Cartwright Consolidated culture when it was supplied with benzoate as an electron donor and carbon source...... 202 Table H.4. Genes that were transcribed in Cartwright Consolidated culture during growth on benzoate ...... 203 Table H.5. Genes that were transcribed in cells of Cartwright Consolidated culture during growth on benzene ...... 230
xi List of Figures Figure 2.1. Metabolic pathway of aerobic benzene degradation (Habe and Omori 2003)………18 Figure 2.2. Carboxylation as the initial step of anaerobic benzene degradation...... 27
Figure 2.3.!Hydroxylation as the first step in mineralization of benzene… ...... 29 Figure 2.4. Proposed methylation of benzene to toluene as the activation mechanism of benzene ring...... 31 Figure 2.5. Fumarate addition as a mechanism for activating the benzene ring (Coates et al. 2002) ...... 33 Figure 2.6. Benzene ring reduction as the initial step of benzene mineralization (Coates et al. 2002) ...... 33 Figure 3.1. Parent cultures and subcultures that were maintained in our laboratory...... 47 Figure 3.2. Nitrate consumption and nitrite production are shown during biodegradation of benzene in Cartwright 1b enrichment culture...... 52 Figure 3.3. Mineralization of benzene by Cartwright 1b after nitrite removal...... 53 Figure 3.4. Effect of FeS and resazurin on the observed lag time...... 56 Figure 3.5. Benzene degradation curves and nitrite concentrations for transfers into a) regular medium and b) medium with tungstate...... 58 Figure 3.6. Comparison between benzene biodegradation of transfers into a) regular medium, b) medium without trace minerals, and c) medium with 10 times more vitamins………………… 60 Figure 3.7. Benzene degradation curves for treatments cultivated into a) regular medium and b) autoclaved culture...... 61 Figure 3.8. Comparison between anaerobic benzene degradation in dilution cultures, a) Swamp Consolidated 10-2 and b) Swamp Consolidated 10-5, both with and without autoclaved culture. .63 Figure 4.1. Parent cultures and subcultures characterized in this study...... 75 Figure 4.2. Phylogenetic tree showing relationship between observed OTUs in Swamp Consolidated culture and other classified bacteria...... 80 Figure 4.3. Different bacterial species 16S rRNA gene copies in a) Cartwright and Swamp cultures and b) dilutions prepared from Cartwright and Swamp microbial consortia...... 82 Figure 4.4. Benzene degradation curves for a) 2008 and b) 2009 time course experiments...... 84 Figure 4.5. Peptococcaceae, Azoarcus, Chlorobi, Dechloromonas, and Anammox organism growth in individual culture bottles during benzene degradation...... 87 Figure 4.6. Proposed role of different bacterial species in the flow of carbon and electrons within the benzene-degrading nitrate-reducing enrichment cultures...... 93 Figure 4.7. The free energy of fermentation of benzene to acetate and hydrogen calculated at different hydrogen partial pressures...... 94
xii Figure 5.1. Fluorescence microscopy image of 10-5 dilution of Swamp Consolidated culture at a 40X magnification...... 108 Figure 5.2. Phylogenetic tree showing relationship between bacteria isolated from our benzene- degrading nitrate-reducing enrichment cultures and other classified microorganisms...... 110 Figure 6.1. Benzene hydroxylation to phenol (1), carboxylation to benzoate (2) and methylation to toluene (3) as proposed activation mechanisms of benzene ring (Coates et al. 2002)...... 120 Figure 6.2. Schematic of different steps taken for analysis and identification of rRNA and mRNA sequences in the pool of total RNA...... 125 Figure 6.3. Degradation of benzoate (a) and benzene (b) by benzene-degrading nitrate-reducing Cartwright Consolidated culture...... 126 Figure 6.4. The MEGAN tree showing comparison between community compositions of Cartwright Consolidated culture during growth on benzoate (red colors) versus growth on benzene (blue colors)...... 131 Figure 6.5. Anaerobic pathway of benzoate degradation (Carmona et al, 2009)...... 133 Figure 6.6. Genes that were transcribed in the cells grown on benzoate (red) and the ones grown on benzene (blue)...... 143 Figure C.1. Anaerobic benzene mineralization in dilution cultures, a) Cartwright pw1 10-5 and b) Cartwright consolidated 10-3, both with and without autoclaved culture...... 182 Figure E.1. Calibration curve for Azoarcus qPCR...... 188 Figure G.1. Cartwright 1b 10-2, Cartwright pw1 10-5, and Cartwright Consolidated 10-4 microscopy images taken at a 40X magnification...... 194 Figure H.1. Benzoate and benzene degradation curves...... 197 Figure H.2. Agarose gel images of individual RNA samples...... 198 Figure H3. The MEGAN tree showing comparison between taxonomic profiles of Cartwright Consolidated culture grown with benzoate (red colors) and with benzene (blue colors)...... 199 Figure H.4. Steps for assembly of a gene by Newbler...... 200
xiii Nomenclature
! change in " efficiency of energy transfer in microorganisms A electron equivalent of the substrate converted for energy
ACC Azoarcus co-culture
ACC1 Azoarcus organism in the Azoarcus co-culture bp base pair
BTEX Benzene, Toluene, Ethylbenzene and Xylenes
CT threshold cycle
DCC Dechloromonas co-culture
DCC1 Dechloromonas organism in Dechloromonas co-culture
DCh Dechloromonas-like microorganisms
DNA deoxyribonucleic acid
Dsoma Dechlorosoma-like microorganisms e-eq electron equivalents fe minimum fraction of electrons used for energy production fs maximum fraction of electrons used for cell synthesis
G free energy
Go` standard free energy (-25oC, reactants & products at unit activity, pH 7)
!Gp free energy of conversion of one electron equivalent of cell carbon source to intermediate
!Gpc free energy of conversion of one electron equivalent of intermediate to one electron equivalent of cells
!Gr free energy per electron equivalent of substrate converted for energy is calculated as the !G for electron donor less the !G for the electron acceptor
xiv GC gas chromatograph mRNA messenger RNA
NCBI National Center for Biotechnology Information
OTU operational taxonomic unit
PCR polymerase chain reaction
R Overall reaction
Ra half-reaction for the electron acceptor
Rc half-reaction for cell synthesis
Rd half-reaction for electron donor
RDP Ribosomal Database Project rRNA ribosomal RNA
SIP Stable Isotope Probing t time
Taq DNA polymerase from Thermus aquaticus
TCE Trichloroethene
V Volume
Y Yield
# Density
xv ! !
Chapter 1: Introduction and Objectives
! 1! ! ! 2!
1.1. Introduction 1.1.1. Benzene: a groundwater contaminant
Human activities have resulted in the release of toxic chemicals into the environment and contamination of groundwater and soil worldwide. In the USA, there are approximately 294,000 sites that are in need of remediation because of the risks pollutants impose on human and the ecosystem health (EPA 2004). Among the major environmental contaminants, simple aromatic compounds such as benzene, toluene, ethylbenzene, and xylene are of great concern due to their relatively high water solubility and toxicity. These compounds enter the environment primarily through leakage of underground petroleum storage tanks, spills at petroleum wells, industrial effluents, wood processing, and manufacturing of pesticides, detergents, chemicals and paints. Benzene is of the greatest concern because it is the most soluble, the most persistent, and the most toxic of all. Several studies have shown a trend in mortality figures because of myelogeneous leukemia as a result of increased accumulative exposure to benzene (Rinsky et al. 1987). Based on these studies, benzene is classified as carcinogenic to humans in the Canadian Environmental Protection Act (CEPA).
According to Environment Canada, 30% of Canadians rely on ground water as their only source of water for domestic use. As a result, access to clean water is crucial. Nevertheless, petroleum products such as benzene frequently contaminate water resources. Canadian Environmental Quality Guidelines allow a maximum acceptable concentration of 5 µg/l of benzene in drinking water (Allen-King et al. 1994). Most of the contaminated fields exceed this limit and therefore must be treated.
1.1.2. Benzene bioremediation
Studies conducted during several past decades have shown that benzene readily biodegrades under aerobic conditions (Gibson et al. 1968; Ridgeway et al. 1990; Harwood and Parales 1996; Gulensoy and Alvarez 1999; Nicholson and Fathepure 2005; Fahy et al. 2006; Witzig et al. 2006). A large number of microorganisms capable of mineralizing benzene aerobically have been identified. Pseudomonas species constitute a major class of these microorganisms (Ridgeway et al. 1990). The pathway of aerobic benzene degradation is also well documented (Wilson and Bouwer 1997; Reardon et al. 2000; Jindrova et al. 2002; Andreoni and Gianfreda
! ! ! 3!
2007). In contaminated sites, aerobic microbes utilize oxygen for metabolism of benzene, which in turn results in the depletion of oxygen and transformation of the sites from aerobic to anaerobic (Anderson and Lovley 1997). The frequent occurrence of anoxic conditions in contaminated sites resulting from rapid consumption of oxygen by aerobic bacteria and low solubility of oxygen in water has generated interest in anaerobic biodegradation of benzene.
Anaerobic degradation of benzene has long been observed in polluted fields and microcosms studied under different electron accepting conditions, including nitrate-reducing (Nales et al. 1998; Burland and Edwards 1999; Coates et al. 2001; Ulrich and Edwards 2003; Kasai et al. 2006; Kasai et al. 2007), sulfate-reducing (Grbi!-Gali! and Vogel 1987; Edwards and Grbi!- Gali! 1992; Lovley et al. 1995; Phelps et al. 1996; Kazumi et al. 1997; Nales et al. 1998; Phelps and Young 1999; Caldwell and Suflita 2000; Vogt et al. 2007; Kleinsteuber et al. 2008; Musat and Widdel 2008; Abu Laban et al. 2009; Herrmann et al. 2010), iron-reducing (Lovley et al. 1996; Anderson and Lovley 1997; Kazumi et al. 1997; Nales et al. 1998; Rooney-Varga et al. 1999; Jahn et al. 2005; Botton and Parsons 2007; Kunapuli et al. 2007), and methanogenic conditions (Grbi!-Gali! and Vogel 1987; Kazumi et al. 1997; Ulrich and Edwards 2003; Chang et al. 2005). In spite of the significance of anaerobic benzene degradation, very little is known about the microorganisms involved. So far, only four bacteria that oxidize benzene anaerobically have been isolated. They are related to the genus Dechloromonas and Azoarcus in the beta- subclass of Proteobacteria and couple oxidation of benzene to nitrate reduction (Coates et al. 2001; Chakraborty and Coates 2004; Kasai et al. 2006; Kasai et al. 2007). Anaerobic degradation of benzene has been studied in detail for one of these isolated bacteria called Dechloromonas aromatica strain RCB (Coates et al. 2001; Chakraborty and Coates 2005; Chakraborty et al. 2005; Salinero et al. 2009). A striking finding about Dechloromonas aromatica is the absence of genes that encode for key enzymes involved in anaerobic degradation of monoaromatic compounds such as toluene, phenol, and benzoate from the genome of this bacterium (Salinero et al. 2009). Therefore, the mechanisms and genes employed by Dechloromonas aromatica strain RCB to mineralize benzene and other aromatic compounds remain unidentified and uncharacterized. Recently, Holmes et al. (2011) reported that the hyperthermophilic archaeon Ferroglobus placidus could grow anaerobically on benzene using Fe (III) as an electron acceptor. To date, this is the only microorganism in pure culture that is capable of growth on benzene under strict anaerobic conditions.
! ! ! 4!
To confirm that in situ mineralization of a pollutant through microbial activities can occur at a site and to verify the efficiency of this process for remediation, a comprehensive characterization of site-specific biodegradation processes is necessary. This can be accomplished via detection of metabolites and functional genes responsible for the decomposition of contaminants. Metabolites formed during oxidation of benzene can be used as an evidence for in situ biodegradation. Examining functional genes encoding key enzymes of degradation pathway can provide the most conclusive evidence of anaerobic mineralization of benzene by microbial communities. A prerequisite for employing these approaches as a direct evidence of in situ bioremediation is the knowledge of the degradation pathway and genes and enzymes involved in this process. Despite significant efforts in the past, little is known about anaerobic benzene mineralization pathway, genes, and enzymes.
1.2. Research objectives
Microbial consortia that degrade benzene under a variety of anaerobic conditions have been enriched in our laboratory for several years. The nitrate-reducing cultures are the focus in this study because of their relatively fast-growing nature. In addition, similar to benzene, nitrate is a common groundwater contaminant due to its use in fertilizers (Weil et al. 1990; Starr and Gillham 1993). During benzene mineralization coupled to nitrate reduction, both pollutants will simultaneously be eliminated from groundwater.
While anaerobic degradation of benzene has been extensively studied in the past, little is known about microorganisms and metabolic pathways involved in this process. This is in contrast to toluene, which is a very similar compound to benzene. Toluene can be degraded under different electron-accepting conditions such as nitrate-reducing, sulfate-reducing, iron- reducing, and methanogenic conditions (Carmona et al. 2009). Most of what is known about the anaerobic catabolism of toluene is based on studies carried out with isolated denitrifying Thauera aromatica strains K172 and T1 and with Azoarcs sp. strain T (Carmona et al. 2009; Dolfing et al. 1990; Evans et al. 1991; Schocher et al. 1991; song et al. 1998). The initial step of anaerobic mineralization of toluene is addition of methyl group of toluene to fumarate catalyzed by benzylsuccinate synthase. This enzyme is conserved among all microorganisms with the ability to anaerobically degrade toluene (Carmona et al. 2009). Enhancing our knowledge of microbial communities mediating benzene mineralization can potentially lead to the
! ! ! 5! development of well-characterized cultures with high degradation capacities and tolerance to a broad range of environmental, chemical or physical stresses. These cultures can subsequently be used for remediation of polluted fields. Successful application of bioremediation to contaminated sites requires identification of parameters that hinder or facilitate the biodegradation process. Such information can be utilized to supply the microorganisms with necessary compounds to enhance the rate of remediation. Understanding the fate of benzene under anaerobic conditions is as important as identifying microorganisms that participate in benzene degradation. Since benzene has the highest C-H bond dissociation energy among all hydrocarbons, its activation requires novel reactions (Carmona et al. 2009), which are potentially useful in several areas including chemical synthesis, green chemistry, and biotechnology. For example, elucidating activation mechanism of benzene ring could contribute to our understanding of decomposition of other compounds such as poly aromatic hydrocarbons (PAHs).
This thesis seeks to approach the aforementioned knowledge gaps. To this end, the overall aim of this work is to identify microorganisms and metabolic steps involved in benzene mineralization in benzene-degrading nitrate-reducing cultures.
The three main hypotheses of this study are as follows:
1) The medium composition used for maintaining the anaerobic benzene-degrading enrichment cultures is not currently optimal. Some components required by benzene-degrading bacteria are missing from the medium. This medium contains ingredients that have an inhibitory impact on growth of key microorganisms present in the culture. 2) Unlike the case for similar compounds such as toluene, anaerobic degradation of benzene is mediated by several distinct microbial genera, even within a single enrichment culture. 3) Pure cultures are not required to predict the metabolic pathway of anaerobic benzene degradation; metatranscriptomic and “next generation sequencing” approaches enable such analyses in mixed cultures.
To address these hypotheses, four research objectives were investigated as follows:
1) To increase the growth rates and shorten lag times in anaerobic benzene-degrading nitrate- reducing enrichment cultures.
! ! ! 6!
2) To identify microorganisms present in the microcosms and to monitor the abundance of specific phylotypes within the benzene-degrading nitrate-reducing cultures during biodegradation of benzene using molecular approaches. 3) To isolate pure cultures of benzene-degrading denitrifying microbes. 4) To investigate the metabolic pathway of anaerobic benzene oxidation and genes and enzymes that are involved in this process and exist in the enrichment cultures.
1.3. Thesis outline
Chapter 2 provides the theoretical background and an overview of the research conducted in this field. Chapter 3 addresses the first objective and describes the investigation of parameters affecting the growth of nitrate-reducing cultures. The second objective is covered in Chapter 4 where molecular biology approaches are employed to identify and quantify bacterial species in the cultures. Chapter 4 is prepared in the format of a paper to be submitted to Applied and Environmental Microbiology. The authors of this paper and their contributions are as follows: Roya Gitiafroz designed and carried out the experiments, analyzed data, and drafted the manuscript; Cheryl E. Devine participated in performing qPCR experiments; Lutgarde Raskin and Elizabeth A. Edwards helped conceived of the study and to draft the manuscript. Chapter 5 describes the attempts made to isolate benzene-degrading nitrate-reducing microbes from the enriched cultures. Identification of key metabolic steps and genes involved in anaerobic degradation of benzene is provided in Chapter 6, which is presented in a paper format and is intended for eventual submission to Applied and Environmental Microbiology. The following is the list of authors and authors’ contributions: Roya Gitiafroz designed and performed all the experiments, analyzed RNA sequences obtained using 454 pyrosequencing, and wrote the first draft of the manuscript (which is the thesis chapter); Fei Luo and Yunchen Gong are currently analyzing additional RNA sequences obtained using Illumina sequencing; eventually, the data from these sequences will be added to the current manuscript draft as these sequences help confirm results. Lutgarde Raskin and Elizabeth A. Edwards conceived of the study, participated in its design and coordination and will help write the final manuscript. A general discussion and synthesis of this work is provided in Chapter 7. Finally, conclusions and engineering significance of this study are described in Chapter 8.
! ! !
1.4. References Chapter 1
Abu Laban, N., D. Selesi, C. Jobelius and R. U. Meckenstock (2009). "Anaerobic benzene degradation by gram-positive sulfate-reducing bacteria." FEMS Microbiol. Ecol. 68: 300-311.
Allen-King, R. M., J. F. Barker, R. W. Gillham and B. K. Jensen (1994). "Substrate-limited and nutrient-limited toluene biotransformation in sandy soil." Environ. Toxicol. Chem. 13: 693-705.
Anderson, R. T. and D. R. Lovley (1997). "Ecology and biochemistry of in situ groundwater bioremediation." Adv. Microb. Ecol. 15: 289-350.
Andreoni, V. and L. Gianfreda (2007). "Bioremediation and monitoring of aromatic-polluted habitats." Appl. Microbiol. Biotechnol. 76: 287-308.
Botton, S. and J. R. Parsons (2007). "Degradation of BTX by dissimilatory iron-reducing cultures." Biodegradation 18: 371-381.
Burland, S. M. and E. A. Edwards (1999). "Anaerobic benzene biodegradation linked to nitrate reduction." Appl. Environ. Microbiol. 65(2): 529-533.
Caldwell, M. E. and J. M. Suflita (2000). "Detection of phenol and benzoate as intermediates of anaerobic benzene biodegradation under different terminal electron accepting condition." Environ. Sci. Technol. 34: 1216-1220.
Carmona, M., M. T. Zamarro, B. Blázquez, G. Durante-Rodríguez, J. F. Juárez, J. A. Valderrama, M. J. Barragán, J. L. García and E. Díaz (2009). "Anaerobic catabolism of aromatic compounds: a genetic and genomic view." Microbiol. Mol. Biol. Rev. 73(1).
Chakraborty, R. and J. D. Coates (2004). "Anaerobic degradation of monoaromatic hydrocarbons." Appl. Microbiol. Biotechnol. 64: 437-446.
Chakraborty, R. and J. D. Coates (2005). "Hydroxylation and carboxylation-two crucial steps of anaerobic benzene degradation by Dechloromonas Strain RCB." Appl. Environ. Microbiol. 71(9): 5427-5432.
Chakraborty, R., S. M. O’Connor, E. Chan and J. D. Coates (2005). "Anaerobic biodegradation of benzene, toluene, ethylbenzene, and xylene compounds by Dechloromonas strain RCB." Appl. Environ. Microbiol. 71(12): 8649-8655.
Chang, W., Y. Um and T. R. P. Holoman (2005). "Molecular charecterization of anaerobic microbial communities from benzene-degrading sediments under methanogenic conditions." Biotechnol. Prog. 21: 1789-1794.
! 7! ! ! 8!
Coates, J. D., R. Chakraborty, J. G. Lack, S. M. O’Connor, K. A. Cole, K. S. Bender and L. A. Achenbach (2001). "Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas." Nature 411: 1039-1043.
Dolfing, J., J. Zeyer, P. Binder-Eicher, and R. P. Schwarzenbach (1990). "Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen." Arch. Microbiol. 154: 336–341.
Edwards, E. A. and D. Grbi!-Gali! (1992). "Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions." Appl. Environ. Microbiol. 58: 2663-2666.
EPA (2004). Cleaning up the nation's waste sites: markets and technology trends. Office of Solid Waste and Emergency Response, Washington DC.
Evans, P. J., D. T. Mang, K. S. Kim, and L. Y. Young (1991). "Anaerobic degradation of toluene by a denitrifying bacterium."Appl. Environ. Microbiol. 57: 1139–1145.
Fahy, A., T. J. McGenity, K. N. Timmis and A. S. Ball (2006). "Heterogeneous aerobic benzene- degrading communities in oxygen-depleted groundwaters." FEMS Microbiol. Ecol. 58: 260-270.
Gibson, D. T., J. R. Koch and R. E. Kallio (1968). "Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene." Biochemistry 7: 2653-2658.
Grbi!-Gali!, D. and T. M. Vogel (1987). "Transformation of toluene and benzene by mixed methanogenic cultures." Appl. Environ. Microbiol. 53: 254-260.
Gulensoy, N. and P. J. J. Alvarez (1999). "Diversity and correlation of specific aromatic hydrocarbon biodegradation capabilities." Biodegradation 10: 331-340.
Harwood, C. S. and R. E. Parales (1996). "The B-Ketoadipate pathway and the biology of self- identity." Ann. Rev. Microbiol. 50: 553-590.
Herrmann, S., S. Kleinsteuber, A. Chatzinotas, S. Kupparsdt, T. Lueders, H. H. Richnow and C. Vogt (2010). "Functional charecterization of an anaerobic benzene-degrading enrichment culture by DNA stable isotope probing." Environ. Microbiol. 12(2): 401-411.
Holmes, D. E., C. Risso, J. A. Smith and D. R. Loveley (2011). "Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus." Appl. Environ. Microbiol. 77(17): 5926-5933.
Jahn, M. K., S. B. Haderlein and R. U. Meckenstock (2005). "Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures." Appl. Environ. Microbiol. 71: 3355-3358.
! ! ! 9!
Jindrova, E., M. Chocova, K. Demnerova and V. Brenner (2002). "Bacterial aerobic degradation of benzene, toluene, ethylbenzene and xylene." Folia Microbiol. 47(2): 83-93.
Kasai, Y., Y. Kodama, Y. Takahata, T. Hoaki and K. Watanabe (2007). "Degradative capacities and bioaugmentation potential of anaerobic benzene-degrading bacterium strain DN11." Environ. Sci. Technol. 41: 6222-6227.
Kasai, Y., M. Manefield and K. Watanabe (2006). "RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater." Appl. Environ. Microbiol. 72 (5): 3586-3592.
Kazumi, J., M. E. Cadwell, J. M. Sulfita, D. R. Lovley and L. Y. Young (1997). "Anaerobic degradation of benzene in diverse anoxic environments." Environ. Sci. Technol. 31: 813-818.
Kleinsteuber, S., K. M. Schleinitz, J. Breitfeld, H. Harms, H. H. Richnow and C. Vogt (2008). "Molecular charecterization of bacterial communities mineralizing benzene under sulfate- reducing conditions." FEMS Microbiol. Ecol. 66: 143-157.
Kunapuli, U., T. Lueders and R. U. Meckenstock (2007). "The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation." ISME J. 1: 643-653.
Lovley, D. R., J. D. Coates, J. C. Woodward and E. J. Phillips (1995). "Benzene oxidation coupled to sulfate reduction." Appl. Environ. Microbiol. 61: 953-958.
Lovley, D. R., J. C. Woodward and F. H. Chapelle (1996). "Rapid anaerobic benzene oxidation with a variety of chelated Fe (III) forms." Appl. Environ. Microbiol. 62: 288-291.
Musat, F. and F. Widdel (2008). "Anaerobic degradation of benzene by a marine sulfate- reducing enrichment culture, and cell hybridization of the dominant phylotype." Environ. Microbiol. 10(1): 10-19.
Nales, M., B. J. Butler and E. Edwards (1998). "Anaerobic benzene biodegradation: a microcosm survey." Bioremediat. J. 2: 125-144.
Nicholson, C. A. and B. Z. Fathepure (2005). "Aerobic biodegradation of benzene and toluene under hypersaline conditions at the great salt plains, Oklahoma." FEMS Microbiol. Lett. 245: 257-262.
Phelps, C. D., J. Kazumi and L. Y. Young (1996). "Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction." FEMS Microbiol. Lett. 145: 433-437.
Phelps, C. D. and L. Y. Young (1999). "Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments." Biodegradation 10: 15-25.
! ! ! 10!
Reardon, K. F., D. C. Mosteller and J. D. B. Rogers (2000). "Biodegradation kinetics of benzene, toluene, and phenol as single and mixed substrates for Pseudomonas putida F1." Biotechnol. Bioeng. 69(4): 385-400.
Ridgeway, H. F., J. Safarik, D. Phipps, P. Carl and D. Clark (1990). "Identification and catabolic activity of well-derived gasoline-degrading bacteria and a contaminated aquifer." Appl. Environ. Microbiol. 56: 3565-3575.
Rinsky, R. A., A. B. Smith, R. Hornung, T. G. Filloon, R. J. Young, A. H. Okun and P. J. Landrigan (1987). "Benzene and Leukemia - An epidemiologic risk assessment." N. Engl. J. Med. 316: 1044-1050.
Rooney-Varga, J. N., R. T. Anderson, J. L. Fraga, D. Ringelberg and D. R. Lovley (1999). "Microbial communities associated with anaerobic benzene degradation in a petroleum- contaminated aquifer." Appl. Environ. Microbiol. 65: 3056-3063.
Salinero, K. K., K. Keller, W. S. Feil, H. Feil, S. Trong, G. D. Bartolo and A. Lapidus (2009). "Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation " BMC Genomics 10: 351-74.
Schocher, R. J., B. Seyfried, F. Vazquez, and J. Zeyer (1991). "Anaerobic degradation of toluene by pure cultures of denitrifying bacteria." Arch. Microbiol. 157: 7–12.
Song, B., L. Y. Young, and N. J. Palleroni (1998). "Identification of denitrifier strain T1 as Thauera aromatica and proposal for emendation of the genus Thauera definition." Int. J. Syst. Bacteriol. 48: 889–894.
Starr, R. C. and R. W. Gillham (1993). "Denitrification and Organic Carbon Availability in Two Aquifers." Ground Water 31(6): 934-947.
Ulrich, A. C. and E. A. Edwards (2003). "Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures." Environ. Microbiol. 5(2): 92-102.
Vogt, C., S. Gödeke, H. C. Treutler, H. Weiß, M. Schirmer and H. H. Richnow (2007). "Benzene oxidation under sulfate-reducing conditions in columns simulating in situ conditions." Biodegradation 18: 625-636.
Weil, R. R., R. A. Weismiller and R. S. Turner (1990). "Nitrate Contamination of Groundwater Under Irrigated Coastal Plain Soils." J. Environ. Qual. 19: 441-448.
Wilson, L. P. and E. J. Bouwer (1997). "Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review." J. Ind. Microbiol. Biotechnol. 18: 116-130.
Witzig, R., H. Junca, H. J. Hecht and D. H. Pieper (2006). "Assessment of toluene/biphenyl dioxygenase gene diversity in benzene-polluted soils: links between benzene biodegradation and
! ! ! 11! genes similar to those encoding isopropylbenzene dioxygenases." Appl. Environ. Microbiol. 72: 3504-3514.
! ! !
Chapter 2. Background and Literature Review
! 12! ! 13! ! 2.1. Background 2.1.1. Bacterial energetics and growth
Microorganisms gain their energy for growth (cell synthesis) and cell maintenance from oxidation-reduction reactions. These reactions involve transfer of electrons from the electron donor to the electron acceptor to produce energy. This energy is expressed as change in free energy of reaction (!G) and is calculated by subtracting the free energy of formation of products from that of reactants. The free energy of formation is the energy required for the formation of a given molecule from its constituting elements. The change in the free energy of a reaction is usually calculated under standard physiological conditions (25°C, pH=7, and all products and reactants at unit activity) and is annotated as !G°". The more negative the value of !G°" of an oxidation-reduction reaction, the more energy is released.
Table 2.1 shows the redox reactions and the change in standard free energy of benzene oxidation coupled to reduction of nitrate to nitrite and nitrogen gas (refer to Appendix A for detail calculations). For the mineralization of each mole of benzene, fifteen moles of nitrate are required if nitrate is reduced to nitrite, or six moles of nitrate are needed if nitrate is converted to nitrogen gas. To calculate these stoichiometric ratios, it is assumed that all of the electrons of the electron donor are used for energy production. However, bacteria employ some of these electrons for cell synthesis. The fraction of electrons that is required for energy production is designated as fe and the portion consumed for cell synthesis as fs. Both fe and fs can be estimated by the method proposed by Rittman and McCarty (McCarty 1971; Rittmann and McCarty 2001) which is described in Appendix A. Taking into account values of fe and fs, and the equation for cell synthesis, the stoichiometric ratios for relevant redox couples are calculated as outlined in Appendix A. Table 2.2 provides the summary of the balanced stoichiometric equations allowing for cell growth. It is assumed that the efficiency of electron transfer (#) is 0.6 for calculation of these stoichiometric ratios. Studies by Ulrich and Edwards (2003) indicated that the observed yields for our benzene-degrading denitrifying Cartwright and Swamp enrichment cultures were 8.6±1.8 and 22±7.7 g of cells/mole of benzene, respectively. These values are much lower than theoretical yields of 101.7 and 79.1 g of cells/mole of benzene predicted for benzene degradation coupled to reduction of nitrate to nitrogen or to nitrite based on the stoichiometric equations in Table 2.2 (it is assumed that each mole of cell is 113 g). This suggests that in our cultures, the
! ! 14! ! cell yield and fs values are lower than those obtained by assuming a value of 0.6 for !. It means that the efficiency of electron transfer (!) is lower than 0.6. In this case more energy is lost in the form of heat or due to other electron transfer inefficiencies than when ! is 0.6. Therefore, the observed yields of 8.6 and 22 g of cells/mole of benzene were used to calculate fs and fe and to derive the stoichiometric equations for mineralization of benzene in the presence of nitrate (refer to Appendix A for details). These equations are given in Table 2.3.
Since benzoate and acetate are used in this study as a carbon source and electron donor, the redox equations for these compounds are also provided in Table 2.1 and Table 2.2.
Table 2.1. Energy-yielding equations and standard free energy changes for oxidation of various electron donors coupled to reduction of nitrate to nitrogen gas or nitrate to nitrite.
Electron Electron "G°# acceptor Overall energy equation donor (KJ/mol) (ox/red)
NO -/N " " -2987 3 2 C 6 H6 + 6NO3 # 6 HCO3 +3N2 Benzene NO -/NO - " " " + -2070 3 2 C 6 H6 + 15 NO3 +3H2 O # 6 HCO3 + 15 NO2 + 6 H NO -/N " " + " -2990 !3 2 C 6 H5 COO + 6NO3 + 6H # 6CO2 + HCO3 + 3N2 + 5H2 O Benzoate NO -/NO - " " " " -2070 !3 2 C 6 H5 COO + 15 NO3 # 6CO2 + HCO3 + 15 NO2 + 2 H2 O
!- " 8 " 8 + " 4 9 NO3 /N2 CH3 COO + NO3 + H # CO2 + HCO3 + N2 + H2 O -800 Acetate 5 5 5 5 ! NO -/NO - " " " " -552 3 2 CH 3 COO + 4 NO3 # CO2 + HCO3 + 4 NO2 + H2 O !
!
! ! 15! ! Table 2.2. Theoretical stoichiometric equations for oxidation of various electron donors coupled to either reduction of nitrate to nitrite or reduction of nitrate to nitrogen gas considering cell growth. These equations were derived based on thermodynamic calculations assuming an efficiency of electron transfer of !=0.6.
Electron Electron a acceptor Stoichiometry equation fe fs donor (ox/red)
" + C6 H6 + 2.6NO3 + 0.9NH4 +3.4CO2 +2.6H2 O# - NO3 /N2 " + 0.43 0.57 1.3N2 +5.1HCO3 +0.9C5 H7 O2 N+3.4H Benzene " + C6 H6 +7.7 NO3 + 0.7NH4 +2.9CO2 +3.7H2 O# NO -/NO - ! 0.52 0.48 !3 2 " " + 7.7NO2 +5.3HCO3 +0.7C5 H7 O2 N+6H " " + + C6 H5 COO + 2.6NO3 + 0.9NH4 +2.6H # - NO3 /N2 " 0.43 0.57 1.3N2 +0.1HCO3 +0.9C5 H7 O2 N+2.4H2 O+2.6CO2 ! Benzoate " " + C6 H5 COO + 7.8NO3 + 0.7NH4 # - - NO!3 /NO2 " " 0.52 0.48 7.8NO2 +0.3HCO3 +0.7C5 H7 O2 N+1.3H2 O+3.1CO2
" " + + CH3COO +0.7NO3 + 0.2NH 4 +0.7H # - NO!3 /N 2 " 0.42 0.58 0.3N2 +0.8HCO3 +0.2C5 H7O2N+1.1H2O+0.07CO2
Acetate " " + CH3COO + 2.1NO3 + 0.2NH 4 # - - NO!3 /NO2 " " 0.52 0.48 2.1NO2 +0.8HCO3 +0.2C5 H7O2N+0.8H2O+0.2CO2
! a To evaluate if reduction! of an electron acceptor is coupled to oxidation of an electron donor, one could compare the measured values of electron acceptor reduced per values of electron donor mineralized in the cultures to the values shown in bold.
!
! ! 16! ! Table 2.3. Experimentally derived stoichiometric equations for oxidation of benzene coupled to either reduction of nitrate to nitrite or reduction of nitrate to nitrogen gas. fs was calculated from the observed yields of 8.6 and 22 g of cells/mole of benzene for Cartwright and Swamp enrichment cultures, respectively.
Observed Electron Electron Yield a acceptor Stoichiometry equation fe fs donor (g cells/mole (ox/red) benzene)
" + C H +5.7NO3 + 0.08NH4 +0.3 CO +0.23H O# - 6 6 2 2 ! 0.95 0.05 NO3 /N2 8.6 " + 2.9N +5.9HCO3 +0.08C H O N+0.3H Benzene 2 5 7 2
" + C6 H6 +14.3 NO3 + 0.08 NH4 +0.3 CO2 +3.1 H2 O# - - NO3 /NO2 8.6 " " + 0.95 0.05 ! 14.3NO2 +5.9HCO3 +0.08C5 H7 O2 N+6H !
" + C H +5.2NO3 + 0.2NH4 +0.78CO +0.59H O# NO -/N 22 6 6 2 2 ! 0.87 0.13 3 2 ! " + 2.6N2 +5.8HCO3 +0.2C5 H7 O2 N+0.78H Benzene " + C6 H6 +13.1 NO3 + 0.2 NH4 +0.78 CO2 +3.2 H2 O# NO -/NO - 22 " " + 0.87 0.13 3 2 13.1NO2 +5.8HCO3 +0.2C5 H7 O2 N+6H ! a To evaluate if reduction of benzene! in our cultures is coupled to oxidation of nitrate to nitrogen gas or to nitrite, we should compare the measured amounts of electron acceptor reduced per amounts of electron donor mineralized in the cultures to the values shown in bold.
2.2. Literature review 2.2.1. Aerobic benzene degradation
Oxygen is the most common electron acceptor for bacterial respiration and growth. Aromatic compounds such as benzene are readily biodegradable in the presence of oxygen. Metabolism of benzene by aerobic microorganisms has extensively been studied for several decades (Gibson et al. 1968; Ridgeway et al. 1990; Harwood and Parales 1996; Gulensoy and Alvarez 1999; Nicholson and Fathepure 2005; Fahy et al. 2006; Witzig et al. 2006). The key model organisms that have provided us with the details about biochemical and genetic information regarding aerobic benzene mineralization include Pseudomonas, Comamonas, Alcaligenes, Acinetobacter, and Burkholderia spp. (Liou et al. 2008).
! ! 17! ! Aerobic biodegradation of benzene involves its oxidation by molecular oxygen resulting in the formation of intermediates, which enter central metabolic pathways such as Krebs cycle and !-oxidation (Wilson and Bouwer 1997; Andreoni and Gianfreda 2007; Cao et al. 2009). The initial step in degradation pathway of benzene is incorporation of two atoms of oxygen into the benzene ring by a multicomponent dioxygenase enzyme system (Jindrova et al. 2002; Habe and Omori 2003). This results in hydroxylation of benzene and in production of an unstable metabolite called cis-benzene dihydrodiol (Figure 2.1). cis-benzene dihydrodiol is dehydrogenated by cis-diol dehydrogenase to catechol (Habe and Omori 2003). As it is shown in Figure 2.1 the next step is opening of the ring. Ring cleavage occurs by the action of either intradiol (ortho) or extradiol (meta) dioxygenases, which break the aromatic ring between the two hydroxyl groups (ortho cleavage) or proximal to one of the two hydroxyl groups (meta cleavage) by addition of molecular oxygen (Williams and Sayers 1994; Whiteley and Lee 2006; Andreoni and Gianfreda 2007; Cao et al. 2009). The intradiol dioxygenase is catechol 1,2- dioygenase (EC 1.13.11.1), which converts catechol to cis, cis-muconic acid, and the extradiol dioxygenase is catechol 2,3-dioxygenase (EC 1.13.11.2), which oxidizes catechol to 2- hydroxymuconic semialdehyde (Williams and Sayers 1994; Whiteley and Lee 2006). The products of ring cleavage are further converted to small aliphatic compounds such as simple acids and aldehydes (TCA cycle intermediates) that can enter central metabolic pathways.
! ! 18! !
O2 H OH OH
Dioxygenase OH H OH Benzene cis-benzene dihydrodiol Catechol
OH
OH Ortho or intradiol Catechol Meta or extradiol pathway Catechol 1, 2- Catechol 2, 3- pathway Dioxygenase Dioxygenase O2 O2 OH
COOH COOH COOH CHO
cis, cis-Muconic acid 2-Hydroxymuconic semialdehyde
O COOH COOH COOH
!-Ketoadipic acid OH O 4-Hydroxy-2- O oxovaleric acid COSCoA OH CH3 CH3 COSCoA OH C=O + -Ketoadipyl-CoA CHO ! Acetaldehyde COOH Pyruvic acid COSCoA CH3 CH3 OH + COSCoA COSCoA COSCoA Succinyl-CoA Acetyl-CoA Acetyl-CoA
! Figure 2.1. Metabolic pathway of aerobic benzene degradation (Habe and Omori 2003). ! 19! ! 2.2.2. Anaerobic benzene degradation
Prior to 1986 there was no evidence for benzene degradation under anaerobic conditions. The early studies conducted by Vogel and Grbi!-Gali! (Vogel and Grbi!-Gali! 1986; Grbi!-Gali! and Vogel 1987) with methanogenic enrichment cultures, which were originally established from sewage sludge, indicated the possibility of anaerobic benzene mineralization. This finding provided the impetus for a wealth of studies carried out later in this area. Subsequently, anaerobic benzene oxidation has been linked to nitrate reduction, ferric iron reduction, sulfate reduction, and methanogenesis. The following sections provide an overview of research performed in this field.
Mineralization of benzene under different electron accepting conditions Nitrate
Nitrate is a preferable alternative to oxygen among other electron acceptors available to subsurface microorganisms. This is because reduction of nitrate provides the microbes with an energy yield close to that of oxygen, it has a relatively high water solubility (660 g/l), it is inexpensive, it is nontoxic to microorganisms at concentrations below 500 mg/l, and it does not form precipitate oxides as oxygen does (Hutchins 1991; Wilson and Bouwer 1997). In addition nitrate-reducing bacteria are nutritionally versatile and widely distributed in the environment, in the subsurface and in association with contaminated aquifer materials (Wilson and Bouwer 1997; Caldwell et al. 1999), which make them good candidates for remediation purposes.
Microorganisms use nitrate as an electron acceptor in energy metabolism. The first step in dissimilative nitrate reduction is conversion of nitrate to nitrite mediated by an enzyme called nitrate reductase (Wilson and Bouwer 1997; Madigan et al. 2009). Nitrate reductase is a molybdenum-containing enzyme whose synthesis is repressed by molecular oxygen (Madigan et al. 2009). Further reduction of nitrite by nitrite reductase results in the formation of nitric oxide. Nitric oxide is then converted to nitrous oxide in a reaction catalyzed by nitric oxide reductase. Finally, nitrogen gas is produced from nitrous oxide in the presence of nitrous oxide reductase. All of these steps are collectively called denitrification and bacteria involved in this process are called denitrifiers. Several studies have shown that the presence of molecular oxygen suppresses denitrification and the synthesis and/or activity of denitrifying enzymes especially those of
! ! 20! ! nitrate reductase (Wilson and Bouwer 1997; Madigan et al. 2009). As a result, anoxic conditions are required for the reduction of nitrate to occur. We note that there are also some organisms that reduce nitrite to ammonium in a dissimilative process, but this is not the focus of present study.
For a long time, benzene was considered to be recalcitrant in the field under nitrate-reducing conditions (Johnson et al. 2003). Early studies by Major et al. (1988) and Morgan et al. (1993) indicted the nitrate-dependent removal of benzene in anaerobic incubations of aquifer sediments and groundwater. Since the ratio of benzene consumed per nitrate reduced was not measured, it was unclear whether oxidation of benzene was coupled to nitrate reduction. Nales et al. (1998) provided the first firm evidence for benzene mineralization linkage to the reduction of nitrate. They also found that presence of toluene, ethylbenzene, and xylene competitively inhibited degradation of benzene. Subsequently, the experiments conducted by Burland and Edwards (1999), where 14C-benzene was supplied to the enrichment cultures established from soil and 14 14 groundwater microcosms, showed that 92% to 95% of C-benzene was converted into CO2 with concomitant disappearance of nitrate. In these enrichments, benzene oxidation was more tightly coupled to incomplete reduction of nitrate to nitrite rather than its complete reduction to nitrogen gas.
To date, only four bacteria capable of mineralizing benzene anaerobically have been reported to be isolated. The first two isolates are Dechloromonas sp. JJ and Dechloromonas strain RCB (Coates et al. 2001). These strains are closely related to each other and are members of Dechloromonas genus in the beta subclass of Proteobacteria. Dechloromonas sp. JJ was isolated using a humic-substance analog (2,6-anthrahydroquinone disulphonate) as the electron donor, nitrate as the electron acceptor and acetate as the carbon source. Dechloromonas sp. JJ was reported to completely oxidize benzene in the absence of oxygen and couple benzene oxidation to nitrate reduction. Dechloromonas strain RCB was enriched with 4-chlorobenzoate as an electron donor and chlorate as an electron acceptor, can also couple growth and benzene mineralization to the reduction of nitrate. The two other bacterial isolates are strains AN9 and DN11, which are affiliated with the members of Azoarcus genus (Kasai et al. 2006). Both bacteria grow on benzene using nitrate as an electron acceptor. Molecular analysis of a benzene- degrading denitrifying enrichment culture by Ulrich and Edwards (2003) indicated the presence of bacteria that were similar to both members of Dechloromonas and Azoarcus genus. This
! ! 21! ! further supports the importance of these species in anaerobic benzene degradation under nitrate- reducing conditions.
Ferric iron
The first evidence for complete oxidation of benzene to carbon dioxide coupled to the reduction of ferric iron to ferrous iron came from studies by Lovley et al. (1994) with sediments from a petroleum-contaminated aquifer. Since then, there have been several reports on the degradation of benzene under iron-reducing conditions in microbial consortia (Lovley et al. 1996; Anderson and Lovley 1997; Kazumi et al. 1997; Nales et al. 1998; Caldwell et al. 1999; Rooney-Varga et al. 1999; Jahn et al. 2005; Botton and Parsons 2007; Kunapuli et al. 2007).
Thus far, only one pure culture of a hyperthermophilic archaeon called Ferroglobus placidus capable of mineralizing benzene with iron as an electron acceptor has been obtained (Holmes et al. 2011). Therefore, the current understanding of this process is mostly based on enrichment cultures, with little insight into the roles of different microorganisms in decomposition of benzene. A comparison between 16S rRNA gene sequences of the cultures established with sediments from a petroleum-contaminated aquifer in Bemidji, MN, USA indicated that sediments with benzene degradation ability had significantly higher numbers of sequences related to the Geobacter cluster of Geobacteraceae than those without benzene degradation activity (Anderson et al. 1998; Rooney-Varga et al. 1999). Geobacteraceae were also identified as the dominant phylotype in benzene-degrading consortia obtained from the contaminated aquifer located downstream of the Banisveld landfill, Boxtel, the Netherlands (Botton et al. 2007). These results collectively suggest a role for Geobacteraceae in benzene degradation. More recently, Kunapuli et al. (2007) employed DNA-stable isotope probing to link metabolism of benzene in an iron-reducing enrichment culture to bacteria related to Peptococcaceae and Desulfobulbaceae. They proposed a syntrophic mode of interaction during anaerobic decomposition of benzene. In this culture, members of the Peptococcaceae appeared to be responsible for the initial attack on benzene and assimilation of carbon from 13C-benzene.
Desulfobulbaceae appeared to be thriving primarily on the H2 produced by the primary degrader using iron as electron acceptor, thereby pulling the initial reaction towards completion (Kunapuli et al. 2007).
! ! 22! ! Sulfate
Initial studies suggesting that benzene mineralization could be coupled to the reduction of sulfate were the findings of Edwards and Grbi!-Gali! (1992) that benzene was completely oxidized to carbon dioxide in the presence of sulfate as a potential electron acceptor in microcosms prepared with gasoline-contaminated subsurface sediments from Seal Beach, California. Following this study, there have been several reports on the oxidation of benzene linked to the reduction of sulfate both in laboratory settings and in the field (Lovley et al. 1995; Phelps et al. 1996; Kazumi et al. 1997; Nales et al. 1998; Phelps and Young 1999; Caldwell and Suflita 2000; Vogt et al. 2007; Herrmann et al. 2008; Kleinsteuber et al. 2008; Abu Laban et al. 2009).
No pure culture of a benzene-degrading sulfate-reducing microorganism has been isolated to date. There is little information available regarding the phylotypes that are responsible for sulfate dependent degradation of benzene in enriched cultures. Molecular community analysis of a sulfate-reducing consortium by Phelps et al. (1998) indicated the presence of clones (SB-9, 21, 29, and 30) in the culture that fell within the family Desulfobacteriaceae, whose members are known to degrade aromatic hydrocarbons. DNA-based stable isotope probing and terminal restriction fragment length polymorphism (TRFLP) analysis performed on this culture distinguished SB-21 as the active benzene-degrading microorganism (Oka et al. 2008). A methanogenic benzene-degrading culture was predominantly comprised of an Operational Taxonomic Unit (OR-M2), which was closely related to clone SB-21 (Ulrich and Edwards 2003). In 2008, Musat and Widdel (2008) also identified a Desulfobacteriaceae-related phylotype (designated as clone BznS295) as the dominant bacteria within a sulfidogenic culture and postulated that this organism was solely responsible for benzene mineralization. All these findings support a functional role for bacteria within Desulfobacteriaceae family to anaerobically degrade benzene. Laboratory and field stable isotope probing (SIP) experiments conducted by Liou et al. (2008) showed the dominance of sequences related to Pelomonas in sediment microcosms incubated with benzene under anoxic conditions. Pelomonas was hypothesized to be involved in anaerobic benzene biodegradation. A member of the Cryptanaerobacter/Pelotomaculum group within the Peptococcaceae, and a phylotype related to the genus of the Epsilonproteobacteria showed significant increase in their population in a
! ! 23! ! benzene-degrading sulfate-reducing laboratory enrichment culture after repeated spiking with benzene (Kleinsteuber et al. 2008). Further analysis revealed that the relative abundance of the terminal restriction fragments of these two bacteria increased greatly in heavy fractions of 13C- benzene incubated microcosms compared to controls supplied with 12C-benzene (Herrmann et al. 2010). Based on the possible functions of community members and thermodynamics calculations, authors proposed a syntrophic association for mineralization of benzene. In this process, the Cryptanaerobacter/Pelotomaculum was responsible for initial steps of benzene degradation and the release of reduced metabolites such as hydrogen or other low molecular weight fermentation products usable for a syntrophic partner (Kleinsteuber et al. 2008; Herrmann et al. 2010). Recently, Abu Laban et al. (2009) described a Pelotomaculum-related microorganism within the gram-positive family Peptococcaceae as the dominant member of a sulfidogenic benzene-mineralizing enrichment culture. Since Pelotomaculum was the only prominent species within the culture, it was hypothesized to utilize benzene as the sole carbon source with sulfate as an electron acceptor.
Methanogenic conditions
As explained earlier, Vogel and Grbi!-Gali! (Vogel and Grbi!-Gali! 1986; Grbi!-Gali! and Vogel 1987) provided the first evidence for the mineralization of benzene under methanogenic conditions. Further clue for benzene oxidation under methanogenic conditions was the production of significant quantities of methane after addition of benzene to aquifer sediments by 14 14 14 Kazumi et al. (1997). C-benzene added to these sediments was converted to CH4 and CO2. Subsequently, there have been several reports on benzene mineralization linked to methanogenesis (Weiner and Lovley 1998; Ulrich and Edwards 2003; Chang et al. 2005; Kasai et al. 2005).
Molecular characterization of a methanogenic culture by Ulrich and Edwards (2003) showed the predominant presence of Desulfobacterium-related bacteria (named as OR-M2) and Desulfosporosinus-related strains within the culture (designated as OR-M1). Based on the phylogenetic classification of these two species, they suggested that OR-M2 initiated the attack on benzene while OR-M1 fulfilled the role of a syntrophic bacterium by further breaking the putative metabolites produced by OR-M2 (Ulrich and Edwards 2003). An anaerobic aquifer column was bioaugmented with the aforementioned methanogenic consortium. The quantitative
! ! 24! ! polymerase chain reaction performed using primers targeting OR-M2 on the DNA extracted from this column indicated a strong correlation between benzene degradation activity and the concentration of targeted species (Da Silva and Alvarez 2007). Sakai et al. (2009) employed stable isotope probing to identify microorganisms involved in anaerobic mineralization of benzene in a methanogenic culture established with the soil from Lotus field, in Tsuchiura, Ibaraki, Japan. A bacterium (named as Hasda-A) within the class of Deltaproteobacteria and closely related to OR-M2 was identified as the one assimilating 13C-labled benzene. These results further provided an evidence for OR-M2 like microbes to be involved in degradation of benzene.
Anaerobic benzene degradation pathway
The biochemical pathway of anaerobic benzene degradation is currently unknown but there are several studies that propose initial carboxylation, hydroxylation, methylation, and reduction of benzene ring, followed by subsequent transformation to the central aromatic intermediate benzyl-CoA and ring cleavage as the pathway for benzene oxidation.
Carboxylation
Benzoate was detected for the first time as a metabolite of benzene degradation in a sulfate- reducing culture by Caldwell and Suflita (2000). They considered the direct carboxylation of benzene ring to benzoate as a possible initial step in metabolism of benzene (Figure 2.2). 13 However, experiments performed using C6-benzene revealed that the carboxyl group of benzoate was 13C-labeled. This observation suggested that the carboxyl group might originate from benzene itself and that this process involved a complex reaction. A separate study also showed that addition of 13C-labeled bicarbonate to an enriched sulfate-reducing culture that exhibited transient accumulation of benzoate during benzene degradation did not result in the incorporation of 13C-labeled bicarbonate into the aromatic ring of benzene (Phelps et al. 2001). Hence, bicarbonate did not appear as the origin of carboxyl group. The authors speculated that transformation of benzene to benzoate relied on a more complex set of reactions than simple 13 carboxylation through addition of carbonate (Phelps et al. 2001). Recently, using C6-benzene as 13 a growth substrate for an iron-reducing enriched culture, Kunapuli et al. (2008) detected C6- benzoate in culture supernatant. Additional experiments conducted with this iron-reducing
! ! 25! ! culture and 13C-labeled bicarbonate buffer identified bicarbonate buffer as the source of carboxyl group of benzoate, supporting a direct carboxylation as the mechanism of benzene ring activation (Kunapuli et al. 2008). The proteomes of benzene-, phenol- and benzoate-grown cells of this culture were compared using SDS-PAGE gels. Several carboxylase genes were expressed specifically with benzene as the carbon source and electron donor (Abu Laban et al. 2010). These genes were proposed to be different subunits of an enzyme responsible for carboxylating benzene. Holmes et al. (2011) reported accumulation of benzoate in the pure cultures of hyperthermophilic archaeon Ferroglobus placidus during growth on benzene as the sole electron donor and Fe III as the electron acceptor. Analysis of gene transcript levels of this culture revealed an increase in the expression of genes that encode enzymes of anaerobic benzoate degradation in the cells grown on benzene versus those grown on acetate. Moreover, a putative benzene carboxylase gene similar to the one identified by Abu Laban et al. (2010) was highly expressed in cells supplied with benzene compared to those fed with benzoate. All of these results supported carboxylation of benzene to benzoate as the activation mechanism of benzene ring. Benzoate was also identified as a metabolite of benzene degradation in a sulfate-reducing culture, providing further evidence for the carboxylation of benzene as the initial step in its activation (Abu Laban et al. 2009).
The anaerobic catabolism of benzoate occurs via benzoyl-CoA pathway, which has been studied at a molecular level in facultative anaerobes such as denitrifying Azoarcus spp. (López Barragán et al. 2004a; Kasai et al. 2007), Thauera aromatica (Breese et al. 1998; Harwood et al. 1999), and Magnetospirillum spp. (López Barragán et al. 2004b; Shinoda et al. 2005), in the strictly anaerobic iron-reducing Geobacter spp. (Wischgoll et al. 2005; Kung et al. 2009; Aklujkar et al. 2010), in the fermentative Syntrophus aciditrophicus (McInerney et al. 2007), and in the photosynthetic Rhodopseudomonas palustris (Egland et al. 1997). Degradation of benzoate starts with a one-step peripheral pathway through activation of benzoate to benzoyl-CoA, which is catalyzed by benzoate-CoA ligase. This enzyme couples conversion of benzoate to benzoyl- CoA to the hydrolysis of ATP to AMP and diphosphate (Figure 2.2). The ATP-mediated activation of benzoate is a general feature shared by anaerobic microorganisms regardless of the redox potential of the electron-accepting system (Fuchs 2008; Carmona et al. 2009). Benzoyl- CoA is further metabolized to yield an aliphatic C7-dicarboxyl-CoA derivative through several steps that are collectively called the upper benzoyl-CoA pathway. This pathway consists of two
! ! 26! ! major metabolic steps: 1) the dearomatization/reduction of benzoyl-CoA to a cyclic, conjugated diene and 2) a modified !-oxidation of diene resulting in ring cleavage and formation of the C7- dicarboxylic CoA ester. In anaerobic facultative and phototrophic bacteria, the reductive dearomatization of benzoyl-CoA to cyclohex-(di)ene-carbonyl-CoA is catalyzed by benzoyl- CoA reductase. This enzyme couples transfer of electrons to benzene ring to the stoichiometric ATP hydrolysis (1 ATP/electron) (Carmona et al. 2009). The reduction of benzoyl-CoA by benzoyl-CoA reductase is energetically expensive. Unlike facultative and phototrophic microorganisms, obligate anaerobes and fermenting bacteria cannot afford a stoichiometric ATP- dependent ring reduction (Harwood et al. 1999; Gibson and Harwood 2002; Boll 2005). In addition, the genomes of iron-reducing Geobacter metallireducens and the fermentating Syntrophus aciditrophicus lack the genes encoding for benzoyl-CoA reductase (Butler et al. 2007; McInerney et al. 2007). It appears that obligate anaerobes and fermenters use a different enzyme for the reduction of benzene ring. In these bacteria, an enzyme encoded by BamB-I genes is likely involved in this step and the electron transfer is hypothesized to be driven by a membrane potential (Figure 2.2) (Wischgoll et al. 2005).
After formation of the cyclic (di)enoyl-CoA, a modified !-oxidation pathway is employed to convert this compound to an aliphatic C7-dicarboxyl-CoA. This process involves the addition of water to a double bond by acyl-CoA hydratase, followed by a dehydrogenation by hydroxyacyl- CoA dehydrogenase, and finally a hydrolytic ring cleavage catalyzed by oxoacyl-CoA hydrolase. As it is shown in Figure 2.2, in the modified !-oxidation pathway for both facultative and obligate anaerobes cylohex-1, 5-diene-carbonyl-CoA is transformed to 3-hydroxy-pimelyl-CoA as the final product via several enzymatic reactions (Carmona et al. 2009). Phototrophic bacteria employ another type of modified !-oxidation in which the cyclic monoenoyl-CoA produced from reduction of benzoyl-CoA is used as the substrate resulting in production of pimelyl-CoA (Pelletier and Harwood 1998; Harwood et al. 1999; Pelletier and Harwood 2000). Further degradation of aliphatic C7-dicarboxyl-CoA to three acetyl-CoA and one CO2 occurs through !- oxidation, which is also referred to as the lower benzoyl-CoA pathway (Figure 2.2).
! ! 27! ! ! COO- COSCoA CoA, ATP AMP, PPi CO2
Benzoyl-CoA ligase (1. BzdA, "#!$%&' (#!$)*+,!!!!!!!! Benzene Benzoate -#!$)*+, .#!$)/'0 $12345&674'! ! Obligate Facultative & ! anaerobes &! Phototrophic fermenting bacteria bacteria Dearomatization Benzene ring
5. 2.BcrCBADFdx 1.BzdNOPQBzdM reductase Benzoyl 4 3.BamB 2ATP, 2H .
bacterBamB ia 2H BadDEFGBadB
- - -
CoA I ? I ? 2ADP, 2P i Phototrophic bacteria
COSCoA COSCoA COSCoA
Upper Pathway
2H Cyclohex-1,5-diene-1carbonyl-CoA Cyclohex-1ene-1- Cyclohexadienoyl- carbonyl-CoA H2O
5. CoA hydratase
BadK H2O 1.BzdW, 2.Dch 3.BamR, 4.BamR
Modified COSCoA COSCoA HO HO
! - oxidation 6-Hydroxycyclohex-1-ene-1-carbonyl-CoA 2-Hydroxycyclohexane- + Hydroxyenoyl-CoA NAD 1-carbonyl-CoA dehydrogenase 5. + + Bad NAD 1.Bzdx, 2.Had
NADH+H + 3.BamQ, 4.BamQ H NADH+H COSCoA COSCoA COSCoA COSCoA O HO - O 2H2O COO COO- H2O Oxoenoyl-CoA hydrolase 5.BadI 6-Ketocyclohex-1- 1.BzdY, 2.Oah 3-Hydroxy Pimelyl-CoA 2-Ketocyclohexane- ene-1-carbonyl-CoA 3.BamA, 4.BamA -pimelyl-CoA 1-carbonyl-CoA Lower Lower 3Acetyl-CoA + CO pathway 2 pathway
Figure 2.2. Carboxylation as the initial step of anaerobic benzene degradation. Benzoate is metabolized through benzoyl-CoA pathway. Genes encoding the enzymes involved in various steps of the pathway for different bacteria are given either next to or below the arrows in the figure. The numbers next to the name of each gene correspond to the gene identified in Azoarcus spp. (1), Thauera aromatica (2), Geobacter spp. (3), Syntrophus aciditrophicus (4), and Rhodopseudomonas palustris (5), respectively (Carmona ! et al. 2009). ! 28! ! Hydroxylation
In 1987, phenol, cyclohexanone, and propanoic acid were detected as intermediates of benzene degradation in a methanogenic enrichment culture (Grbi!-Gali! and Vogel 1987). It was proposed that the benzene degradation pathway involved hydroxylation of benzene to phenol followed by the subsequent reduction of phenol to cyclohexanone, which was then cleaved to form aliphatic acids (Figure 2.3.a). Subsequently, experiments conducted using 18O-labled water, suggested that the oxygen group derived from water was incorporated into benzene, converting it to phenol (Vogel and Grbi!-Gali! 1986). Weiner and Lovley (1998) also reported the production of phenol, propionate and acetate in methanogenic cultures during isotope trapping experiments with [14C]- benzene. This provided another piece of evidence for hydroxylation of the benzene ring to phenol. In 2000, Caldwell and Suflita (2000) observed accumulation of [13C-UL]-phenol and [13C-UL]-benzoate in sulfate-reducing enrichments supplemented with [13C-UL]-benzene. Chakraborty et al. (2005) also identified phenol and benzoate as metabolites of benzene mineralization by denitrifying Dechloromonas strain RCB. They proposed that the benzene degradation pathway starts with hydroxylation of benzene to phenol followed by loss of the hydroxyl group and formation of benzoate. It was hypothesized that hydroxylation of benzene ring was mediated through a hydroxyl free radical that did not originate from water but rather, formed on the outer membrane or in the periplasm of this bacterium. Phenol and benzoate were also detected in a methanogenic culture as well as an iron-reducing consortium supplied with benzene (Ulrich et al. 2005; Botton and Parsons 2007). The appearance of phenol suggested a pathway involving hydroxylation of benzene to phenol as it was proposed in previous studies. Figure 2.3.b shows a possible degradation pathway for anaerobic benzene mineralization based on these studies. In this pathway, the hydroxylation of the aromatic ring occurs either through incorporation of hydroxyl group of water or free hydroxyl radicals into the benzene ring to form phenol. Phenol catabolism by pure cultures of Thauera aromatica has been studied in detail and involves several steps (Narmandakh et al. 2006). In the first step, phenol is phosphorylated in an ATP-dependant mechanism to form phenyl phosphate (Figure 2.3.b). This process is catalyzed by a phenyl phosphate synthase that transfers the !-phosphoryl group from ATP to phenol resulting in formation of phenyl phosphate, AMP, and Pi (Carmona et al. 2009). Phenyl phosphate is carboxylated by phenyl phosphate carboxylase to form 4-hydroxybenzoate (Schühle and Fuchs 2004). A specific CoA ligase (4-hydroxybenzoyl-CoA ligase) then activates 4-
! ! 29! ! hydroxybenzoate and converts it to 4-hydroxybenzoyl-CoA. Finally, the hydroxyl group of 4- hydroxybenzoyl-CoA is reductively removed, resulting in the production of benzoate or benzoylCoA (Figure 2.3.b) (Heider and Fuchs 1997; Coates et al. 2002). The 4-hydroxybenzoyl- CoA reductase is the enzyme that catalyzes this process. Further mineralization of benzoyl-CoA takes place via the pathway explained in carboxylation section.
! (a) OH O
+ H2 H2O H Aliphatic acids
Benzene Phenol CyclohexanoneCyclohexanon e
OH (b) O P O-
OH O COOH + H2O/OH• H ATP AMP+Pi
CO2
Phenyl phosphate Phenyl phosphate synthase carboxylase 4.PpsABC 4.PpcBCAD OH Benzene Phenol Phenyl phosphate 4-Hydroxybenzoate
COSCoA COSCoA H O 2H+ AMP+PP ATP+CoA 2 i 3 Acetyl -CoA 4-Hydroxybenzoate (4- + CO2 4-Hydroxybenzoyl- CoA reductase HBA)-CoA ligase 1.HcrCBA, 2.HbaBCD, 1.HcrL, 2.HbaA, 4.HcrL 3.HcrCAB, 4.HcrCAB, OH 5.PcmRST Benzoyl-CoA 4-Hydroxybenzoyl CoA
Figure 2.3. Hydroxylation as the first step in mineralization of benzene. Two pathways have been proposed for transformation of phenol. One of these pathways is based on detection of phenol and cyclohexanone during benzene degradation in a methanogenic culture (Grbi!- Gali! and Vogel 1987) (a) and the other pathway is based on what is known about anaerobic mineralization of phenol by isolated bacteria (b) (Carmona et al. 2009). The name of genes coding for corresponding enzymes of the pathway for various bacteria are provided below each arrow. The numbers next to the name of each gene correspond to the gene identified in Thauera aromatica (1), Rhodopseudomonas palustris (2), Magnetospirillum magneticum (3), Azoarcus sp. EbN1 (4), and Geobacter metallireducens (5), respectively. ! ! 30! ! Recently Kunapuli et al. (2008) and Abu Laban et al. (2009) reported the abiotic formation of phenol from benzene due to the exposure of culture samples to air. Therefore, caution should be exercised in interpreting phenol as a metabolite of benzene degradation (Abu Laban et al. 2010).
Methylation
In 2002, Coates et al. (2002) proposed alkylation of benzene to toluene as the first step in anaerobic benzene degradation (Figure 2.4). Biologically mediated alkylation of benzene to toluene as well as alkylation of toluene to the various isomers of xylene has previously been observed with bone marrow (Flesher and Myers 1991). When the methyl donors are methyl- tetrahydrofolate or S-adenosyl-methionine, the free energy of alkylation of benzene to toluene is negative i.e., the alkylation reaction is energetically favorable (Coates et al. 2002). The corresponding reactions are given below:
Benzene + methyl-tetrahydrofolate ! toluene + tetrahydrofolate
"G° = -32.31 KJ/mol
Benzene + S-adenosyl-methionine ! toluene + S-adenosyl-homocysteine
"G° = -112.33 KJ/mol
13 In 2005, Ulrich et al. (2005) showed that C6-benzene supplied to benzene-oxidizing nitrate- 13 reducing and methanogenic enrichment cultures resulted in the formation of C6-labeled toluene and benzoate. This observation supported methylation of benzene as the activation mechanism of benzene ring.
After transformation of benzene to toluene, this compound may be mineralized through the anaerobic metabolic pathway of toluene, which has been intensively investigated over the past several decades (Heider et al. 1999; Spormann and Widdel 2000; Widdel and Rabus 2001; Boll et al. 2002; Chakraborty and Coates 2004; Fuchs 2008). As shown in Figure 2.4, the initial step in the catabolism of toluene is addition of the methyl group of toluene onto fumarate to form benzylsuccinate (Biegert et al. 1996; Beller and Spormann 1997a; Beller and Spormann 1997b; Chakraborty and Coates 2004). A glycyl radical enzyme called benzylsuccinate synthase catalyzes this process (Coschigano et al. 1998; Leuthner et al. 1998; Leuthner and Heider 2000;
! ! 31! !
- X=S-adenosyl-methionine, Benzylsuccinate COO Tetrahydrofolate, synthase or Cobalamin CH3 1.BssABCD, 2.BssABCD, COO- CH3-X X Benzylsuccinate3.BssABCD, synthase4.TutDEFG
COO-
- Benzene Toluene COO Benzylsuccinate Fumarate COSCoA 1.BbsEF CoA benzylsuccinate Succinyl 2H - Succinate COO- transferase
dehydehydrogenasdrogenase - - CoA:(R) e COO
- COO Succinate - COSCoA COSCoA COSCoA HO
- - - COO 2H COO COO H2O
Benzylsuccinyl-CoA Phenylitaconyl-CoA dehydrogenase hydratase 1.BbsH 1.BbsG 2-[hydroxyl(phenyl)methyl]- Phenylitaconyl-CoA Benzylsuccinyl-CoA succinyl-CoA
Benzoate or 2-[hydroxyl(phenyl)methyl]- CoA derivative succinyl-CoA dehydrogenase 2H 1.BbsCD
COSCoA COSCoA O COSCoA
CoA - Succinyl-CoA COO- COO
3 Acetyl-CoA Benzoylsuccinyl-CoA thiolase + CO2 1.BbsAB Benzoylsuccinyl-CoA Benzoyl-CoA
Figure 2.4. Proposed methylation of benzene to toluene as the activation mechanism of benzene ring. Toluene may further be metabolized via fumarate addition (Coates et al. 2002;
Carmona et al. 2009). The name of genes encoding the enzymes of the pathway for different bacteria is written either above or below each arrow in figure. The numbers next to the name of each gene correspond to the gene identified in Thauera aromatica K172, Azoarcus sp. T and EbN1, and Geobacter metallireducens (1), Magnetospirillum sp. TS-6 (2), Thauera aromatica sp. DNT-1 ! (3) and Thauera aromatica T1 (4), respectively.
! ! 32! ! Achong et al. 2001; Selmer et al. 2005). Further degradation of benzylsuccinate to benzoyl-CoA and succinyl-CoA occurs via !-oxidation pathway (Carmona et al. 2009). The first step of benzylsuccinate oxidation involves the activation of this compound to its CoA thioester by succinyl-CoA:benzylsuccinate-CoA transferase (Leutwein and Heider 2001) (Figure 2.4). Benzylsuccinyl-CoA is then converted into phenyl-itaconyl-CoA by a benzylsuccinyl-CoA dehydrogenase (Leutwein and Heider 2002). The subsequent three steps are catalyzed by phenyl- itaconyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, and benzoylsuccinyl-CoA thiolase and result in the generation of benzoyl-CoA and succinyl-CoA. Recycling of the fumarate, co- substrate of benzylsuccinate synthase, from succinate completes the reaction cycle. This process is catalyzed by succinate dehydrogenase (Figure 2.4).
Fumarate addition
A possible pathway for anaerobic oxidation of benzene is fumarate addition, which is one of the common mechanisms for the activation of hydrocarbons (Figure 2.5). In this reaction, the carbon atoms of hydrocarbon are added to the double bond of fumarate to form a succinyl derivative (Coates et al. 2002). Fumarate addition has been observed in the metabolism of xylenes (Krieger et al. 1999), alkanes (Rabus et al. 2001), ethylbenzene (Kniemeyer et al. 2003), methylnaphthalene (Annweiler et al. 2000), cresols (Muller et al. 2001), and toluene (Heider et al. 1999; Spormann and Widdel 2000; Krieger et al. 2001). Addition of toluene to fumarate involves the formation of an enzyme-base radical. This enzyme-base radical separates one hydrogen atom from the methyl group of toluene and transforms toluene to a benzyl radical. The benzyl radical is then added to the fumarate. The energy required for abstraction of a hydrogen atom from benzene to form a phenyl radical is much higher than that of alkanes and alkylbenzenes (Widdel and Rabus 2001). Due to the high activation energy requirement, it seems unlikely for fumarate addition to be the initial step for the activation of benzene (Figure 2.5). Moreover, potential metabolites of fumarate addition have never been detected during anaerobic benzene degradation.
! ! 33!
! - COO - - COOH COO OOC
COO-
3 Acetyl-CoA Fumarate + CO2
Benzene Benzoate or CoA derivative Figure 2.5. Fumarate addition as a mechanism for activating the benzene ring (Coates et al. 2002).
Ring Reduction
One other mechanism for the activation of benzene is through reduction of its ring (Figure 2.6). This process has been proposed for the metabolism of phenol (Bakker 1977; Grbi!-Gali! and Vogel 1987). Although this pathway, which is mechanistically and energetically difficult, might be possible for benzene, an initial ring reduction step is not consistent with the detection of toluene, phenol, and benzoate as intermediates of anaerobic benzene degradation in previous studies.
H2 and ATP ?
Benzene ! Figure 2.6. Benzene ring reduction as the initial step of benzene mineralization (Coates et al. 2002).
2.3. Syntrophic mineralization of benzene
Syntrophy is a type of microbial interaction in which two or more microorganisms cooperate to degrade a compound that neither microorganism can degrade alone (Madigan et al. 2009). In methanogenic cultures, degradation of benzene to methane and carbon dioxide occurs by concerted action of three groups of microorganisms, including primary fermenting bacteria, secondary fermenting bacteria (syntrophic bacteria), and methanogens (Schink 1997). In this process, primary fermenters convert the substrate to products such as alcohols, organic acids, and hydrogen. Some of these fermentation products, especially acetate, hydrogen and carbon dioxide
! ! 34! ! are utilized by methanogens generating methane and carbon dioxide. Other reduced fermentation intermediates are degraded by secondary fermenters. These bacteria convert their substrate to acetate, carbon dioxide, and hydrogen, which are subsequently used by methanogens (Schink 1997). This reaction is thermodynamically unfavorable under standard conditions (25°C, pH=7, and all compounds at unit activity) and is only energetically favorable under low concentrations of hydrogen. As a result, methanogens exert a significant influence on the earlier members in the chain by removing hydrogen and reducing the concentrations of hydrogen to low levels. Earlier members in turn provide methanogens with their required substrates.
Recently there have been several reports on mineralization of benzene to occur through a syntrophic cooperation between metabolically different microorganisms in iron-reducing and sulfate-reducing enrichment cultures (Kunapuli et al. 2007; Kleinsteuber et al. 2008; Herrmann et al. 2010). In a sulfate-reducing microcosm benzene was degraded through syntrophic association between members of Peptococcaceae, Epsilonproteobacteria, and Deltaproteobacteria (Kleinsteuber et al. 2008; Herrmann et al. 2010). It was suggested that Peptococcaceae was involved in initial attack on benzene and converting it to hydrogen, acetate, or low molecular weight fermentation products. Fermentation of benzene to acetate and hydrogen is endergonic and thermodynamically unfavorable under standard conditions but it becomes exergonic when the hydrogen and acetate concentrations are low (Kleinsteuber et al. 2008). Therefore, the role of Delta- and Epsilonproteobacteria in this sulfate-reducing culture was proposed to be consumption of intermediates such as hydrogen and acetate produced by primary benzene degrader (Kleinsteuber et al. 2008; Herrmann et al. 2010). This in turn reduces hydrogen and acetate concentrations and makes degradation of benzene feasible. In an iron- reducing enrichment culture, Peptococcaceae and Desulfobulbaceae-related bacteria were identified as key-players in mineralization of benzene (Kunapuli et al. 2007). A syntrophic mode of interaction between these phylotypes was proposed during anaerobic benzene degradation. In this interaction, Peptococcaceae seemed to be responsible for initial attack on benzene while Desulfobulbaceae appeared to be thriving on the hydrogen produced by Peptococcaceae. The relationship between these two microorganisms could be based on hydrogen transfer. In this interaction hydrogen, which is released by the primary degrader, is utilized by syntrophic partner and therefore, pulling the initial reaction towards completion (Kunapuli et al. 2007).
! ! 35! ! 2.4. Conclusions
In spite of significant efforts in the past several decades, microorganisms responsible for degradation of benzene under anaerobic conditions remain largely unknown. To date, phenol, toluene, and benzoate have been identified as intermediates of benzene in various anaerobic enrichment cultures and benzene hydroxylation, methylation, and carboxylation have been proposed as three major processes for the initial step in degradation of this substrate. Nevertheless, key metabolic steps, genes and enzymes remain largely unknown. There are now two studies that describe a putative benzene carboxylase enzyme involved in carboxylation of benzene to benzoate (Abu Laban et al. 2010; Holmes et al. 2011). But, the enzymatic activity of this protein has not been elucidated yet. Recently, the complete genome of isolated benzene- degrading denitrifying Dechloromonas RCB was sequenced. However, it did not provide information regarding the benzene degradation pathway. The genome of this bacterium lacked the key enzymes for the mineralization of toluene, phenol, and benzoyl-CoA (Salinero et al. 2009). As a result, in this study we focus on identifying the microorganisms, key metabolic steps, genes, and enzymes involved in anaerobic degradation of benzene. Accomplishing these objectives will mark a significant progress in the field of anaerobic benzene mineralization.
! ! ! 2.5. References Chapter 2 Abu Laban, N., D. Selesi, C. Jobelius and R. U. Meckenstock (2009). "Anaerobic benzene degradation by gram-positive sulfate-reducing bacteria." FEMS Microbiol. Ecol. 68: 300-311.
Abu Laban, N., D. Selesi, T. Rattei, P. Tischler and R. U. Meckenstock (2010). "Identification of enzymes involved in anaerobic benzene degradation by strictly anaerobic iron-reducing enrichment culture." Environ. Microbiol. 12(10): 2783-2796.
Achong, G. R., A. M. Rodriguez and A. M. Spormann (2001). "Benzylsuccinate synthase of Azoarcus sp. strain T: cloning, sequencing, transcriptional organization, and its role in anaerobic toluene and m-xylene mineralization." J. Bacteriol. 183: 6763-6770.
Aklujkar, M., N. D. Young, D. Holmes, M. Chavan, C. Risso, H. E. Kiss, C. S. Han, M. L. Land and D. R. Lovley (2010). "The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments." BMC Genomics 11: 490.
Anderson, R. T. and D. R. Lovley (1997). "Ecology and biochemistry of in situ groundwater bioremediation." Adv. Microb. Ecol. 15: 289-350.
Anderson, R. T., J. N. Rooney-Varga, C. V. Gaw and D. R. Lovely (1998). "Anaerobic benzene oxidation in the Fe(III) reduction zone of petroleum-contaminated aquifers." Environ. Sci. Technol. 32: 1222-1229.
Andreoni, V. and L. Gianfreda (2007). "Bioremediation and monitoring of aromatic-polluted habitats." Appl. Microbiol. Biotechnol. 76: 287-308.
Annweiler, E., A. Materna, M. Safinowski, A. Kappler, H. H. Richnow, W. Michaelis and R. U. Meckenstock (2000). "Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture." Appl. Environ. Microbiol. 66: 5329-5333.
Bakker, G. (1977). "Anaerobic degradation of aromatic compounds in the presence of nitrate." FEMS Microbiol. Lett. 1: 103-107.
Beller, H. R. and A. M. Spormann (1997a). "Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T." J. Bacteriol. 179: 670-676.
Beller, H. R. and A. M. Spormann (1997b). "Benzyl-succinate formation as a means of anaerobic toluene activation by sulfate-reducing strain PRTOL1." Appl. Environ. Microbiol. 63: 3729- 3731.
Biegert, T., G. Fuchs and J. Heider (1996). "Evidence that anaerobic oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate." Eur. J. Biochem 238: 661-668.
! 36! ! ! 37! ! Boll, M. (2005). "Key enzymes in the anaerobic metabolism catalysing Birch-like reductions." Biochem. Biophys. Acta 1707: 34-50.
Boll, M., G. Fuchs and J. Heider (2002). "Anaerobic oxidation of aromatic compounds and hydrocarbons." Curr. Opin. Chem. Biol. 6: 604-611.
Botton, S., M. v. Harmelen, M. Braster, J. R. Parsons and W. F. Röling (2007). "Dominance of Geobacteraceae in BTX-degrading enrichments from an iron-reducing aquifer." FEMS Microbiol. Ecol. 62: 118-130.
Botton, S. and J. R. Parsons (2007). "Degradation of BTX by dissimilatory iron-reducing cultures." Biodegradation 18: 371-381.
Breese, K., M. Boll, J. Alt-Mörbe, H. Schägger and G. Fuchs (1998). "Genes coding for the benzoyl-CoA pathway of anaerobic aromatic metabolism in the bacterium Thauera aromatica." Eur. J. Biochem 256(1): 148-154.
Burland, S. M. and E. A. Edwards (1999). "Anaerobic benzene biodegradation linked to nitrate reduction." Appl. Environ. Microbiol. 65(2): 529-533.
Butler, J. B., Q. He, K. P. Nevin, Z. He, J. Zhou and D. R. Lovley (2007). "Genomic and microarray analysis of aromatics degradation in Geobacter metallireducens and comparison to a Geobacter isolate from a contaminated field site." BMC Genomics 8: 180-196.
Caldwell, M. E. and J. M. Suflita (2000). "Detection of phenol and benzoate as intermediates of anaerobic benzene biodegradation under different terminal electron accepting condition." Environ. Sci. Technol. 34: 1216-1220.
Caldwell, M. E., R. S. Tanner and J. M. Suflita (1999). "Microbial metabolism of benzene and the oxidation of ferrous iron under anaerobic conditions: implications for bioremediation." Environ. Microbiol. 5(6): 595-603.
Cao, B., K. Nagarajan and K. C. Loh (2009). "Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches " Appl. Microbiol. Biotechnol. 85: 207- 228.
Carmona, M., M. T. Zamarro, B. Blázquez, G. Durante-Rodríguez, J. F. Juárez, J. A. Valderrama, M. J. Barragán, J. L. García and E. Díaz (2009). "Anaerobic catabolism of aromatic compounds: a genetic and genomic view." Microbiol. Mol. Biol. Rev. 73(1).
Chakraborty, R. and J. D. Coates (2004). "Anaerobic degradation of monoaromatic hydrocarbons." Appl. Microbiol. Biotechnol. 64: 437-446.
Chakraborty, R. and J. D. Coates (2005). "Hydroxylation and carboxylation-two crucial steps of anaerobic benzene degradation by Dechloromonas Strain RCB." Appl. Environ. Microbiol. 71(9): 5427-5432.
! ! ! 38! ! Chang, W., Y. Um and T. R. P. Holoman (2005). "Molecular charecterization of anaerobic microbial communities from benzene-degrading sediments under methanogenic conditions." Biotechnol. Prog. 21: 1789-1794.
Coates, J. D., R. Chakraborty, J. G. Lack, S. M. O’Connor, K. A. Cole, K. S. Bender and L. A. Achenbach (2001). "Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas." Nature 411: 1039-1043.
Coates, J. D., R. Chakraborty and M. J. McInerney (2002). "Anaerobic benzene biodegradation-a new era." Res. Microbio. 153: 621-628.
Coschigano, P. W., T. S. Wehrman and L. Y. Young (1998). "Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: Putative role of a glycine free radical." Appl. Environ. Microbiol. 64: 1650-1656.
Da Silva, M. and P. Alvarez (2007). "Assessment of anaerobic benzene degradation potential using 16S rRNA gene-targeted real-time PCR." Environ. Microbiol. 9: 72-80.
Edwards, E. A. and D. Grbi!-Gali! (1992). "Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions." Appl. Environ. Microbiol. 58: 2663-2666.
Egland, P. G., D. A. Pelletier, M. Dispensa, J. Gibson and C. S. Harwood (1997). "A cluster of bacterial genes for anaerobic benzene ring biodegradation." Proc. Natl. Acad. Sci. USA.
Fahy, A., T. J. McGenity, K. N. Timmis and A. S. Ball (2006). "Heterogeneous aerobic benzene- degrading communities in oxygen-depleted groundwaters." FEMS Microbiol. Ecol. 58: 260-270.
Flesher, J. W. and S. R. Myers (1991). "Methyl-Substitution of Benzene and Toluene in Preparations of Human Bone Marrow." Life Science 48: 843-850.
Fuchs, G. (2008). "Anaerobic metabolism of anaerobic compounds." Ann. N. Y. Acad. Sci. 1125: 82-99.
Gibson, D. T., J. R. Koch and R. E. Kallio (1968). "Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene." Biochemistry 7: 2653-2658.
Gibson, J. and C. S. Harwood (2002). "Metabolic diversity in aromatic compound utilization by anaerobic microbes." Annu. Rev. Microbiol. 56: 345-369.
Grbi!-Gali!, D. and T. M. Vogel (1987). "Transformation of toluene and benzene by mixed methanogenic cultures." Appl. Environ. Microbiol. 53: 254-260.
Gulensoy, N. and P. J. J. Alvarez (1999). "Diversity and correlation of specific aromatic hydrocarbon biodegradation capabilities." Biodegradation 10: 331-340.
! ! ! 39! ! Habe, H. and T. Omori (2003). "Genetics of polycyclic aromatic hydrocarbon metabolism in diverse aerobic bacteria." Biosci. Biotechnol. Biochem. 67(2): 225-243.
Harwood, C. S., G. Burchhardt, H. Herrmann and G. Fuchs (1999). "Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway." FEMS Microbiol. Rev. 22: 439-458.
Harwood, C. S. and R. E. Parales (1996). "The B-Ketoadipate pathway and the biology of self- identity." Ann. Rev. Microbiol. 50: 553-590.
Heider, J. and G. Fuchs (1997). "Anaerobic metabolism of aromatic compounds." Eur. J. Biochem 243: 577-596.
Heider, J., A. M. Spormann, H. R. Beller and F. Widdel (1999). "Anaerobic bacterial metabolism of hydrocarbons." FEMS Microbiol. Rev. 22: 459-473.
Herrmann, S., S. Kleinsteuber, A. Chatzinotas, S. Kupparsdt, T. Lueders, H. H. Richnow and C. Vogt (2010). "Functional charecterization of an anaerobic benzene-degrading enrichment culture by DNA stable isotope probing." Environ. Microbiol. 12(2): 401-411.
Herrmann, S., S. Kleinsteuber, T. R. Neu, H. H. Richnow and C. Vogt (2008). "Enrichment of anaerobic benzene-degrading microorganisms by in situ microcosms." FEMS Microbiol. Ecol. 63(1): 94-106.
Holmes, D. E., C. Risso, J. A. Smith and D. R. Loveley (2011). "Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus." Appl. Environ. Microbiol. 77(17): 5926-5933.
Hutchins, S. R. (1991). "Optimizing BTEX biodegradation under denitrifyng conditions." Environ. Toxicol. Chem. 10: 1437-1448.
Jahn, M. K., S. B. Haderlein and R. U. Meckenstock (2005). "Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures." Appl. Environ. Microbiol. 71: 3355-3358.
Jindrova, E., M. Chocova, K. Demnerova and V. Brenner (2002). "Bacterial aerobic degradation of benzene, toluene, ethylbenzene and xylene." Folia Microbiol. 47(2): 83-93.
Johnson, S. J., K. J. Woolhouse, H. Prommer, D. A. Barry and N. Christofi (2003). "Contribution of anaerobic microbial activity to natural attenuation of benzene in groundwater " Engineering Geology 70: 343-349.
Kasai, Y., Y. Kodama, Y. Takahata, T. Hoaki and K. Watanabe (2007). "Degradative capacities and bioaugmentation potential of anaerobic benzene-degrading bacterium strain DN11." Environ. Sci. Technol. 41: 6222-6227.
! ! ! 40! ! Kasai, Y., M. Manefield and K. Watanabe (2006). "RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater." Appl. Environ. Microbiol. 72 (5): 3586-3592.
Kasai, Y., Y. Takahata, T. Hoaki and K. Watanabe (2005). "Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil." Environ. Microbiol. 7(6): 806-818.
Kazumi, J., M. E. Cadwell, J. M. Sulfita, D. R. Lovley and L. Y. Young (1997). "Anaerobic degradation of benzene in diverse anoxic environments." Environ. Sci. Technol. 31: 813-818.
Kleinsteuber, S., K. M. Schleinitz, J. Breitfeld, H. Harms, H. H. Richnow and C. Vogt (2008). "Molecular charecterization of bacterial communities mineralizing benzene under sulfate- reducing conditions." FEMS Microbiol. Ecol. 66: 143-157.
Kniemeyer, O., T. Fischer, H. Wilkes, F. O. Glockner and F. Widdel (2003). "Anaerobic degradation of ethylbenzene by a new type of marine sulfate-reducing bacterium." Appl. Environ. Microbiol. 69: 760-768.
Krieger, C. J., H. R. Beller, M. Reinhard and A. M. Spormann (1999). "Initial reactions in anaerobic oxidation of m-Xylene by the denitrifying bacterium Azoarcus sp strain T." J. Bacteriol. 181: 6403-6410.
Krieger, C. J., W. Roseboom, S. P. J. Albracht and A. M. Spormann (2001). "A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T." J. Biol. Chem. 276: 12924-12927.
Kunapuli, U., C. Griebler, H. Beller and R. U. Meckenstock (2008). "Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture." Environ. Microbiol. 10: 1703-1712.
Kunapuli, U., T. Lueders and R. U. Meckenstock (2007). "The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation." ISME J. 1: 643-653.
Kung, J. W., C. Löffler, K. Dörner, D. Heintz, A. V. D. S. Galliend, T. Friedrich and M. Boll (2009). "Identification and characterization of the tungsten-containing class of benzoyl- coenzyme A reductases." Proc. Natl. Acad. Sci. USA 106(42): 17687-17692.
Leuthner, B. and J. Heider (2000). "Anaerobic toluene catabolism of Thauera aromatica: the bbs operan codes for enzymes of betaoxidation of the intermediate benzyl suucinate." J. Bacteriol. 182: 272-277.
Leuthner, B., C. Leutwein, H. Schulz, P. Horth, W. Haehnel, E. Schiltz, H. Schagger and J. Heider (1998). "Biochemical and genetic charecterization of benzylsuccinatesynthase from
! ! ! 41! ! Thauera aromatica: a new glycyl radical enzyme catalyzng the first step in anaerobic toluene metabolism." Mol. Microbiol. 28: 615-628.
Leutwein, C. and J. Heider (2001). "Succinyl-CoA:(R)-benzylsuccinate CoA trasferase: an enzyme of the anaerobic toluene catabolic pathway in denitrifying bacteria." J. Bacteriol. 183: 4288-4295.
Leutwein, C. and J. Heider (2002). "(R)-Benzylsuccinyl-CoA dehydrogenase of Thauera aromatica, an enzyme of the anaerobic toluene catabolic pathway." Arch. Microbiol. 178: 517- 524.
Liou, J. S. C., C. M. Derito and E. L. Madsen (2008). "Field-based and laboratory stable isotope probing surveys of the identities of both aerobic and anaerobic benzene-metabolizing microorganisms in freshwater sediment." Environ. Microbiol. 10(8): 1964-1977.
López Barragán, M. J., M. Carmona, M. T. Zamarro, B. Thiele, M. Boll, G. Fuchs, J. L. García and E. Díaz (2004a). "The bzd gene cluster, coding for anaerobic benzoate catabolism, in Azoarcus sp. strain CIB." J. Bacteriol. 186: 5762-5774.
López Barragán, M. J., E. Díaz, J. L. García and M. Carmona (2004b). "Genetic clues on the evolution of anaerobic catabolism of aromatic compounds." Microbiology 150: 2018-2021.
Lovley, D. R., J. D. Coates, J. C. Woodward and E. J. Phillips (1995). "Benzene oxidation coupled to sulfate reduction." Appl. Environ. Microbiol. 61: 953-958.
Lovley, D. R., J. C. Woodward and F. H. Chapel (1994). "Stimulated anoxic biodegradation of aromatic hydrocarbons using Fe(III) ligands." Nature 370: 128-131.
Lovley, D. R., J. C. Woodward and F. H. Chapelle (1996). "Rapid anaerobic benzene oxidation with a variety of chelated Fe (III) forms." Appl. Environ. Microbiol. 62: 288-291.
Madigan, M. T., J. M. Martinko, P. V. Dunlap and D. P. Clark (2009). Brock Biology of Microorganisms. San Francisco, Pearson Benjamin Cummings.
Major, D. W., C. I. Mayfield and J. F. Barker (1988). "Biotransformation of benzene by denitrification in aquifer sand." Ground Water 26: 8-14.
McCarty, P. L. (1971). Energetics and Bacterial Growth, in Organic Compounds in Aquatic Environments, Marcel Dekker.
McInerney, M. J., L. Rohlin, H. Mouttaki, U. Kim, R. S. Krupp, L. Rios-Hernandez, J. Sieber, C. G. Struchtemeyer, A. Bhattacharyya, J. W. Campbell and R. P. Gunsalus (2007). "The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth." Proc. Natl. Acad. Sci. USA 104: 7600-7605.
! ! ! 42! ! Morgan, P., S. T. Lewis and R. J. Watkinson (1993). "Biodegradation of benzene, toluene, ethylbenzene, and xylenes in gas-condensate-contaminated groundwater." Environ. Pollu. 82: 181-190.
Muller, J. A., A. S. Galushko, A. Kappler and B. Schink (2001). "Initiation of anarobic degradation of p-cresol by formation of 4-hydroxybenzylsuccinate in Desulfobacterium cetonicum." J. Bacteriol. 183: 752-757.
Musat, F. and F. Widdel (2008). "Anaerobic degradation of benzene by a marine sulfate- reducing enrichment culture, and cell hybridization of the dominant phylotype." Environ. Microbiol. 10(1): 10-19.
Nales, M., B. J. Butler and E. Edwards (1998). "Anaerobic benzene biodegradation: a microcosm survey." Bioremediat. J. 2: 125-144.
Narmandakh, A., N. God'on, F. Drepper, B. Knapp, W. Haehnel and G. Fuchs (2006). "Phosphorylation of phenol by phenyl phosphate synthase: role of histidine phosphate in catalysis." J. Bacteriol. 188: 7815-7822.
Nicholson, C. A. and B. Z. Fathepure (2005). "Aerobic biodegradation of benzene and toluene under hypersaline conditions at the great salt plains, Oklahoma." FEMS Microbiol. Lett. 245: 257-262.
Oka, A. R., C. D. Phelps, L. M. McGuinness, A. Mumford, L. Y. Young and L. J. Kerkhof (2008). "Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing." Appl. Environ. Microbiol. 74: 6476-6480.
Pelletier, D. A. and C. H. Harwood (2000). "2-Hydroxycyclohexanecarbonyl coenzyme A dehydrogenase, an enzyme charecteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris." J. Bacteriol. 182: 2753-2760.
Pelletier, D. A. and C. S. Harwood (1998). "2-ketocyclohexanecarbonyl coenzyme A hydrolase, the ring cleavage enzyme required for anaerobic benzoate degradation by Rhodopseudomonas palutris." J. Bacteriol. 180: 2330-2336.
Phelps, C. D., J. Kazumi and L. Y. Young (1996). "Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction." FEMS Microbiol. Lett. 145: 433-437.
Phelps, C. D., L. J. Kerkhof and L. Y. Young (1998). "Molecular charecterization of a sulfate- reducing consortium which mineralizes benzene." FEMS Microbiol. Ecol. 27: 269-279.
Phelps, C. D. and L. Y. Young (1999). "Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments." Biodegradation 10: 15-25.
! ! ! 43! ! Phelps, C. D., X. Zhang and L. Y. Young (2001). "Use of stable isotopes to identify benzoate as a metabolite of benzene degradation in a sulphidogenic consortium." Environ. Microbiol. 3: 600- 603.
Rabus, R., H. Wilkes, A. Behrends, A. Armstroff, T. Fischer, A. J. Pierik and F. Widdel (2001). "Anaerobic Initial Reaction of n-Alkanes in a Denitrifying Bacterium: Evidence for (1- Methylpentyl)succinate as Initial Product and for Involvement of an Organic Radical in n- Heaxane Metabolism." J. Bacteriol. 183: 1707-1715.
Ridgeway, H. F., J. Safarik, D. Phipps, P. Carl and D. Clark (1990). "Identification and catabolic activity of well-derived gasoline-degrading bacteria and a contaminated aquifer." Appl. Environ. Microbiol. 56: 3565-3575.
Rittmann, B. E. and P. L. McCarty (2001). Environmental Biotechnology: Principles and Applications. New York, McGraw-Hill.
Rooney-Varga, J. N., R. T. Anderson, J. L. Fraga, D. Ringelberg and D. R. Lovley (1999). "Microbial communities associated with anaerobic benzene degradation in a petroleum- contaminated aquifer." Appl. Environ. Microbiol. 65: 3056-3063.
Sakai, N., F. Kurisu, O. Yagi, F. Nakajima and K. Yamamoto (2009). "Identification of putative benzene-degrading bacteria in methanogenic enrichment cultures." J. Biosci. Bioeng. 108: 501- 507.
Salinero, K. K., K. Keller, W. S. Feil, H. Feil, S. Trong, G. D. Bartolo and A. Lapidus (2009). "Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation " BMC Genomics 10: 351-74.
Schink, B. (1997). "Energetics of syntrophic cooperation in methanogenic degradation." Microbiol. Mol. Biol. Rev. 61(2): 262-280.
Schühle, K. and G. Fuchs (2004). "Phenylphosphate Carboxylase: a New C-C Lyase involved in Anaerobic Phenol Metabolism in Thauera aromatica." J. Bacteriol. 186: 4556-4567.
Selmer, T., A. J. Pierik and J. Heider (2005). "New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria." Biol. Chem. 386: 981-988.
Shinoda, Y., J. Akagi, Y. Uchihashi, A. Hiraishi, H. Yukawa, H. Yurimoto, Y. Sakai and N. Kato (2005). "Anaerobic degradation of aromatic compounds by Magnetospirillum Strains: isolation and degradation genes." Biotechnol. Biochem. 69: 1483-1491.
Spormann, A. M. and F. Widdel (2000). "Metabolism of alkylbenzenes, alkanes, and other hydrocarbons in anaerobic bacteria." Biodegradation 11: 85-105.
! ! ! 44! ! Ulrich, A. C., H. R. Beller and E. A. Edwards (2005). "Metabolites detected during 13 biodegradation of C6-benzene in nitrate-reducing and methanogenic enrichment cultures." Environ. Sci. Technol 39: 6681-6691.
Ulrich, A. C. and E. A. Edwards (2003). "Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures." Environ. Microbiol. 5(2): 92-102.
Vogel, T. M. and D. Grbi!-Gali! (1986). "Incorporation of oxygen from water into toluene and benzene during anaerobic fermentative transformation." Appl. Environ. Microbiol. 52: 200-202.
Vogt, C., S. Gödeke, H. C. Treutler, H. Weiß, M. Schirmer and H. H. Richnow (2007). "Benzene oxidation under sulfate-reducing conditions in columns simulating in situ conditions." Biodegradation 18: 625-636.
Weiner, J. M. and D. R. Lovley (1998). "Rapid benzene degradation in methanogenic sediments from a petroleum-contaminated aquifer." Appl. Environ. Microbiol. 64: 1937-1939.
Whiteley, C. G. and D. J. Lee (2006). "Enzyme technology and biological remediation." Enzyme Microb. Technol. 38: 291-316.
Widdel, F. and R. Rabus (2001). "Anaeorobic biodegradation of saturated and aromatic hydrocarbons." Curr. Opin. Biotech 12: 259-276.
Williams, P. A. and J. R. Sayers (1994). "The evolution of pathways for aromatic hydrocarbon oxidation in Pseudomonas." Biodegradation 5: 195-217.
Wilson, L. P. and E. J. Bouwer (1997). "Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review." J. Ind. Microbiol. Biotechnol. 18: 116-130.
Wischgoll, S., D. Heintz, F. Peters, A. Erxleben, E. Sarnighausen, R. Reski, A. V. Dorsselaer and M. Boll (2005). "Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens." Mol. Microbiol. 58(5): 1238-1252.
Witzig, R., H. Junca, H. J. Hecht and D. H. Pieper (2006). "Assessment of toluene/biphenyl dioxygenase gene diversity in benzene-polluted soils: links between benzene biodegradation and genes similar to those encoding isopropylbenzene dioxygenases." Appl. Environ. Microbiol. 72: 3504-3514.
! ! !
Chapter 3: Maintenance and Optimization of Growth of Benzene- Degrading Nitrate-Reducing Enrichment Cultures
! 45! ! 46!
3.1. Introduction
Benzene-degrading nitrate-reducing microcosms were originally prepared 16 years ago using samples from three distinct sites: a decommissioned retail gasoline station on Cartwright avenue in Toronto, Ontario (Cartwright), an uncontaminated swamp in Perth, Ontario (Swamp), and a petrochemical landfarm in Ontario (Landfarm) (Figure 3.1) (Nales et al. 1998). The enrichment cultures have been maintained over the years by transferring into an anaerobic mineral medium (Burland and Edwards 1999) and adding benzene as the sole source of carbon and electron donor and nitrate as an electron acceptor.
Currently, biodegradation of benzene and microbial growth in laboratory microcosms is relatively slow compared to other anaerobic enrichment cultures degrading, for example, Toluene or Trichloroethene (TCE). Maintenance of the nitrate-reducing cultures in our laboratory has been challenging due to various problems such as stalling for unknown reasons, presence of significant lag times (no benzene degradation) after transferring the cultures into fresh medium (refer to Appendix B for a detailed list of the medium constituents), and nitrite accumulation that inhibits benzene degradation (Burland and Edwards 1999). A likely reason for the presence of lag periods is that some components of growth medium have inhibitory impacts on mineralization of benzene. These compounds should be removed by microorganisms prior to benzene degradation. Another possible explanation for observing lag time upon transfer of cultures into fresh medium is that some essential compounds such as cofactors (a cofactor is an atom, organic molecule, or molecular group needed for the catalytic activity of an enzyme) necessary for the mineralization of benzene are missing from the medium and that synthesis of these substances by microorganisms is a pre-requisite for benzene degradation. Thus, one of the objectives of this study was to gain a better insight into conditions that promote or inhibit benzene mineralization in these cultures and help optimize the growth of microorganisms by shortening the lag times and minimizing nitrite accumulation and inhibition.
! ! 47!
Site #1 Site #2 Site #3 Cartwright gasoline station Uncontaminated Swamp Landfarm
Parent Cultures
Cartwright 1b Swamp 1a, 1ab, 1b, Landfarm 1bb, and 1bc
Subcultures
Cartwright Cartwright Cartwright Swamp pw1 Consolidated 8acw1 Consolidated th (8th generation (Prepared by addition (8 generation (Established by transfer) of several cultures at transfer) addition of several different levels of cultures at different enrichment) levels of enrichment)
-5 10 10-3 10-2 10-5 dilution dilution dilution dilution Dilution cultures
Cartwright Cartwright Swamp Swamp -5 -3 -2 pw1 10 Consolidated 10 Consolidated 10 Consolidated 10-5
Figure 3.1. Parent cultures and subcultures that were maintained in our laboratory. The benzene-degrading nitrate-reducing parent cultures originally came from three distinct sites: a decommissioned retail gasoline station on Cartwright avenue in Toronto, Ontario, a pristine swamp in Perth, Ontario, and a petrochemical landfarm in Ontario (Nales et al. 1998). Dilution cultures were prepared by serially diluting the subcultures.
3.2. Materials and Methods 3.2.1. Maintenance of benzene-degrading nitrate-reducing enrichment cultures
Benzene was supplied to the enrichment cultures at concentrations ranging from 128 to 256 !M (aqueous concentration) from a neat benzene stock and nitrate was added at a concentration of 2 mM from a sterile anaerobic stock solution of NaNO3. Benzene and nitrate concentrations were monitored consistently. Cultures were re-fed with benzene and nitrate when their concentrations dropped below 64 !M and 0.5 mM, respectively.
! ! 48!
Microbial cultures were maintained statically in the dark inside an anaerobic chamber (Coy
Laboratory Products, Madison, WI) with a gas composition of 80% N2, 10% CO2, and 10% H2.
3.2.2. Nitrite removal
In this study, the strategy to counteract the inhibitory impact of nitrite on the mineralization of benzene was to remove this compound from the bottles. The entire volume of culture was centrifuged at 9900 ! g at 4°C for about 30 minutes. The supernatant that contained nitrite was discarded and replaced with fresh medium. Subsequently, the cell suspension and fresh medium were returned into the original bottle. Culture was then supplied with benzene and nitrate.
3.2.3. Medium omission and addition experiments
The effect of different components of medium including resazurin (redox indicator), FeS (reducing agent), vitamins, sodium tungstate, and trace minerals on lag times was studied. For this purpose, transfer cultures of Cartwright Consolidated were diluted by 50%, 70% or 90% into a medium that either lacked the component of interest (medium omission experiments) or contained it (medium addition experiments). Transfers into the regular medium having all of the customary ingredients were set as positive controls. To ensure that benzene mineralization does not occur abiotically, sterile controls were also prepared. All experiments were performed in triplicates. Benzene and nitrate were supplied at a concentration of 10 to15 mg/l (128 to 192 µM) and 2 mM, respectively. For the tungstate experiment, this compound was added to the medium at a final concentration of 0.75 µm from an anaerobic stock solution of sodium tungstate. Previous work with various concentrations of sodium tungstate by Afshar et al. (1998) showed that addition of this chemical to a denitrifying culture at 0.75 µm resulted in the fastest doubling time and reduced nitrite accumulation. Therefore, this concentration was selected for this study.
To investigate whether the cause of observed lags was the lack of some compounds necessary for the mineralization of benzene from the medium, the lag times for transfers of Cartwright Consolidated into an autoclaved enrichment culture continuously degrading benzene and presumably containing all of the ingredients required for benzene oxidation, were compared with those of transfers into regular medium. In a parallel experiment, four cultures, a 10-5 dilution of Cartwright pw1, 10-2 and 10-5 dilutions of Swamp Consolidated, and a 10-3 dilution of Cartwright Consolidated that were prepared in the course of this study were selected (Figure
! ! 49!
3.1). Before this set of experiments, these cultures were showing a long lag time of 97-137 days after addition of fresh medium. Each of the cultures was subdivided into two portions. One portion was kept as it was. To provide the missing components, an autoclaved enrichment culture, which consistently degrades benzene and has the necessary compounds for benzene mineralization, was added to the other portion. It should be noted that no filtration or centrifugation was performed on the autoclaved enrichment culture prior to adding it to the experimental bottles. These cultures were fed with 128 µM benzene and 2 mM nitrate. Benzene and nitrate concentrations were monitored over time.
3.2.4. Analytical methods Benzene
Benzene concentration was measured by removing a 300 µl sample from the headspace of benzene-degrading nitrate-reducing cultures with a 500 µl Pressure-Lok gastight syringe (Precision Sampling, Baton Rouge, LA) and injecting it onto a Hewlett-Packard 6890 Series gas chromatograph equipped with a HP-5, 30 m ! 0.32 mm I.D. column and a flame ionization detector. Nitrogen at a flow rate of 3 ml/min was used as the carrier gas. The injector, the FID detector, and the oven temperatures were at 200°C, 250°C, and 85°C, respectively. Under these conditions, the retention time for benzene was 2.5 minutes.
Nitrate and nitrite
Nitrate and nitrite concentrations were analyzed by injecting 20 µl of diluted liquid samples taken from the culture into a Dionex DX-100 Series Ion Chromatograph. This Ion Chromatograph was equipped with an IonPac AS9 ion exchange column and an AG9 guard column. The mobile phase consisted of 5 mM sodium bicarbonate and 12.25 mM sodium carbonate. The eluent flow rate was 1 ml/min.
3.3. Results and discussion 3.3.1. Monitoring benzene degradation and nitrate reduction in enrichment cultures
Since their inception, benzene-degrading nitrate-reducing enrichment cultures have been maintained on benzene as an electron donor and the sole source of carbon and energy and nitrate as an electron acceptor. To correlate the oxidation of benzene to the reduction of nitrate and
! ! 50! establish the rate of benzene degradation in these cultures, benzene and nitrate concentrations were monitored in all bottles throughout this study. The status of nitrate-reducing microcosms maintained in the Edwards Laboratory is summarized in Table 3.1. The rate of benzene degradation in the microbial cultures was in the range of 5.2 to 11.6 µM/day. The nitrate to benzene consumption ratio was 9.5 to 13.8 (moles of nitrate reduced/mole of benzene oxidized) (Table 3.1). This ratio is consistent with the range 13.1-14.3 for the degradation of benzene coupled to the incomplete reduction of nitrate to nitrite (refer to section 2.1.1 of chapter 2 and Table 2.3 for details). This result is also supported by the observation that nitrite was accumulating either transiently or permanently in the bottles. Accumulation of nitrite had a detrimental impact on benzene degradation. As it is shown in Table 3.1, in the majority of the microcosms, the mineralization of benzene is significantly inhibited at nitrite concentrations of 5-7 mM. Some of the enrichment cultures appeared to be more resistant to nitrite than others. For example, the inhibitory concentration of nitrite for Cartwright 8acw1 is 6 mM compared to 2 mM for Cartwright pw1. Nitrite accumulated in Cartwright Consolidated transiently. Original benzene-degrading nitrate-reducing microcosms did not build up nitrite (Burland and Edwards 1999). These observations suggest that the original microcosms were less enriched and contained bacterial species that could remove nitrite produced during mineralization of benzene. Enrichment of cultures over time resulted in either the extinction or reduction in the population of these microorganisms in the cultures and consequently, loss or partial loss of nitrite removal ability. However, Cartwright Consolidated might still contain microbes that can reduce and efficiently remove nitrite. As it is discussed in chapter 4, Cartwright Consolidated culture in fact has a large population of Anammox bacteria, which are known to reduce nitrite to the nitrogen gas during oxidation of ammonium (Mulder 1992; Graaf et al. 1995; Graaf et al. 1997; You et al. 2009; Kumar and Lin 2010). This explains the transient accumulation of nitrite in this particular culture.
! ! 51!
Table 3.1. Benzene mineralization rates and nitrite accumulation in benzene-degrading nitrate-reducing cultures.
Average Supplied Inhibitory benzene Ratio of nitrate concentration Accumulation concentration Culturea degradation reduced/benzene nf of benzene of nitrite d of nitrite e rate b oxidized c (µM) (mM) (µM/day) Cartwright 192 8.1 (2.9) 10.7 (0.7) Accumulates 5 5 1b Cartwright Transient 192 11.3 (3.7) 10.8 (2.4) - 13 Consolidated accumulation Cartwright 256 5.5 (2.0) 11.6 (3.7) Accumulates 2 3 pw1 Cartwright 192 9.2 (4.7) 13.6 (0.6) Accumulates 6 4 8acw1
Swamp 1a 256 11.6 (4.5) 10.7 (0.3) Accumulates 5 4 Swamp 1ab 256 10 (2.8) 11.5 (3.3) Accumulates 5 5 Swamp 1b 256 11.2 (1.9) 10.2 (2.3) Accumulates 7 6 Swamp 1bb 128 5.2 (2.0) 9.5 (0.8) Accumulates 7 5 Swamp 1bc 128 7.1 (2.5) 13.8 (1.1) Accumulates 7 4 Swamp 256 9.9 (4.5) 11.4 (2.4) Accumulates 6 7 Consolidated
Landfarm 192 0 - - - - ! a Cartwright 1b and Swamp 1a, 1ab, 1b, 1bb and 1bc are parent cultures; Cartwright and Swamp Consolidated are established by addition of several cultures at different levels of enrichment; Cartwright pw1 and 8acw1 are more enriched than other consortia (8th generation). b Rate of benzene degradation for each culture bottle was calculated by taking an average of benzene mineralization rates observed between January 2009 and May 2010. c The theoretical number of moles of nitrate consumed per moles of benzene mineralized for benzene oxidation coupled to reduction of nitrate to nitrite is 7.7-15, while this ratio is 2.6-6 for benzene oxidation coupled to reduction of nitrate to nitrogen gas (refer to Appendix A). d Accumulates means nitrite is consistently accumulating in the culture; Transient accumulation means that concentration of nitrite increases periodically and then decreases by activity of bacteria present in the culture. e Maximum concentration of nitrite at which benzene degradation is completely inhibited. f Numbers in the parenthesis represent the standard deviations of n values corresponding to n rounds of benzene degradation.
3.3.2. Preventing nitrite accumulation and inhibition
The assessment of the enrichment microcosms revealed that nitrate is reduced to nitrite in the culture bottles. In these microbial consortia, nitrite accumulates to a level (2-7 mM) that represses benzene degradation. Nitrite may inhibit the nitrate reduction pathway and prevent
! ! 52! active mineralization of benzene. Another possible explanation for the loss of benzene degradation activity in the presence of nitrite could be toxicity of this compound to the microorganisms that play important roles in mineralization of benzene in the enrichment cultures. Figure 3.2 shows nitrate utilization and nitrite production during biodegradation of benzene in Cartwright 1b. As benzene was oxidized, nitrate was reduced to nitrite, which accumulated in the bottle. When the nitrite concentration reached 4 mM, no benzene mineralization was observed up to 40 days (Figure 3.2). At this point, the strategy explained in section 3.2.2 was employed to remove nitrite. The benzene degradation curve as well as nitrate and nitrite concentrations after elimination of nitrite are provided in Figure 3.3. As it is shown in this figure, removal of nitrite causes the culture to regain its ability to mineralize benzene. The culture experienced a lag time of about 20 days prior to benzene degradation. During this lag time, no reduction in nitrate or increase in nitrite concentrations was evident. The observed lag time was probably due to the addition of fresh medium. Thereafter, nitrate was reduced to nitrite concomitantly with benzene mineralization causing nitrite to be built up in the culture bottle. This strategy was implemented and utilized throughout this research for successfully removing nitrite from enrichment cultures and reviving their benzene degradation capacity.
250 4
200 3 150 Benzene 2
Nitrate (mM) 100 Nitrite
1 50 Fed benzene
Benzene concentration (µM) and nitrate Nitrate and nitrite concentrations 0 0 0 20 40 60 80 100
Time (day) Figure 3.2. Nitrate consumption and nitrite production are shown during biodegradation of benzene in Cartwright 1b enrichment culture. Arrows indicate time points at which the culture was supplied with benzene and nitrate.
! ! 53!
250 4
200 3
150 Benzene 2 (mM) 100 Nitrate
Nitrite 1 50 Benzene concentration (µM) Nitrate and nitrite concentrations
0 0 0 10 20 30 Time (day)
Figure 3.3. Mineralization of benzene by Cartwright 1b after nitrite removal. As benzene is being degraded nitrate is reduced to nitrite.
3.3.3. The effect of different components of medium on the lag time
Most of cultures maintained in our laboratory show significant lag times (no benzene degradation) after transferring them into fresh medium. One of the goals of this study was to optimize the growth of benzene-degrading microorganisms through shortening of these lag times. The study of the observed lag times was based on two hypotheses: 1. Some constituents of growth medium are at concentrations that pose inhibitory effects on the degradation of benzene. The activity of microorganisms present in the culture should reduce the concentration of these compounds to a lower level before degradation can start; 2. Some components are missing from the medium and must be synthesized before benzene mineralization can start.
Medium components of particular interest are resazurin (redox indicator), FeS (reducing agent), vitamins, sodium tungstate, and trace minerals for the following reasons: (i) In previous microcosm experiments, it was shown that resazurin had an inhibitory effect on the degradation of other compounds (Edwards 2006). (ii) The growth medium of Dechloromonas aromatica RCB (Bruce et al. 1999), which is an isolated microorganism with a high benzene degradation rate of 32 µM/day, does not contain FeS and has 10 times more vitamins than our medium. (iii)
! ! 54!
Comparing our medium and several other anaerobic media (Loffler et al. 1996; Plugge 2005) indicates that our medium consists of much higher concentrations of trace minerals. (iv) In the past decade, isolation of several tungsten-containing enzymes has shown a very important biological role for this element in various metabolic reactions (L'vov et al. 2002). Tungsten also stimulates the growth of a variety of microorganisms (Andreseen and Ljungdahl 1973; Andreesen et al. 1974; Taya et al. 1985; Widdel 1986; Wanger and Andreesen 1987; Zindel et al. 1988; Hensgens et al. 1994; Huber et al. 1995; Rosner and Schink 1995; Afshar et al. 1998). In most cases, presence of tungsten greatly improves growth yields or activities of certain enzymes 2- (Afshar et al. 1998). Moreover, addition of tungstate (the metal oxyanion WO4 ) to the medium results in a decrease in nitrite accumulation in some nitrate-reducing cultures (Afshar et al. 1998; L'vov et al. 2002). In the following sections, the effects of these compounds on lag time in benzene-degrading nitrate-reducing cultures are presented.
FeS and Resazurin
The effect of omission of FeS and resazurin from the medium was examined on the lag time and growth of microorganisms. Figure 3.4 compares the mineralization of benzene and lag times for transfers into regular medium, medium without resazurin, FeS, or both. The onset of benzene degradation in the regular medium was about 45 days from the transfer and it took another 12 days for benzene to be degraded (Figure 3.4a). Eliminating resazurin from the culture reduced the lag time to about 25 days and consumption of benzene by microorganisms was completed within 12 days (Figure 3.4b). Transfers into the FeS-free medium performed almost similar to those into the regular medium. Benzene mineralization started on day 43 and it was depleted in about 13 days (Figure 3.4c). These observations indicated that resazurin had a negative effect on the mineralization of benzene by increasing the lag time. The highest performing cultures were those cultivated into the medium with both resazurin and FeS omitted. In these treatments benzene was degraded within few days once the process started around day 15. Overall, it appears that concomitant presence of resazurin and FeS resulted in longer lag periods. During preparation of medium, resazurin, which is blue in color, is reduced to resorufin (a pink compound) and then to dihydroresorufin (a colorless compound) by FeS. We have observed that the colorless medium turns into pink when nitrate is added to the active cultures. It suggests that microorganisms in the culture are involved in re-oxidation of dihydroresorufin to resurofin using
! ! 55! nitrate as an electron acceptor. The interaction and shuttling of electrons between FeS and resazurin may increase the toxicity of resazurin and result in the observed lags.
The average number of moles of nitrate consumed per moles of benzene oxidized was 11.3 ± 0.3 (n=11 individual bottles, one measurement per bottle), which is consistent with oxidation of benzene coupled to incomplete reduction of nitrate to nitrite.
! ! 56! 200
(a) 150
100 Regular medium
50
Benzene concentration (µM) 0 200 (b) 150
Without Resazurin 100
50
Benzene concentration (µM) 0 200 ) (c) 150
100 Without FeS
50
0 Benzene concentration (µM 200
(d) 150
100 Without FeS and Resazurin
50
Benzene concentration (µM) 0 0 20 40 60 80 Time (day) Figure 3.4. Effect of FeS and resazurin on the observed lag time. Benzene degradation curves for transfers of Cartwright Consolidated culture into a) regular medium, b) medium without resazurin, c) medium without FeS, and d) medium without FeS and resazurin. Three replicates were set for each treatment. Each line in figure panels represents one replicate. One of the transfers into the medium without FeS did ! not show benzene degradation activity up to 80 days. ! 57!
Tungstate
2- It has been reported previously that addition of tungstate (the metal oxyanion WO4 ) to the culture medium improves the growth yield or certain enzyme activities of different microorganisms (Andreseen and Ljungdahl 1973; Andreesen et al. 1974; Taya et al. 1985; Widdel 1986; Wanger and Andreesen 1987; Zindel et al. 1988; Hensgens et al. 1994; Huber et al. 1995; Rosner and Schink 1995; Afshar et al. 1998). The isolation of a number of tungsten- containing enzymes such as formate dehydrogenase, aldehyde:ferredoxin-oxidoreductase, formaldehyde:ferredoxin-oxidoreductase in the past decade has proven the biological importance of this element (L'vov et al. 2002). It has also been suggested that tungstate affects the activity or synthesis of enzymes involved in nitrate reduction (Afshar et al. 1998; L'vov et al. 2002) and that it can potentially be used to decrease the amount of nitrite build-up. In a nitrate-reducing culture, an increase in nitrate reductase activity was observed in cells grown at low tungstate concentrations (0.1-0.3 µM) (Afshar et al. 1998). In this study, the effect of addition of 0.75 µM tungstate on the lag times and nitrite accumulation was examined. This concentration was selected based on previous studies by Afshar et. al (1998) who showed that 0.75 µM is the optimum concentration to result in the shortest doubling time and reduced accumulation of nitrite in a denitrifying culture. Benzene degradation curves and nitrite concentration for transfers into regular medium and medium with sodium tungstate are shown in Figure 3.5. There is essentially no difference between the performance of transfers into the regular and sodium tungstate-added media. In addition, nitrite accumulation in both treatments suggests that tungstate at concentration of 0.75 µM does not prevent nitrite build-up in these cultures. It should be noted that sodium tungstate was added to the medium only at a single concentration. However, this may not be the optimum concentration for the benzene-degrading nitrate-reducing microbial consortia.
! ! 58!
2.0 160 (a)
1.5 120 Regular medium 1.0 80
40 0.5
Nitrite concentration (mM) Benzene concentration (µM)
0 0.0
2.0 160 (b)
1.5 120
Medium with tungstate 80 1.0
40 0.5 Nitrite concentration (mM) Benzene concentration (µM)
0 0.0 0 10 20 30 40 Time (day) Figure 3.5. Benzene degradation curves and nitrite concentrations for transfers into a) regular medium and b) medium with tungstate. This experiment was performed in triplicates. Closed symbols represent benzene concentration for each individual treatment and open symbols represent the corresponding nitrite concentrations. Each of the lines in each figure corresponds to one replicate.
!
! ! 59!
Trace minerals and Vitamins
The mineral medium used for maintaining the enrichment cultures has higher concentrations of trace minerals (!5 to 100 times higher) but ten times lower vitamin concentrations compared to other anaerobic media such as the one employed for cultivating denitrifying benzene- degrading Dechloromonas aromatica RCB (Loffler et al. 1996; Bruce et al. 1999; Plugge 2005; Kumar and Lin 2010). Therefore, the effect of omission of trace minerals and addition of 10 times more vitamins on the performance of benzene-degrading nitrate-reducing cultures was studied. Figure 3.6 shows that transfers into the medium without trace minerals have a much longer lag time (about 20 days longer) (panel b) compared to the transfers into regular medium (panel a). This result indicates that trace minerals at the concentration present in the medium indeed promote benzene degradation and facilitate the mineralization process. Treatments cultivated into the medium with 10 times more vitamins behave similar to those into regular medium (Figure 3.6c). Most likely, the regular medium contains sufficient concentration of vitamins and addition of 10 times more vitamins does not improve the reduction of the lag time further.
! ! 60!
160
(a) 120
Regular medium 80
40
Benzene concentration (µM) 0 0 10 20 30 40 50 60
160
(b) 120
80 Without trace minerals
40
Benzene concentration (µM) 0
0 10 20 30 40 50 60
160
120 (c)
With 10 times more vitamins 80
40 Benzene concentration (µM) 0 0 10 20 30 40 50 60
Time (day) Figure 3.6. Comparison between benzene degradation of transfers into a) regular medium, b) medium without trace minerals, and c) medium with 10 times more vitamins. This experiment was prepared in triplicates. Each of the lines in each figure represents one replicate. One of the transfers into 10 times more vitamins did not ! degrade benzene up to 70 days. ! ! 61!
Components such as cofactors
One possible explanation for the lag time observed after addition of fresh medium is either the absence or dilution of some components such as cofactors that are necessary for benzene degradation. These compounds must be synthesized to certain concentrations before benzene degradation can start. To study if this may contribute to the observed patterns, the lag time for transfers into an autoclaved culture degrading benzene without delay, which most probably contains all the components required for the mineralization of benzene, was compared with the lag time for transfers into a regular medium. Transfers into the autoclaved culture have slightly shorter lag times than those into the regular medium (Figure 3.7). The slight reduction in the lag time for treatments cultivated into autoclaved culture is most likely due to the presence of cofactors or minerals in the autoclaved culture medium.
160 (a) 120
80 Regular medium
40
Benzene concentration (µM) 0 0 5 10 15 20 25 30
160 (b) 120
80 Transfers into autoclaved culture 40
Benzene concentration (µM) 0
0 5 10 15 20 25 30 Time (day) Figure 3.7. Benzene degradation curves for treatments cultivated into a) regular medium and b) autoclaved culture. This experiment was performed in triplicate. Each line in each figure represents one replicate. ! ! 62!
In a similar experiment, four of the dilution cultures Cartwright pw1 10-5, Swamp Consolidated 10-2 and 10-5, and Cartwright Consolidated 10-3 (Figure 3.1) that had been stalled for a long time after addition of fresh medium, were each subdivided into two bottles. One bottle was kept as it was. An autoclaved enrichment culture, which continuously degrades benzene, was added to the other bottle to provide missing medium components. This strategy proved to be useful and resulted in significant enhancement in benzene degradation capability of two of the cultures, Swamp Consolidated 10-2 and Cartwright pw1 10-5. As an example, benzene degradation curves for Swamp Consolidated 10-2 with and without addition of autoclaved culture are shown in Figure 3.8a. In the presence of autoclaved enrichment culture, microorganisms effectively degraded benzene five times over a course of 150 days, whereas in its absence, only two degradation cycles were observed and a fairly long lag time of >60 days was developed after the second degradation. Most likely, the autoclaved culture provides the microorganisms with the components (minerals, cofactors, and etc) that are either missing from the medium or are present at suboptimal concentrations. Furthermore, autoclaving the culture results in destruction of the cells and cellular components, which could be then used as a carbon or nutrient source by benzene-degrading bacteria to promote their growth. Analysis of autoclaved culture can provide information regarding its constituents. The other two cultures, Cartwright Consolidated 10-3 and Swamp Consolidated 10-5, however, did not exhibit much improvement in benzene degradation after the addition of autoclaved culture, as shown in Figure 3.8b for Swamp Consolidated 10-5. Benzene mineralization curves for the two other cultures, Cartwright pw1 10-5 and Cartwright Consolidated 10-3 are provided in Appendix C. In these experiments bottles that only contained autoclaved enrichment culture were not used as the controls. Therefore, it was unclear whether the autoclaved culture had any benzene degradation activity on its own. However, it is unlikely that autoclaved culture would show any activity.
! ! 63!
160 (a) Without autoclaved culture With autoclaved culture 120
80
40 Benzene concentration (µM)
0 0 20 40 60 80 100 120 140 160
(b) Without autoclaved culture 120 With autoclaved culture
80
40
Benzene concentration (µM)
0 0 20 40 60 80 100 120 140 160 Time (day) Figure 3.8. Comparison between anaerobic benzene degradation in dilution cultures, a) Swamp Consolidated 10-2 and b) Swamp Consolidated 10-5, both with and without autoclaved culture. Arrows indicate time points at which benzene is added to the culture.
! ! 64!
3.4. Conclusions
Benzene degradation in the enrichment cultures is coupled to incomplete reduction of nitrate to nitrite and not to its complete reduction to the nitrogen gas. Nitrite accumulates in the cultures and prevents benzene mineralization. Nitrite removal (by dilution or centrifugation) is then required for the culture to regain its degradation capacity. Omitting both FeS and resazurin from the medium greatly decreases lag times observed upon transfer of culture into fresh medium. Addition of autoclaved culture results in the reduction of the lag time probably by providing sufficient concentrations of compounds required for the mineralization of benzene as in the original culture. Although this strategy was not helpful to all cultures used in this study, it did not induce any inhibitory effect either. Therefore, this strategy can be employed as a means of improving benzene degradation in microbial cultures.
3.5. Recommendations for future work
The strategy employed in this study for removing nitrite proved useful to counteract the inhibitory effect of nitrite. Therefore, it can be used to revive the ability of cultures to oxidize benzene.
Addition of tungstate at a concentration of 0.75 µM does not seem to benefit benzene degrading capability of the nitrate-reducing cultures. Therefore, this compound should be added to the medium at various concentrations to substantiate its effect on the lag time, nitrite accumulation, and microbial growth.
In order to obtain an optimized growth strategy, it is important to reduce the lag time upon transfer of cultures to fresh media. Removal of resazurin and FeS from the medium and addition of autoclaved culture will help to reduce the observed lag times.
It has been observed that metabolism of aromatic hydrocarbons such as benzoate for sulfate- reducing Desulfococcus multivorans and iron-reducing Geobacter metallireducens is strictly dependent on the presence of selenium, and molybdenum (or tungstate) (Peters et al. 2004; Wischgoll et al. 2005). The effect of these compounds on the growth of the microbial consortia should be studied in the future.
! ! 65!
Amberlite-XAD7 ion exchange resin has been previously used for enrichment of the cultures on benzene (Morasch et al. 2001; Jahn et al. 2005; Herrmann et al. 2008). Amberlite-XAD7 adsorbs benzene and keeps the concentration of this compound and possibly toxic metabolites produced during the mineralization process low. With time, the substrate pool adsorbed to the resin is steadily released and becomes available to the organisms leading to a better growth and higher cell densities (Morasch et al. 2001; Jahn et al. 2005; Herrmann et al. 2008). This set up more closely resembles in situ conditions at contaminated sites where usually low concentrations of substrate are predominant. Therefore, we recommend employing Amberlite-XAD7 ion exchange resin for further enrichment of the cultures on benzene.
Herrmann et al. (2008) showed that solid supports such as sand and lava granules were required for enrichment of benzene-degrading sulfate-reducing microorganisms. Control microcosms without filling material initially degraded benzene, but the benzene-degrading capacity was sustained only for a short period of time. On the other hand, benzene was repeatedly degraded in cultures prepared using solid materials. The authors suggested that the use of solids could be favorable for the enrichment of bacteria involved in degradation of benzene. This could be used as a means of improving the growth of benzene degraders within enrichment cultures.
A study conducted by Musat et al. (2008) showed that an increase in concentration of trace elements specifically that of iron resulted in shortening lag-phase prior to benzene degradation and stimulating growth of bacteria in a sulfate-reducing culture. Employing a similar strategy may also be helpful for reducing the lag times and enhancing the growth of bacteria in the cultures.
Duldhardt et al. (2007) recently reported a decrease in the growth rate and degradation kinetics of anaerobic bacteria due to exposure to high concentrations of organic hydrocarbons such as benzene and toluene. They correlated this effect to toxicity of these compounds. Increasing the feeding concentration of benzene from 10 ppm to 200 ppm in an anaerobic benzene-degrading culture caused the rate and extent of benzene mineralization to severely diminish (Liou et al. 2008). Kasai et al. (Kasai et al. 2007) found that benzene degradation rates for denitrifying pure culture of Azoarcus strain DN11 improved by increasing the benzene concentration to 3 µM. A study conducted by Junfeng et al. (2008) with a BTEX-degrading
! ! 66! nitrate-reducing enriched culture also indicated that concentration of benzene affected rate of degradation. Therefore, it is suggested that the impact of addition of benzene at different concentrations on the benzene mineralization rate and growth of microorganisms should be studied in the benzene-degrading nitrate-reducing cultures. This will allow identify the optimum concentrations at which cultures can grow and degrade benzene with an optimal rate.
! !
3.6. References Chapter 3
Afshar, S., C. Kim, H. G. Monbouquette and I. Schroder (1998). "Effect of tungstate on nitrate reduction by hyperthermophilic archaeon Pyrobaculum aerophilum." Appl. Environ. Microbiol. 64: 3004-3008.
Andreesen, J. R., E. E. Ghazzawi and G. Gottschalk (1974). "The effect of ferrous ions, tungstate and selenite on the level of formate dehyrogenase in Clostridium formicoaceticum and formate synthesis from CO2 during pyrovate fermentation." Arch. Microbiol. 96: 103-118.
Andreseen, J. R. and L. G. Ljungdahl (1973). "Formate dehydrogenase of Clostridium thermoaceticum: incorporation of selenium-75, and the effects of selenite, molybdate, and tungstate on the enzyme." J. Bacteriol 116: 867-873.
Bruce, R. A., L. A. Achenbach and J. D. Coates (1999). "Reduction of (per)chlorate by a novel organism isolated from paper mill waste." Environ. Microbiol. 1(4): 319-329.
Burland, S. M. and E. A. Edwards (1999). "Anaerobic benzene biodegradation linked to nitrate reduction." Appl. Environ. Microbiol. 65(2): 529-533.
Duldhardt, I., I. Nijenhuis, F. Schauer and H. J. Heipieper (2007). "Anaerobically grown Thauera aromatica, Desulfococcus multivorans, Geobacter sulfurreducens are more sensitive towards organic solvents than aerobic bacteria." Appl. Microbiol. Biotechnol. 77(3): 705-711.
Edwards, E. A. (2006). Personal Communication.
Graaf, A. A. v. d., P. D. Bruijn, L. A. Robertson, M. S. M. Jetten and J. G. Kuenen (1997). "Metabolic pathway of anaerobic ammonium oxidation on the basis of I5N studies in a fluidized bed reactor." Microbiology 143: 2415-2421.
Graaf, A. A. v. d., A. Mulder, P. D. Bruijn, M. S. Jetten, L. A. Robertson and J. G. Kuenen (1995). "Anaerobic oxidation of ammonium is a biologically mediated process." Appl. Environ. Microbiol. 61(4): 1246-1251.
Hensgens, C. M. H., M. E. Nienhuiskuiper and T. A. Hansen (1994). "Effects of tungsten on the growth of Desulfovibrio giagas NCIMB 9332 and other sulfate reducing bacteria with ethanol as a substrate." Arch. Microbiol 162: 143-147.
Herrmann, S., S. Kleinsteuber, T. R. Neu, H. H. Richnow and C. Vogt (2008). "Enrichment of anaerobic benzene-degrading microorganisms by in situ microcosms." FEMS Microbiol. Ecol. 63(1): 94-106.
Huber, C., H. Skopan, R. Feicht and H. Simon (1995). "Pterin cofactor, substrate specificity, and observations on the kinetics of the reversible tungestencontaining aldehyde oxidoreductase from Clostridium thermoaceticum." Arch. Microbiol 164: 110-118.
! 67! ! 68!
Jahn, M. K., S. B. Haderlein and R. U. Meckenstock (2005). "Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures." Appl. Environ. Microbiol. 71: 3355-3358.
Junfeng, D., L. Xiang and H. Zhifeng (2008). "Anaerobic BTEX degradatin in soil bioaugmented with mixed consortia under nitrate-reducing conditions." J. Environ. Sci. 20: 585-592.
Kasai, Y., Y. Kodama, Y. Takahata, T. Hoaki and K. Watanabe (2007). "Degradative capacities and bioaugmentation potential of anaerobic benzene-degrading bacterium strain DN11." Environ. Sci. Technol. 41: 6222-6227.
Kumar, M. and J. G. Lin (2010). "Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal-Strategies and issues." J. Hazard. Mater. 178: 1-9.
L'vov, N. P., A. N. Nosikov and A. N. Antipov (2002). "Tungsten-containing enzymes." Biochemistry (Moscow) 67(2): 196-200.
Liou, J. S. C., C. M. Derito and E. L. Madsen (2008). "Field-based and laboratory stable isotope probing surveys of the identities of both aerobic and anaerobic benzene-metabolizing microorganisms in freshwater sediment." Environ. Microbiol. 10(8): 1964-1977.
Loffler, F. E., R. A. Sanford and J. M. Tiedje (1996). "Initial charecterization of a reductive dehalogenase from Desolfitobacterium chlororespirans Co23." Appl. Environ. Microbiol 62(10): 3809-3813.
Morasch, B., E. Annweiler, R. J. Warthmann and R. U. Meckenstock (2001). "The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, O-, and m-xylene by sulfate-reducing bacteria." J. Microbiol. Methods 44(2): 183-191.
Mulder, A. (1992). Anoxic ammonia oxidation. U. S.
Musat, F. and F. Widdel (2008). "Anaerobic degradation of benzene by a marine sulfate- reducing enrichment culture, and cell hybridization of the dominant phylotype." Environ. Microbiol. 10(1): 10-19.
Nales, M., B. J. Butler and E. Edwards (1998). "Anaerobic benzene biodegradation: a microcosm survey." Bioremediat. J. 2: 125-144.
Peters, F., M. Rother and M. Boll (2004). "Selenocysteine-containing proteins in anaerobic benzoate metabolism of Desulfococcus multivorans." J. Bacteriol. 186: 2156-2163.
Plugge, M. C. (2005). "Anoxic media design, preparation, and consideration." Methods Enzymol. 397: 3-16.
! ! 69!
Rosner, B. M. and B. Schink (1995). "Purification and charecterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein." J. Bacteriol 177: 5767-5772.
Taya, M. H., H. Hinoki and T. Kobayashi (1985). "Tungsten requirement of an extremely thermophilic cellulytic anaerobe (strain NA 10)." Agric. Biol. Chem. 49: 2513-2515.
Wanger, R. and J. R. Andreesen (1987). "Accumulation and incorporation of W-tungsten into protein of Clostridium acidurici cylindrosporum." Arch. Microbiol 147: 295-299.
Widdel, F. (1986). "Growth of methangenic bacteria in pure culture with 2-propanol and other alcohols as hydrogen donors." Appl. Environ. Microbiol 51: 1056-1062.
Wischgoll, S., D. Heintz, F. Peters, A. Erxleben, E. Sarnighausen, R. Reski, A. V. Dorsselaer and M. Boll (2005). "Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens." Mol. Microbiol. 58(5): 1238-1252.
You, J., A. Das, E. M. Dolan and Z. Hu (2009). "Ammonia-oxidizing archaea involved in nitrogen removal." Water Res. 43(7): 1801-1809.
Zindel, U., W. Freudenberg, M. Rieth, J. R. Andreesen, J. Schnell and F. Widdel (1988). "Eubacterium acidaminophilum sp. nov., a versatile amino acid degrading anaerobe producing or utilizing H2 or formate. Description and enzymatic studies." Arch. Microbiol 150: 254-266.
! !
Chapter 4: Multiple Syntrophic Associations in Nitrate-Reducing Benzene-Degrading Cultures
Prepared for publication as:
Multiple Syntrophic Associations in Nitrate-Reducing Benzene-Degrading Cultures Roya Gitiafroz, Cheryl E. Devine, Lutgarde Raskin, and Elizabeth A. Edwards. 2011.
! 70! ! 71!
4.1. Introduction
! The monoaromatic hydrocarbons Benzene, Toluene, Ethylbenzene, and the Xylenes, collectively called BTEX, are a major source of groundwater contamination from petroleum sources. Benzene in particular is of major concern due to its toxicity and relatively high water- solubility compared to other components of petroleum. Fortunately, benzene is readily biodegradable in the presence of oxygen and is degraded by a large number of aerobic benzene- degrading microorganisms, most notable of which are the Pseudomonas species (Ridgeway et al. 1990). However, in contaminated aquifers, aerobic microorganisms deplete oxygen and generate extensive anaerobic zones. The prevalence of anoxic environments at contaminated sites has motivated research to determine the fate of benzene under these conditions. Anaerobic benzene degradation has been demonstrated under nitrate-reducing (Nales et al. 1998; Burland and Edwards 1999; Coates et al. 2001; Ulrich and Edwards 2003; Kasai et al. 2006; Kasai et al. 2007), sulfate-reducing (Grbi!-Gali! and Vogel 1987; Edwards and Grbi!-Gali! 1992; Lovley et al. 1995; Phelps et al. 1996; Kazumi et al. 1997; Nales et al. 1998; Phelps and Young 1999; Caldwell and Suflita 2000; Vogt et al. 2007; Kleinsteuber et al. 2008; Musat and Widdel 2008; Abu Laban et al. 2009; Herrmann et al. 2010), iron-reducing (Lovley et al. 1996; Anderson and Lovley 1997; Kazumi et al. 1997; Nales et al. 1998; Rooney-Varga et al. 1999; Jahn et al. 2005; Botton and Parsons 2007; Kunapuli et al. 2007), and methanogenic conditions (Grbi!-Gali! and Vogel 1987; Kazumi et al. 1997; Ulrich and Edwards 2003; Chang et al. 2005). Yet despite almost two decades of research, very little is known about the microorganisms and mechanisms involved in the anaerobic degradation process, primarily because active pure cultures are difficult to obtain and maintain (Coates et al. 2001; Kasai et al. 2006). In addition to this, the diversity of microbes identified in anaerobic benzene-degrading enrichment cultures (Phelps et al. 1998; Ulrich and Edwards 2003) makes attribution of function challenging. A survey of benzene-degrading anaerobic cultures (Table 4.1) reveals that certain phyla tend to recur in strictly anaerobic enrichments, suggesting that the ability to degrade benzene under these conditions may be restricted to certain types of microbes. Bacteria related to families Peptococcaceae, Geobacteraceae, Desulfobacteraceae, and the order Desulfuromonadales have been found in iron-reducing, sulfate-reducing and methanogenic enrichment cultures (Phelps et al. 1998; Rooney-Varga et al. 1999; Ulrich and Edwards 2003; Chang et al. 2005; Da Silva and Alvarez 2007; Kunapuli et al. 2007; Kleinsteuber et al. 2008; Musat and Widdel 2008; Oka et al.
! ! 72!
2008). In addition, one obligate anaerobic isolate, the hyperthermophilic archaeon Ferroglobus placidus, has recently been shown to couple benzene mineralization to iron reduction (Holmes et al. 2011). A few isolates related to the genera Dechloromonas and Azoarcus have been obtained under nitrate-reducing conditions (Table 4.1) (Coates et al. 2001; Chakraborty and Coates 2004; Kasai et al. 2006; Kasai et al. 2007).
We have been maintaining nitrate-reducing benzene-degrading enrichment cultures in our laboratory for over a decade. These cultures are derived from a microcosm study initiated in 1995 (Nales et al. 1998). Previous work identified major phylogenetic groups in these enrichment cultures (Ulrich and Edwards 2003; Nandi 2006). To attribute functional roles to the community members, and in particular to identify the organism responsible for the initial attack on benzene, we quantified the abundance of the dominant phylotypes in several different enrichment cultures, as well as the change in the abundance of these phylotypes during the course of benzene degradation. This study enhances the current understanding of the bacteria responsible for oxidation of benzene under nitrate-reducing conditions and thus, will contribute to the successful future application of bioremediation to contaminated sites.
! ! 73!
Table 4.1. Microorganisms in enriched or pure cultures that are involved in anaerobic degradation of benzene under different electron accepting conditions.
Microorganisms Source of inoculum Pure Reference culture Iron-reducing Geobacteraceae Sediment from the U. S. (Anderson et al. (Geobacter) Geological Survey Groundwater No 1998; Rooney- Toxics Sites, Bemidji, Minn Varga et al. 1999)
Ferroglobus placidus Hydrothermally heated marine Yes (Holmes et al. 2011) sediment, Vulcano, Italy Sulfate- Desulfobacteraceae Sediment from an area of deep- No (Phelps et al. 1998; reducing Clone SB-21 water hydrocarbon seeps, in the Oka et al. 2008) Guaymas Basin, Gulf of Calfornia, Mexico
Desulfobacterium Sediment from a stagnant part of No (Musat and Widdel Clone BznS295 a Mediterranean laggon, Etang 2008) de Berre, France
Clostridia Groundwater from a BTEX No (Kleinsteuber et al. (Peptococcaceae-related) contaminated aquifer located in 2008; Herrmann et Zietz, Saxonia-Anhalt, Germany al. 2010)
Soil from a former coal No (Abu Laban et al. gasification site in Gliwice, 2009) Poland Nitrate- Dechloromonas strain RCB Sediments from the Potomac Yes (Coates et al. 2001) reducing River, Maryland, USA
Dechloromonas strain JJ Sediments from Campus Lake, Yes (Coates et al. 2001) Southern Illinois University, USA
Azoarcus strain DN11 BTX-contaminated subsurface Yes (Kasai et al. 2006) aquifer, Kumamoto, Japan
Azoarcus strain AN9 BTX-contaminated subsurface Yes (Kasai et al. 2006) aquifer, Kumamoto, Japan Methanogenic Desulfuromonadales Soil and groundwater from a No (Ulrich and Edwards Clone OR-M2 decommissioned retail gasoline 2003; Da Silva and station on Cartwright Avenue, Alvarez 2007) Toronto, Canada
Deltaproteobacteria Lotus field soil from Tsuchiura, No (Sakai et al. 2009) Clone Hasda-A Ibaraki, Japan
! ! 74!
4.2. Materials and Methods 4.2.1. Benzene-degrading nitrate-reducing cultures and subcultures
Transfer cultures from original microcosms were maintained for over 16 years in ferrous sulfide-reduced defined anaerobic mineral medium, as previously described (Burland and Edwards 1999; Ulrich and Edwards 2003). The microcosms and subsequent transfer cultures were supplied with benzene (130-250 !M doses from neat stocks) as the sole exogenous source of carbon and electron donor and nitrate (NaNO3; 2 mM doses) as electron acceptor. Cultures were fed every 2-4 weeks and maintained statically inside an anaerobic chamber (Coy
Laboratory Products, Madison, WI) supplied with a gas mix of 80% N2, 10% CO2, and 10% H2. The parent cultures (Swamp and Cartwright, whose names derive from the source site material) and subcultures investigated herein are summarized in Figure 4.1. Dilution cultures were retained in a slightly different medium than the one used for maintaining the parent cultures and subcultures. This medium did not contain FeS and resazurin. While the parent cultures originally came from distinct sites, over the many years of maintenance of multiple sub-cultures in the labratory with many different researchers, it is likely that these cultures are no longer completely distinct.
! ! 75!
Site #1 Site #2 Cartwright gasoline station Uncontaminated Swamp
Parent Cultures
Cartwright 1b Swamp
Subcultures
Cartwright pw1 Cartwright Consolidated Swamp Consolidated th (8 generation (Prepared by addition of (Established by addition of transfer) several cultures at different several cultures at different levels of enrichment) levels of enrichment)
10-5 10-2 10-4 10-2 10-5 dilution dilution dilution dilution dilution Dilution cultures
Cartwright Cartwright Cartwright Swamp Swamp -5 pw1 10 1b 10-2 Consolidated 10-4 Consolidated 10-2 Consolidated 10-5
Figure 4.1. Parent cultures and subcultures that were characterized in this study. The benzene-degrading nitrate-reducing parent cultures originally came from two distinct sites: a decommissioned retail gasoline station on Cartwright avenue in Toronto, Ontario, and a pristine swamp in Perth, Ontario (Nales et al. 1998). Dilution cultures were established through serially diluting the subcultures.
4.2.2. DNA extraction, 16S rRNA gene cloning and sequencing
A 16S rRNA gene clone library was prepared from 8 mL of the Swamp Consolidated culture. Cells were pelleted by centrifugation at 13000 ! g for 15 minutes at 4°C, and DNA was extracted using the UltraClean Soil DNA Kit (Mo Bio Laboratories, Inc., Solana Beach, CA) according to the manufacturer’s alternative protocol for maximum yields. Purified DNA was subjected to PCR to selectively amplify bacterial 16S rRNA genes using the forward primer 27f (5’ AGAGTTTGATCCTGGCTCAG 3’) and the reverse primer 1492r (5’
! ! 76!
GGTTACCTTGTTACGACTT 3’) (Weisburg et al. 1991). PCR was performed in six replicate reactions. Each 50 !l PCR reaction contained 1! ThermoPol PCR buffer (New England Biolabs, Mississauga, Ontario, Canada), 0.4 µM of each primer, 0.3 mM of deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (New Englands Biolabs), and 1 µl of DNA template. PCR amplification conditions were as follows: initial denaturation at 94°C for 5 minutes, then 28 cycles of denaturation at 94°C for 1 minute, primer annealing at 52°C for 1 minute, and chain extension at 72°C for 1.45 minutes, followed by final chain extension at 72°C for 10 minutes. A PTC-200 Peltier Thermal Cycler (MJ Research, Inc., Waltham, Massachusetts, USA) was employed to perform PCR. PCR products were combined and purified using GenEluteTM PCR Clean-up Kit (Sigma-Aldrich, St. Louis, MO, USA) and cloning was performed using the TOPO TA cloning kit (Invitrogen Corp, Carlsbad, CA) as per manufacturer’s instructions. Thirty clones were sequenced using a Beckman Coulter CEQ 2000 automatic Sequencer (University of Toronto’s Health Network Research DNA Sequencing Facility, Toronto, Canada). The sequencing primers were T7 (5’ TAATACGACTCACTATAGGG 3’) and M13 reverse (5’ CAGGAAACAGCTATGAC 3’), which anneal to the flanking regions located on the TOPO TA cloning vector. The CHECK- CHIMERA program of the ribosomal data base project II (Cole et al. 2005) was employed to identify the chimeric sequences. The NCBI GenBank BLASTn tool (Altschul et al. 1990) and the Ribosomal Database project II, Seqmatch tool (Cole et al. 2005), were used to retrieve 16S rRNA gene sequences similar to those found in the clone library. The sequences were aligned using ClustalW (Larkin et al. 2007) and a phylogenetic tree was constructed using the neighbor- joining method in MEGA 4, with 1000 bootstraps (Tamura et al. 2007). Partial 16S rRNA gene sequences of the major Operational Taxonomic Units (OTUs) that were present in Swamp Consolidated culture are provided in Appendix D.
4.2.3. Quantification of specific species in enrichment cultures
For this analysis, 5 ml of nine different enrichment cultures (Figure 4.1) were transferred to sterile, anaerobic 15 mL centrifuge tubes during active benzene degradation. Samples were centrifuged at 13000 ! g for 15 minutes at 4°C and DNA was extracted from the cell pellet using FastDNA Spin Kit for Soil (MP Biomedical, LLC, Solon, OH, USA) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was used to determine the abundance of
! ! 77!
16S rRNA gene copies of five phylotypes that had been identified in the cultures i.e. Azoarcus, Dechloromonas, Peptococcaceae, Chlorobi, and Anammox organisms. qPCR was performed using an Opticon 2 thermocycler (MJ Reserch, Inc., Waltham, Massachusetts, USA) and SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich Co., St. Louis, MO). Each 25 µl qPCR reaction contained 12.5 µl of SYBR Green JumpStart Taq ReadyMix, each forward and reverse primer at a concentration of 0.4 µM, 9.7 µl of water, and 2 µl of DNA template. The species-specific primers used in this study and their annealing temperatures are given in Table 4.2. The qPCR condition was as follows: initial denaturation at 94°C for 5 minutes, 44 cycles of denaturation at 94°C for 30 seconds, annealing at an appropriate annealing temperature for 30 seconds, and chain extension at 72°C for 45 seconds. Calibration curves were prepared using serial dilutions of a known quantity of appropriate 16S rRNA gene-containing plasmid inserts, which were amplified using T7 and M13 reverse primers. A sample calculation for quantification of 16S rRNA gene copies per ml of culture is provided in Appendix E.
4.2.4. Time course experiments
This experiment was performed twice (August 2008 and June 2009). In both cases, 792 ml of anaerobic mineral medium was transferred into each of six sterile, anaerobic glass media bottles. The Cartwright Consolidated culture was added to each bottle at a 1% (vol/vol) inoculum. The headspace of bottles was sparged with a gas mix of 80% N2 and 20% CO2. Bottles were supplemented either with neat benzene (14 mg/l) plus nitrate (2 mM) (active treatments, three replicates), or with 2 mM nitrate only (negative controls, three replicates). At the start of each experiment (T=0), 50 mL was removed from each bottle for DNA extraction. For the August 2008 experiment, 50 mL samples were also taken when 50 % of the benzene was degraded (T=1), and again when 80-90% was degraded (T=2). For the June 2009 experiment, samples were collected at the beginning of the experiment (T=0), 63 days after setting up the bottles (T=1), and when 70-100% of the benzene was degraded (T=2). Two of the active treatments were supplied with benzene again and samples were taken after 75-90% of the benzene was degraded (T=3). In each case, samples were transferred into 50 mL conical centrifuge tubes (Fisher Scientific, Toronto, Ontario, Canada) and centrifuged at 3,250 ! g for 60 minutes at 4°C. DNA was extracted using the FastDNA Spin Kit for Soil (MP Biomedical, LLC, Solon, OH, USA) and 16S rRNA gene copies of the five phylotypes of interest were enumerated as above.
! ! 78!
Table 4.2. List of qPCR primer sets used in this study with their sequences, annealing temperatures, and specificity. Size of amplicon amplified by these primer sets is also provided.
16S rRNA Name Sequence Annealing Amplic Specificitya Ref. Gene Target 5’ to 3’ Temp. -on size (°C) Azoarcus ACC1fb CCAGTCGTGGGGGATAACTA 55 325 bp 84/372 (Ulrich 2004) ACC1rb CACCCGATTTCTTTCCGTCT 27/372 Dechloromnas DCC1fb CGCATATTCTGTGAGCAGGA 55 314 bp 578/947 (Ulrich 2004) DCC1rb GGTACCGTCATCCACACTGG 278/947e Peptococcaceae qSHA824fc CCCCTTCTGTGCCGTAGTTA 55 210 bp 134/175 (Nandi 2006) qSHA1034rc CACCACCTGTCTCCCTGTCT 85/175 Anammox Pla46f GACTTGCATGCCTAATCC 59 322 bp -f (Neef et al. Amx368r CCTTTCGGGCATTGCGAA -f 1998; Schmid et al. 2003; Nandi 2006) Chlorobi Chlo260fd CCATTAGGTAGTTGGCGG 60 114 bp 57/947e This study Chlo374rd CCATTGAGCAATATTCCTTA 33/947e CTGC
! a Specificity is determined using the Probe Match function of RDP (http://rdp.cme.msu.edu) as of June 2010 and is described as the “Number of target species sequences in data base which are perfect match to the corresponding primer” / “Total Number of target-species sequences in database”. The clone library was searched for the non-target sequences with no mismatches to the corresponding primers. These non-target sequences were not present in the clone library. As a result there should not be any amplification of unwanted sequences. b These primers are perfect match to active benzene-degrading Azoarcus (ACC1) and Dechloromonas (DCC1) species in two co-cultures established from one of our enrichment cultures by Ulrich (2004). c These primers are perfect match to Peptococcaceae-related sequences identified in this study (Swampcons-N1) and in previous molecular investigation of our enrichment cultures (SwampNC1/CartNC1). d Primers specific to the Chlorobi sequences found in our cultures were designed by aligning these sequences with other species 16S rRNA gene sequences and identifying regions that are unique to Chlorobi. These primers are perfect match to two Chlorobi sequences identified in our culture. Primers hairpin formation, self-dimerization, and hetero-dimerization were determined using IDT oligoanalizer (Owczarzy et al. 2008). e One mismatch is allowed. f Pla46f is a general Planctomycetals forward primer (Neef et al. 1998) and Amx368r is a primer specific to all Anammox bacteria (Schmid et al. 2003).
4.2.5. Analytical methods
Benzene concentration was measured by removing 300 µl sample from the headspace of culture with a 500 µl Pressure-Lok gastight syringe (Precision Sampling, Baton Rouge, LA) and
! ! 79! injecting it onto a Hewlett-Packard 6890 Series gas chromatograph equipped with a HP-5, 30 m ! 0.32 mm I.D. column and a flame ionization detector. Nitrogen at a flow rate of 3 ml/min was used as the carrier gas. The injector temperature was 200°C, the FID detector temperature was 250°C, and the oven temperature was constant at 85°C. Under these conditions, the retention time for benzene was 2.5 minutes.
Nitrate and nitrite concentrations were analyzed by injecting 20 µl of diluted liquid samples taken from the culture into a Dionex DX-100 Series Ion Chromatograph. This Ion Chromatograph was equipped with an IonPac AS9 ion exchange column and an AG9 guard column. The mobile phase consisted of 5 mM sodium bicarbonate and 12.25 mM sodium carbonate. The eluent flow rate was 1 ml/min.
4.3. Results 4.3.1. Bacterial community structure of the denitrifying benzene-degrading cultures
Five dominant bacterial operational taxonomic units (OTUs; labeled Swampcons-N1 to N5) were identified in the Swamp Consolidated enrichment culture. A Neighbor-Joining tree depicts the phylogenetic position of the observed OTUs compared to known organisms, including other bacteria in aromatic-degrading anaerobic cultures (Figure 4.2). An OTU (Swampcons-N1) whose nearest relative is an uncultured Peptococcaceae clone HT06Ba09 from a benzene-degrading iron-reducing enrichment culture (Kunapuli et al. 2007) was the most abundant OTU observed, and had also been seen in previous clone libraries (Nandi 2006). Two OTUs that were distantly related to known Chlorobi organisms were also found (Swampcons-N3, Sampcons-N4), as they had been before (Ulrich and Edwards 2003; Nandi 2006). "-Proteobacterial 16S rRNA gene sequences related to Ralstonia/Burkholderia (Swampcons-N5), and distantly related to other species in the Rhodocyclaceae family (Swampcons-N2), were also identified. Previous isolation efforts using these cultures had resulted in benzene-degrading, nitrate-reducing co-cultures dominated by other "-Proteobacteria including Azoarcus and Dechloromonas spp. (Ulrich 2005). Finally, though Anammox bacteria could not be detected using the PCR and cloning conditions used in this study, these organisms had previously been found in the culture, and Anammox activity had been observed in the presence of nitrite and ammonium (Whang 2005; Nandi 2006). Based on the above findings, we selected the following five populations to monitor more closely
! ! 80!
using qPCR: Peptococcaceae, Chlorobi, Azoarcus, Dechloromonas, and Anammox organisms.
! [Ferments Propionate] [Ferments Propionate] [Ferments Propionate, Lactate, and!Pyruvate]! [Ferments Benzoate, and all Phthalate isomers] [Ferments Benzoate, Isophthalate, and Terephathalate] ! Clostridiales [Benzoate] {Nitrate and Sulfate} [Lactate, Pyruvate, and Formate] {Sulfate}, [Ferments Lactate] (DQ088773) [CO] {H2O}
[Hydrogen and Acetate] {FeIII}, [CO] {H2O} ((Identified in a 1,2 dichloropropane dechlorinating-bioraector))! ((Identified in a benzene-degrading SwampNC1/CartNC1!!(Nandi, 2006 ! )(Nandi, 2006) iron-reducing enriched culture)) Swampcons"!-30%N1 * 30% !!(Nandi, 2006) [Toluene and Benzoate] {Nitrate} [Benzene, Toluene, m-Xylene, and Benzoate] {Nitrate}
[Benzene, Toluene, and m-Xylene] {Nitrate} [Toluene and Benzoate] {Nitrate} [Benzoate and 3- and 4-Hydroxybenzoate] {Nitrate} [Ethylbenzene, Toluene, and Benzoate] {Nitrate} [Phenol and Benzoate] {Nitrate} ACC1!! (Ulrich, ! (Ulrich, 2004) 2004)
[m- and p-Hydroxybenzoate] {Oxygen} ! ß
- [Toluene, Phenol, and Benzoate] {Nitrate} Proteobacteria Cart-N1 (AY118150)!!(Ulrich, 2003! (Ulrich) and2003) Edwards, 2003) ((Identified in a denitrifying reactor treating quinoline-containing synthetic wastewater)) Swampcons"!9%-N2 * 9% CartNC3!!(Nandi, ! !(Nandi, 2006) 2006) [Autotroph oxidizing FeII] [Benzene, Toluene, and Benzoate] {Nitrate} ! [Benzene, Toluene, Ethylbenzene, Xylenes, and !(Ulrich, 2004) Benzoate] {Nitrate} DCC1! ! (Ulrich, 2004) [Thiosulfate] {Oxygen} [Phenanthrene] {Oxygen} ! [Dehydroabietic acid] {Oxygen} ((Denitrifying reactor for landfill leachate)) [4-Chloro and 4-bromobenzoate] {Nitrate} ((Environmental sample))
! bacteria Acido Swampcons"!-9%N5 * 9% Cart-N4 (AY118153)!!(Ulrich, 2003 ! (Ulrich) and Edwards, 2003) -
[FeII and Hydrogen] ! (Green Sulfur) Chlorobi CartNC2!!(Nandi, ! (Nandi, 2006) 2006) ((Identified in petroleum contaminated!sediments))! Cart-N2 (AY118151)!!(Ulrich, !2003 (Ulrich) and Edwards, 2003) Swampcons"-!16%N3 * 16% Cart-N3 (AY118152! !!(Ulrich,) 2003! (Ulrich) and Edwards, 2003) ! Swampcons"-!N46% * 6% (Out-group)
Figure 4.2. Phylogenetic tree showing relationship between observed OTUs in Swamp Consolidated culture and other classified bacteria. * refers to sequences obtained in this study and ! to sequences found in previous molecular investigation of nitrate-reducing cultures. Numbers at the branching points are bootstrap values. Scale bar represents 10% nucleotide substitution. Numbers in the parenthesis are NCBI accession numbers. Some of the [substrates] and {electron acceptors} utilized by bacteria are given within [] and {}, respectively. The relative abundance of each OTU in the Swamp Consolidate clone library is given as percentage values in bold.
! ! 81!
4.3.2. Quantitative survey of enrichment cultures
16S rRNA gene abundances for the above five groups of microorganisms were measured in one parent culture and eight separate enrichment cultures derived from the parent cultures. Peptococcaceae was detected in all nine cultures at the highest copy numbers in most cases (4.2 ! 106 to 1.8 ! 107 16S rRNA gene copies of Peptococcaceae /ml of culture) (Figure 4.3). Azoarcus was present in all of the cultures except for the 10-5 dilution of Swamp Consolidated (The detection limit for Azoarcus was 8.1 ! 102 gene copies/µl of DNA sample). Dechloromonas was detected in all cultures but often in much lower abundance. Chlorobi-related sequences were detected in all cultures except for Cartwright Consolidated 10-4. Anammox bacteria were only present in three of the nine cultures.
! ! 82!
1.E+08 (a) 1.E+06 Azoarcus Dechloromonas Peptococcaceae 1.E+04 Chlorobi Anammox 1.E+02
1.E+00 16S rRNA copies/ml of culture 16S rRNA Cartwright 1b Cartwright pw1 Swamp Cartwright Consolidated Consolidated
1.E+08 (b)
Azoarcus 1.E+06 Dechloromonas Peptococcaceae 1.E+04 Chlorobi Anammox
1.E+02
1.E+00
16S rRNA gene copies/ml of culture 16S rRNA Cartwright 1b Cartwright Swamp Swamp Cartwright 10E-2 pw1 10E-5 Consolidated Consolidated Consolidated 10E-2 10E-5 10E-4
Figure 4.3. Different bacterial species 16S rRNA gene copies in a) Cartwright and Swamp cultures and b) dilutions prepared from Cartwright and Swamp microbial consortia. 16S rRNA gene copies are averages of two to six qPCR reactions and are expressed as copies per ml of culture, assuming the DNA extraction efficiency to be 100%. Error bars are the range of duplicates or the standard deviations of three to six replicate qPCR reactions. One DNA sample from each culture is used to perform qPCR. The detection limit for Azoarcus, Dechloromonas, Peptococcaceae, Chlorobi, and Anammox are 8.1 ! 102, 2.2 ! 102, 1.4 ! 102, 9.5 ! 102 and 3.0 ! 102 16S rRNA gene copies/µl of DNA sample, respectively.
4.3.3. Time course qPCR The growth of five target populations was monitored over time during benzene degradation. In these experiments, benzene degradation in the triplicate active treatments started after a lag period of 35-38 days (2008) and 58-111 days (2009) (Figure 4.4). These cultures always exhibit
! ! 83! a lag after diluting into new medium. After this lag, 14 mg/l of benzene was completely consumed within 10 to 20 days. When mineralization of benzene was completed, the culture bottles from the 2009 were fed again with benzene and nitrate. Upon re-feeding, benzene degradation started immediately and without a lag (Figure 4.4b). The moles of nitrate consumed per moles of benzene degraded was 11.7±1.6 (n=3 independent measurements) and 12.4±1.0 (n=6 independent measurements) in the first and second experiments, respectively. Both values are close to the expected range of 13.1-14.3 for benzene oxidation coupled to incomplete reduction of nitrate to nitrite (refer to section 2.1.1 of Chapter 2, Table 2.3).
The concentrations of Peptococcaceae, Chlorobi, Azoarcus, Dechloromonas, and Anammox bacteria in active and control bottles over the course of each experiment are shown in are shown in Figure 4.5. It should be noted that in the 2008 experiment, T=1 samples were taken well after benzene degradation had begun, while in the 2009 experiment, the T=1 samples were taken during the lag phase for two of the three replicates, and shortly after degradation had started (15%) for the third. In active treatments, Peptococcaceae 16S rRNA gene copies per ml of culture increased by over two orders of magnitude during benzene degradation, reaching a concentration of 4.3 (± 2.1) ! 106 and 8.2 (± 3.1) ! 106 16S rRNA gene copies/ml at T=2 for the 2008 experiment and at T=3 for the 2009 experiment (n=3), respectively. No significant growth of Peptococcaceae was observed in negative controls that contained only nitrate (Figure 4.5a,b). In addition, Peptococcaceae numbers in the 2009 active treatment replicates 2 and 3 did not increase during the lag time when there was no benzene degradation (i.e. between T=0 and T=1), but only after benzene degradation was apparent. Thus, increases in Peptococcaceae gene copy numbers appear to be directly linked to decreases in benzene. None of the other organisms monitored tracked benzene behaviour so closely.
! ! 84!
15 (a)
August 2008 T=0 10 Benzene+Nitrate Treatment 1 Benzene+Nitrate Treatment 2 T=2 Benzene+Nitrate Treatment 3 5 T=1 Benzene Concentration (mg/l)
0 0 10 20 30 40 50 60 70
15 (b)
10 T=0 June 2009 T=1
Benzene+Nitrate Treatment 1
5 Benzene+Nitrate Treatment 2 T=2 Benzene+Nitrate Treatment 3 Benzene Concentration (mg/l) T=3 Fed benzene 0 0 20 40 60 80 100 120 Time (days) Figure 4.4. Benzene degradation curves for a) 2008 and b) 2009 time course experiments. Arrows indicate the time points at which DNA is extracted from the cultures. O Shows the point at which the culture bottles are fed with benzene and nitrate for the second time.
Azoarcus 16S rRNA gene copies increased substantially in all treatments (active and negative controls) in the 2008 data set, with slightly greater increase in the benzene-fed cultures (Figure 4.5c). In the 2009 data set, Azoarcus growth was more pronounced in the benzene-amended cultures, but in these bottles significant growth was also observed during the lag time before the
! ! 85! onset of benzene biodegradation (Figure 4.5d). The growth of this microorganism in negative controls and during the lag time could be due to endogenous respiration.
Chlorobi increased to a similar extent in both active treatments and negative controls in the 2008 experiment (Figure 4.5e). In the 2009 experiment, Chlorobi did not show significant growth in negative controls, but did increase significantly in benzene-amended cultures, including during the lag time (between T=0 and T=1) (Figure 4.5f). The Chlorobi gene copy numbers at the end of both experiments were an order of magnitude or more lower than those of the Peptococcaceae and Azoarcus. The fact that Chlorobi grew even during the lag before the onset of benzene biodegradation indicates that Chlorobi bacteria were not the primary consumers of benzene.
Dechloromonas was not detected in any of the cultures between T=0 and T=1. Dechloromonas 16S rRNA gene copies per ml of culture reached 102 to 103 in positive treatments at T=2 and T=3 but still remained below detection levels in negative controls. While Dechloromonas 16S rRNA gene copies increased during benzene degradation, their overall numbers were too low to invoke a significant metabolic role.
Anammox populations did not change appreciably in the negative and positive treatments over the course of these experiment (Figure 4.5i,j), although their overall abundance varied between the 2008 and 2009 cultures. Anammox bacteria are therefore not directly linked to the anaerobic benzene oxidation process.
! ! 86!
1.E+08 1.E+08 Peptococcaceae (a) Peptococcaceae (b) 1.E+07 1.E+07
1.E+06 1.E+06 August 2008 June 2009 1.E+05 1.E+05
1.E+04 1.E+04
1.E+03 1.E+03 16S rRNA gene copies/ml of culture 16S rRNA 16S rRNA gene copies/ml of culture 16S rRNA
1.E+02 1.E+02
1.E+08 1.E+08 Azoarcus (c) Azoarcus (d) 1.E+07 1.E+07
1.E+06 1.E+06
1.E+05 1.E+05 August 2008 June 2009 1.E+04 1.E+04
1.E+03 1.E+03 16S rRNA gene copies/ml of culture 16S rRNA 16S rRNA gene copies/ml of culture 16S rRNA 1.E+02 1.E+02
1.E+08 1.E+08 Chlorobi (e) Chlorobi (f) 1.E+07 1.E+07 August 2008 June 2009 1.E+06 1.E+06
1.E+05 1.E+05
1.E+04 1.E+04
1.E+03 1.E+03 16S rRNA gene copies/ml of culture 16S rRNA 16S rRNA gene copies/ml of culture 16S rRNA 1.E+02 1.E+02 0 1 2 0 1 2 3 Time point Time point Lag time Second feeding 0% 50% 100% 0% 0% 100% 100%
Benzene+ 0% 15% 100% 100% Nitrate Treatment 1
! ! 87!
1.E+08 1.E+08 Dechloromonas (g) Dechloromonas (h) 1.E+07 1.E+07
1.E+06 1.E+06 August 2008 June 2009 1.E+05 1.E+05
1.E+04 1.E+04 Below Below Below Below 1.E+03 1.E+03 detection detection detection detection
16S rRNA gene copies/ml of culture 16S rRNA limit limit limit limit 1.E+02 gene copies/ml of culture 16S rRNA 1.E+02
1.E+08 1.E+08 Anammox (i) Anammox (j) 1.E+07 1.E+07 August 2008 June 2009 1.E+06 1.E+06
1.E+05 1.E+05
1.E+04 1.E+04
1.E+03 1.E+03 16S rRNA gene copies/ml of culture 16S rRNA 16S rRNA gene copies/ml of culture 16S rRNA 1.E+02 1.E+02 0 1 2 0 1 2 3 Time point Time point Second feeding 0% 50% 100% Lag time 0% 0% 100% 100%
Benzene+ 0% 15% 100% 100% Nitrate Treatment 1
Figure 4.5. Peptococcaceae, Azoarcus, Chlorobi, Dechloromonas, and Anammox organism growth in individual culture bottles during benzene degradation. Closed symbols and blue color represent positive treatments amended with benzene and nitrate; Open symbols and red color represent negative controls supplied with nitrate only. 0, 1, 2, and 3 on the X-axis represent different time points at which DNA is extracted from the bottles. Percentages below each graph are the approximate amount of benzene degraded at each time point. For the 2009 experiment at T=1, no benzene degradation had yet been observed for two of the benzene+nitrate treatments (closed square and circle), while degradation had just started (15% of benzene removed) in the third treatment (closed triangle). At T=2 cultures were supplied with benzene for second time which is degraded by T=3.
! ! 88!
4.4. Discussion 4.4.1. Phylogenetic analysis and physiological roles of different bacteria in the culture
Swampcons-N1, The most abundant OTU in the 16S rRNA gene clone library, falls within the order Clostridiales, and particularly within the family of Peptococcaceae (Figure 4.2). This sequence was also most abundant organism measured by qPCR in all of the enrichment cultures surveyed (Figure 4.3). As mentioned earlier, the closest GenBank match to this OTU is an uncultured Peptococcaceae bacterium clone from a benzene-degrading iron-reducing enrichment culture (Kunapuli et al. 2007). The closest isolated bacteria to OTU Swampcons-N1 are Thermincola carboxydiphila and Thermincola ferriacetica. These microorganisms are anaerobic, thermophilic bacteria that grow chemolithotrophically on CO (Sokolova et al. 2005; Zavarzina et al. 2007). OTU Swampcons-N1 is also related to other members of Peptococcaceae family including the genus Pelotomaculum. Members of this genus are characterized by their ability to anaerobically metabolize organic substrates such as propionate and alcohols in syntrophic relationships with methanogens (Imachi et al. 2002; de Bok et al. 2005; Imachi et al. 2006; Qiu et al. 2006). Some Pelotomaculum strains can also grow in pure cultures by fermentation (Juteau et al. 2005; Qiu et al. 2006). Several members of this genus are known to be involved in degradation of aromatic compounds. For example, Pelotomaculum terephthalicum and Pelotomaculum isophthalicum are capable of growth on phthalate isomers and benzoate in syntrophic co-culture with hydrogenotrophic methanogens (Qiu et al. 2004; Qiu et al. 2006). Recently, Chen et al. (2008) reported the predominant presence of a bacterial group related to Pelotomaculum in a thermophilic phenol-degrading methanogenic consortium and suggested that these microorganisms mineralize phenol and other aromatic hydrocarbons in close association with methanogens. Furthermore, the community analysis of several enrichment cultures as well as in situ reactor columns degrading benzene under sulfate-reducing conditions revealed the presence of phylotypes affiliated with the family Peptococcaceae (Kunapuli et al. 2007; Kleinsteuber et al. 2008; Abu Laban et al. 2009; Herrmann et al. 2010). The authors speculated that the dominance and increase in the population of Peptococcaceae-related 16S rRNA gene sequences during benzene mineralization were evidence for ability of these species to degrade benzene (Kunapuli et al. 2007; Kleinsteuber et al. 2008; Abu Laban et al. 2009). More recently, Abu Laban et al. (2010) conducted proteomic experiments on an iron-reducing culture mainly composed of Peptococcaceae-related bacteria and identified several putative benzene
! ! 89! carboxylase polypeptides that were specifically expressed in the presence of benzene. It was proposed that these polypeptides were subunits of an enzyme that activates benzene and directly carboxylates it to benzoate. The identified genes are believed to belong to Peptococcaceae. From the experimental data presented herein, we have also found compelling reasons to conclude that Peptococcaceae was responsible for the initial attack on benzene: 1) a specific Peptococcaceae 16S rRNA gene sequence was found historically and currently in all of the cultures at or above 107 copies / mL; 2) increases in gene copy tracked directly with decreases in benzene concentrations; and 3) no changes in copy number were observed during the lag phase before onset of benzene degradation, or in cultures without benzene. None of the other organisms surveyed showed this behavior.
Several years ago, agar shake tubes containing benzene and nitrate were prepared to attempt to isolate benzene-degraders from the Cartwright enrichment cultures (Ulrich 2004). Several colonies were obtained that, upon transfer to liquid medium, degraded benzene coupled to nitrate reduction to nitrite. These cultures were dominated by Azoarcus and Dechloromonas (>90%), however they were not pure but rather co-cultures designated as Azoarcus co-culture (ACC) and Dechloromonas co-culture (DCC) (Ulrich 2004). The evidence at the time strongly linked benzene-degrading activity to the Azoarcus and Dechloromonas species in these co-cultures (Ulrich 2004). This was consistant with the findings of other groups, who were successful in isolating the benzene-degrading Dechloromonas sp. JJ and Azoarcus species (Coates et al. 2001; Kasai et al. 2006), as well as several similar species that can metabolize a variety of aromatic compounds under denitrifying conditions (Figure 4.2). Unfortunately, the ACC and DCC co- cultures are no longer active. The data obtained herein from the time course experiments are consistent with Azoarcus having an important role in benzene biodegradation, possibly being responsible for initial attack on benzene, but even more likely as a consumer of fermentation intermediates, such as acetate or hydrogen, with nitrate as electron acceptor. Evidence for this secondary role includes: 1) the growth of Azoarcus even in the absence of benzene; 2) growth of Azoarcus during the lag period before onset of benzene degradation; and 3) the fact that Azoarcus was undetectable in one of the active benzene-degrading cultures surveyed (Swamp Consolidated 10-5). Dechloromonas may also play an important role in benzene degradation, but in the experiments conducted, the numbers were too low to be conclusive.
! ! 90!
OTUs Swampcons-N3, Swampcons-N4, Cart-N2 and Cart-N3 grouped with the green sulfur bacteria in the phylum Chlorobi (though relationships to known organisms are distant). Most members of Chlorobi are strictly anaerobic and typically photoautotrophic (Figaard et al. 2003; Figaard and Dahl 2009). While our cultures are kept under a dark cloth, light may still reach the bottles from time to time to support the growth of these bacteria. Green sulfur bacteria are known to grow at remarkably low light intensities because of the presence of highly efficient light- harvesting chlorosomes (Figaard et al. 2003; Manske et al. 2005). The majority of characterized Chlorobi strains utilize sulfide, thiosulfide, elemental sulfur, and hydrogen as electron donors during photosynthesis (Drutschmanna and Klemme 1985; Gogotova et al. 1991; Heising et al. 1999; Figaard et al. 2003; Figaard and Dahl 2009; Gregersen et al. 2009). A few strains can oxidize Fe2+ (Heising et al. 1999). In our cultures Chlorobi organisms may use hydrogen, sulfide or Fe2+ (the medium contains FeS) as electron donors for phototrophic growth. The growth of Chlorobi during the lag phase before the onset of benzene degradation in the 2008 and 2009 experiments suggests that this group is not the primary benzene degrader in the cultures. Increase in the gene copies of these organisms in the presence of benzene in the 2009 experiment may suggest that hydrogen, a likely product of benzene fermentation by Peptococcaceae in the culture, can be an electron donor for the Chlorobi. The removal of hydrogen, either by Chlorobi species or by some other organism such as Azoarcus, would drive the benzene fermentaion reactions forward in these cultures. The final 16S rRNA gene copy numbers for the Chlorobi were between 10 and 100 times lower than the Azoarcus sp. in these experiments. Therefore, it is likely that Azoarcus is the primary consumer of benzene fermentation products and Chlorobi species act as opportunistic bacteria.
OTU Swampcons-N5 is identified as a member of Beta-proteobacteria, in the order of Burkholderiales. The closest relative to Swampcons-N5 is Ralstonia sp. P-4CB2 from a 4- bromobenzoate and 4-chlorobenzoate degrading denitrifying consortium (Song et al. 2002). Swampcons-N5 is also phylogenetically related to Burkholderia species, some of which are facultative anaerobes. Burkholderia species that are capable of degrading poly aromatic hydrocarbons have been frequently isolated (Friedrich et al. 2000). The close relationship between Swampcons-N5 and these aromatic hydrocarbon-degrading microorganisms suggests a role for this OTU in the metabolism of intermediates formed during benzene degradation. Since Burkholderia species were present only in low abundance in other clone libraries prepared from
! ! 91! our benzene-degrading nitrate-reducing cultures (Ulrich and Edwards 2003; Nandi 2006), we did not track them in this study.
Anammox bacteria are detected in some of our enrichment cultures. However their abundance does not change significantly during the benzene degradation process indicating that they are not involved in the mineralization of benzene. Anammox organisms couple oxidation of ammonium to reduction of nitrite producing nitrogen gas (Mulder 1992; Graaf et al. 1995; Graaf et al. 1997; You et al. 2009; Kumar and Lin 2010). They are lithotrophs that utilize CO2 as the carbon source while deriving reducing equivalents for carbon-fixation from oxidation of nitrite to nitrate. The presence of Anammox bacteria in the consortia is not surprising because they are provided with all necessary growth components. The mineral medium used for our benzene- degrading cultures contains ammonium and is maintained under a headspace of N2/CO2. The – final element needed by these organisms is NO2 , which is provided by the incomplete reduction of nitrate coupled to the oxidation of benzene (Figure 4.6). Therefore, the role of Anammox bacteria is to recycle nitrite to nitrate and help stabilize the enrichment cultures by reducing inhibitory effect of accumulating nitrite. Nitrate produced during growth of Anammox organisms is used to drive benzene degradation.
4.4.2. Stoichiometric and energetic considerations
Growth yields per mole of benzene degraded were estimated for Peptococcaceae and Azoarcus using the 16S rRNA gene copy and benzene concentration data. The Chlorobi organisms were ignored, since the final gene copy numbers were much lower than those of the other organisms. For yield calculations, the change in the number of 16S rRNA gene copies/ml of culture in negative controls was subtracted from that of active treatments and then this number was divided by the change in concentration of benzene between extraction time points. The Peptococcaceae and Azoarcus yields were 4.41 (±2.07) ! 1013 and 2.39 (± 0.9) ! 1013 16S rRNA gene copies/mol of benzene degraded (n=6 culture bottles), respectively. These values were converted into grams of cells per mole of benzene, assuming that each cell of Peptococcaceae had a mass of 3.43 ! 10-13 g and 2 copies of 16S rRNA gene and that each cell of Azoarcus had a mass of 1.98 ! 10-13 g and 4 copies of 16S rRNA gene (refer to Appendix A for details). The yield for Peptococcaceae and Azoarcus were 7.56 and 1.18 g cells/mole of benzene, respectively. The sum of growth yields of Peptococcaceae and Azoarcus (8.74 cells/mole of
! ! 92!
benzene) was close to the lower end of the yields of 8.6 ± 1.9 to 22 ± 7.7 g cells/mole of benzene previously reported for our benzene-degrading nitrate-reducing cultures (Ulrich and Edwards 2003).
A theoretical yield can also be calculated based on ratio of nitrate to benzene consumption. In our consortium, about 12 moles of nitrate are consumed per each mole of degraded benzene,
which gives a measure of fs (fs= 0.2). Using fs of 0.2, the yield was calculated to be 33.9 g cells per mole of benzene (refer to Appendix A for detail calculations). This value was slightly above the the upper end of observed yields by Ulrich et al. (2003).
The results obtained in this study are consistent with a syntrophic relationship between Peptococcaceae and other organisms in the culture (Figure 4.6). In this syntrophic association, Peptococcaceae ferments benzene, producing metabolites that are subsequently used by other members of the consortium. Hydrogen and acetate are likely interspecies metabolites, but others such as benzoate are possible as well. Benzoate has been identified in several anaerobic benzene- degrading cultures as a metabolite of benzene mineralization (Caldwell and Suflita 2000; Phelps et al. 2001; Chakraborty and Coates 2005; Ulrich et al. 2005; Kunapuli et al. 2008; Abu Laban et al. 2009). The fermentation of benzene to benzoate is energetically favorable (equation 1), meaning that the primary benzene degrader (Peptococcaceae) could thrive by carrying out only this step.
C H CO C H COO- H+ G! 3 kJ mol (1) 6 6 + 2 " 6 5 + # '= $ 3 /
If Peptococcaceae instead fermented benzene to hydrogen and acetate, it would have the ! potential to obtain more energy for growth (equation 2, !G °"= -40 to -90 kJ per mole benzene, depending on the assumed hydrogen partial pressure (10-3 and 10-6 atm)). Fermentation of benzene to acetate and hydrogen is thermodynamically unfavorable (!G °"> 0) at hydrogen partial pressures higher than 0.2 atm (Figure 4.7, refer to Appendix A for detailed calculations). The hydrogen partial pressure can be kept very low by hydrogen-consuming members of the culture, such as Azoarcus, Dechloromonas, or Bulkhorderia (coupled to nitrate reduction) or even members of Chlorobi that may use hydrogen as an electron donor during photosynthesis. Note that Chlorobi species can alternatively use sulfide for their phototrophic growth. Previous studies conducted by Ulrich (2004) and Nandi (2006) with benzene-degrading nitrate-reducing
! ! 93!
cultures in our laboratory indicated that presence of 10% and 80% hydrogen in the headspace of cultures resulted in inhibition of benzene mineralization. This provides further evidence for syntrophic degradation of benzene in our cultures.
C H + 6H O"3CH COO- +3H + 3H+ #G!'= $60 kJ /mol 6 6 2 3 2 -4 (assuming hydrogen partial pressure of 10 atm) (2)
Chlorobi ! Water/Sulfate
Sulfide
Peptococcaceae Or Benzene Low molecular weight fermentation products + Hydrogen
Nitrogen gas - Azoarcus NO3
Anammox Dechloromonas - NO2
Ammonium Carbon dioxide
Figure 4.6. Proposed role of different bacterial species in the flow of carbon and electrons within the benzene-degrading nitrate-reducing enrichment cultures.
The acetate produced in this reaction would be further mineralized by nitrate-reducing members of community such as Azoarcus and Dechloromonas sp. (Equation 3).
" " " ! CH COO " 4NO3 CO 4NO2 HCO3 H O G 552kJ mol 3 3 + # 2 + + + 2 $ '= " / ( )
It is also possible that Peptococcaceae is responsible for complete degradation of benzene to ! carbon dioxide using nitrate as an electron acceptor. However, mineralization of benzene by a single microorganism in the culture cannot justify the persistent presence of different bacterial species within the benzene degrading nitrate-reducing cultures after many years of enrichment. In addition, most members of Peptococcaceae family do not use nitrate as an electron acceptor (De Vos et al. 2009).
! ! 94!
20
0 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00 -20
-40 G' (kJ/mol) ! -60
-80
-100 Hydrogen partial pressure (atm)
Figure 4.7. The free energy of fermentation of benzene to acetate and hydrogen calculated at different hydrogen partial pressures. Benzene and acetate concentrations were assumed to be 0.1 and 0.05 mM, respectively.
4.5. Conclusions
The results of this study provide strong evidence that multiple bacterial genera are maintained in the benzene-degrading nitrate-reducing cultures over many years and successive transfers. Among the monitored species, only a significant increase in the abundance of Peptococcaceae was observed during benzene degradation. Based on these results, it is concluded that mineralization of benzene in the enrichment culture is mediated by syntrophic association between Peptococcaceae, which is responsible for the initial attack on benzene, hydrogen scavenging Chlorobi, and nitrate-reducing bacteria such as Azoarcus and Dechloromonas that oxidize the hydrogen and low molecular weight by-products of benzene oxidation. Anammox bacteria benefit from side reactions in the culture.
4.6. Recommendations for future work
Investigating the effect of elimination of Chlorobi organisms from microbial consortia on benzene degradation will allow to understand whether this microorganism is an essential member
! ! 95! of community and plays an important role in benzene mineralization and in maintaining the benzene degradation capacity of the cultures. To avoid growth of Chlorobi in the cultures, we suggest wrapping the bottles in aluminum foil. Since Chlorobi bacteria are phototrophs, this strategy may result in their extinction. Time course qPCR experiments similar to the ones explained in section 4.3.3 should be employed to identify microorganisms that grow on benzoate as a source of energy and carbon. This would provide clues about the populations that could benefit from a possible metabolite of benzene degradation, i.e. benzoate, and will shed light on the roles these species might play in benzene degradation. To provide further evidence in support of syntrophic benzene degradation in our cultures the effect of hydrogen addition on benzene mineralization should be investigated. If benzene is degraded syntrophically with hydrogen as a central intermediate, based on thermodynamic calculations, presence of hydrogen at partial pressures higher than 0.2 atm should inhibit degradation of benzene.
! !
4.7. References Chapter 4
Abu Laban, N., D. Selesi, C. Jobelius and R. U. Meckenstock (2009). "Anaerobic benzene degradation by gram-positive sulfate-reducing bacteria." FEMS Microbiol. Ecol. 68: 300-311.
Abu Laban, N., D. Selesi, T. Rattei, P. Tischler and R. U. Meckenstock (2010). "Identification of enzymes involved in anaerobic benzene degradation by strictly anaerobic iron-reducing enrichment culture." Environ. Microbiol. 12(10): 2783-2796.
Altschul, S. F., W. Gish, W. Miller, E. W. Myers and D. J. Lipman (1990). "Basic local alignment search tool." J. Mol. Biol. 215: 403-410.
Anderson, R. T. and D. R. Lovley (1997). "Ecology and biochemistry of in situ groundwater bioremediation." Adv. Microb. Ecol. 15: 289-350.
Anderson, R. T., J. N. Rooney-Varga, C. V. Gaw and D. R. Lovely (1998). "Anaerobic benzene oxidation in the Fe(III) reduction zone of petroleum-contaminated aquifers." Environ. Sci. Technol. 32: 1222-1229.
Botton, S. and J. R. Parsons (2007). "Degradation of BTX by dissimilatory iron-reducing cultures." Biodegradation 18: 371-381.
Burland, S. M. and E. A. Edwards (1999). "Anaerobic benzene biodegradation linked to nitrate reduction." Appl. Environ. Microbiol. 65(2): 529-533.
Caldwell, M. E. and J. M. Suflita (2000). "Detection of phenol and benzoate as intermediates of anaerobic benzene biodegradation under different terminal electron accepting condition." Environ. Sci. Technol. 34: 1216-1220.
Chakraborty, R. and J. D. Coates (2004). "Anaerobic degradation of monoaromatic hydrocarbons." Appl. Microbiol. Biotechnol. 64: 437-446.
Chakraborty, R. and J. D. Coates (2005). "Hydroxylation and carboxylation-two crucial steps of anaerobic benzene degradation by Dechloromonas Strain RCB." Appl. Environ. Microbiol. 71(9): 5427-5432.
Chang, W., Y. Um and T. R. P. Holoman (2005). "Molecular charecterization of anaerobic microbial communities from benzene-degrading sediments under methanogenic conditions." Biotechnol. Prog. 21: 1789-1794.
Chen, C. L., J. H. Wu and W. T. Liu (2008). "Identification of important microbial populations in the mesophilic and thermophilic phenol-degrading methanogenic consortia." Water Res. 42: 1963-1976.
! 96! ! 97!
Coates, J. D., R. Chakraborty, J. G. Lack, S. M. O’Connor, K. A. Cole, K. S. Bender and L. A. Achenbach (2001). "Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas." Nature 411: 1039-1043.
Cole, J. R., B. Chai, R. J. Farris, Q. Wang, S. A. Kulam, D. M. McGarrell, G. M. Garrity and J. M. Tiedje (2005). "The Ribosomal Database Project (RDP II): sequences and tools for high- throughput rRNA analysis." Nucleic Acids Res. 33: D294-D296.
De Vos, P., G. M. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K. H. Schleifer and W. L. Whitman (2009). "Bergey's Manual of Systematic Bacteriology: The Firmicutes". New York, Springer.
Da Silva, M. and P. Alvarez (2007). "Assessment of anaerobic benzene degradation potential using 16S rRNA gene-targeted real-time PCR." Environ. Microbiol. 9: 72-80. de Bok, F. A. M., H. J. M. Harmsen, C. M. Plugge, M. C. d. Vries, A. D. L. Akkermans, W. M. d. Vos and A. J. M. Stams (2005). "The first true obligately syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii Sp. nov., co-cultured with Methanospirillum hungatei, and emended description of the genus Pelotomaculum " Int. J. Syst. Evol. Microbiol. 55: 1697-1703.
Drutschmanna, M. and J. H. Klemme (1985). "Sulfide-repressed, membrane-bound hydrogenase in the thermophilic facultative phototroph, Chloroflexus aurantiacus." FEMS Microbiol. Lett. 28(3): 231-235.
Edwards, E. A. and D. Grbi!-Gali! (1992). "Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions." Appl. Environ. Microbiol. 58: 2663-2666.
Figaard, N. U., A. G. M. Chew, H. Li, J. A. Maresca and D. A. Bryant (2003). "Chlorobium tepidum: insights into the structure, physiology, and metabolism of a green sulfur bacterium derived from the complete genome sequence." Photosynth. Res. 78: 93-117.
Figaard, N. U. and C. Dahl (2009). "Sulfur metabolism in phototrophic sulfur bacteria." Adv. Microb. Physiol. 54: 103-200.
Friedrich, M., R. J. Grosser, E. A. Kern, W. P. Inskeep and D. M. Ward (2000). "Effect of model sorptive phases on phenanthrene biodegradation: molecular analysis of enrichments and isolates suggests selection based on bioavailability." Appl. Environ. Microbiol. 66(7): 2703-2710.
Gogotova, I. N., N. A. Zorina and L. T. Serebriakovaa (1991). "Hydrogen production by model systems including hydrogenases from phototrophic bacteria." Int. J. Hydrogen Energy 16(6): 393-396.
Graaf, A. A. v. d., P. D. Bruijn, L. A. Robertson, M. S. M. Jetten and J. G. Kuenen (1997). "Metabolic pathway of anaerobic ammonium oxidation on the basis of I5N studies in a fluidized bed reactor." Microbiology 143: 2415-2421.
! ! 98!
Graaf, A. A. v. d., A. Mulder, P. D. Bruijn, M. S. Jetten, L. A. Robertson and J. G. Kuenen (1995). "Anaerobic oxidation of ammonium is a biologically mediated process." Appl. Environ. Microbiol. 61(4): 1246-1251.
Grbi!-Gali!, D. and T. M. Vogel (1987). "Transformation of toluene and benzene by mixed methanogenic cultures." Appl. Environ. Microbiol. 53: 254-260.
Gregersen, L. H., K. S. Habicht, S. Peduzzi, M. Tonolla, D. E. Canfield, M. Miller, R. P. Cox and N. U. Frigaard (2009). "Dominance of a clonal green sulfur bacterial population in a stratified lake." FEMS Microbiol Ecol 70(1): 30-41.
Heising, S., L. Richter, W. Ludwig and B. Schink (1999). "Chlorobium ferrooxidans sp. nov., a phototrophic green sulfur bacterium that oxidizes ferrous iron in coculture with a “Geospirillum” sp. strain." Arch. Microbiol. 172: 116-124.
Herrmann, S., S. Kleinsteuber, A. Chatzinotas, S. Kupparsdt, T. Lueders, H. H. Richnow and C. Vogt (2010). "Functional charecterization of an anaerobic benzene-degrading enrichment culture by DNA stable isotope probing." Environ. Microbiol. 12(2): 401-411.
Holmes, D. E., C. Risso, J. A. Smith and D. R. Loveley (2011). "Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus." Appl. Environ. Microbiol. 77(17): 5926-5933.
Imachi, H., Y. Sekiguchi, Y. Kamagata, S. Hanada, A. Ohashi and H. Harada (2002). "Pelotomaculum thermopropionicum gen. nov., sp. nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium." Int. J. Syst. Evol. Microbiol. 52: 1729-1735.
Imachi, H., Y. Sekiguchi, Y. Kamagata, A. Loy, Y. L. Qiu, P. Hugenholtz, N. Kimura, M. Wanger, A. Ohashi and H. Harada (2006). "Non-sulfate-reducing, syntrophic bacteria affiliated with Desulfotomaculum Cluster I are widely distributed in methanogenic environments." Appl. Environ. Microbiol. 72(3): 2080-2091.
Jahn, M. K., S. B. Haderlein and R. U. Meckenstock (2005). "Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures." Appl. Environ. Microbiol. 71: 3355-3358.
Juteau, P., V. Côté, M. F. Duckett, R. Beaudet, F. Lépine, R. Villemur and J. G. Bisaillon (2005). "Cryptanaerobacter phenolicus gen. nov., sp. nov., an anaerobe that transforms phenol into benzoate via 4-hydroxybenzoate." Int. J. Syst. Evol. Microbiol. 55: 245-250.
Kasai, Y., Y. Kodama, Y. Takahata, T. Hoaki and K. Watanabe (2007). "Degradative capacities and bioaugmentation potential of anaerobic benzene-degrading bacterium strain DN11." Environ. Sci. Technol. 41: 6222-6227.
! ! 99!
Kasai, Y., M. Manefield and K. Watanabe (2006). "RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater." Appl. Environ. Microbiol. 72 (5): 3586-3592.
Kazumi, J., M. E. Cadwell, J. M. Sulfita, D. R. Lovley and L. Y. Young (1997). "Anaerobic degradation of benzene in diverse anoxic environments." Environ. Sci. Technol. 31: 813-818. Kleinsteuber, S., K. M. Schleinitz, J. Breitfeld, H. Harms, H. H. Richnow and C. Vogt (2008). "Molecular charecterization of bacterial communities mineralizing benzene under sulfate- reducing conditions." FEMS Microbiol. Ecol. 66: 143-157.
Kumar, M. and J. G. Lin (2010). "Co-existence of anammox and denitrification for simultaneous nitrogen and carbon removal-Strategies and issues." J. Hazard. Mater. 178: 1-9.
Kunapuli, U., C. Griebler, H. Beller and R. U. Meckenstock (2008). "Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture." Environ. Microbiol. 10: 1703-1712.
Kunapuli, U., T. Lueders and R. U. Meckenstock (2007). "The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation." ISME J. 1: 643-653.
Larkin, M. A., G. Blackshields, N. P. Brown3, R. Chenna, P. A. McGettigan1, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson and D. G. Higgins (2007). "Clustal W and Clustal X version 2." Bioinformatics 23(1): 2947-2948.
Lovley, D. R., J. D. Coates, J. C. Woodward and E. J. Phillips (1995). "Benzene oxidation coupled to sulfate reduction." Appl. Environ. Microbiol. 61: 953-958.
Lovley, D. R., J. C. Woodward and F. H. Chapelle (1996). "Rapid anaerobic benzene oxidation with a variety of chelated Fe (III) forms." Appl. Environ. Microbiol. 62: 288-291.
Manske, A. K., J. Glaeser, M. M. M. Kuypers and J. Overmann (2005). "Physiology and phylogeny of green sulfur bacteria forming a monospecific phototrophic assemblage at a depth of 100 meters in the Black sea." Appl. Environ. Microbiol. 71: 8049-8060.
Mulder, A. (1992). Anoxic ammonia oxidation. U. S.
Musat, F. and F. Widdel (2008). "Anaerobic degradation of benzene by a marine sulfate- reducing enrichment culture, and cell hybridization of the dominant phylotype." Environ. Microbiol. 10(1): 10-19.
Nales, M., B. J. Butler and E. Edwards (1998). "Anaerobic benzene biodegradation: a microcosm survey." Bioremediat. J. 2: 125-144.
Nandi, M. (2006). Biochemical and molecular charecterization of anaerobic benzene-degrdading cultures. Toronto, University of Toronto. M. A. Sc. thesis.
! ! 100!
Neef, A., R. Arnann, H. Schlesner and K. H. Schleiferl (1998). "Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16s rRNA-targeted probes." Microbiology 144: 3257-3266.
Oka, A. R., C. D. Phelps, L. M. McGuinness, A. Mumford, L. Y. Young and L. J. Kerkhof (2008). "Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing." Appl. Environ. Microbiol. 74: 6476-6480.
Owczarzy, R., A. V. Tataurov, Y. Wu, J. A. Manthey, K. A. McQuisten, H. G. Almabrazi, K. F. Pedersen, Y. Lin, J. Garretson, N. O. McEntaggart, C. A. Sailor, R. B. Dawson and A. S. Peek (2008). "IDT SciTools: a suite for analysis and design of nucleic acid oligomers." Nucleic Acids Res. 36: 163-169.
Phelps, C. D., J. Kazumi and L. Y. Young (1996). "Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction." FEMS Microbiol. Lett. 145: 433-437.
Phelps, C. D., L. J. Kerkhof and L. Y. Young (1998). "Molecular charecterization of a sulfate- reducing consortium which mineralizes benzene." FEMS Microbiol. Ecol. 27: 269-279.
Phelps, C. D. and L. Y. Young (1999). "Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments." Biodegradation 10: 15-25.
Phelps, C. D., X. Zhang and L. Y. Young (2001). "Use of stable isotopes to identify benzoate as a metabolite of benzene degradation in a sulphidogenic consortium." Environ. Microbiol. 3: 600- 603.
Qiu, Y. L., Y. Sekiguchi, S. Hanada, H. Imachi, I. Tseng, S. S. Cheng, A. Ohashi, H. Harada and Y. Kamagata (2006). "Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens." Arch. Microbiol. 185: 172-182.
Qiu, Y. L., Y. Sekiguchi, H. Imachi, Y. Kamagata, I. C. Tseng, S. S. Cheng, A. Ohashi and H. Harada (2004). "Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing." Appl. Environ. Microbiol. 70(3): 1617-1626.
Ridgeway, H. F., J. Safarik, D. Phipps, P. Carl and D. Clark (1990). "Identification and catabolic activity of well-derived gasoline-degrading bacteria and a contaminated aquifer." Appl. Environ. Microbiol. 56: 3565-3575.
Rooney-Varga, J. N., R. T. Anderson, J. L. Fraga, D. Ringelberg and D. R. Lovley (1999). "Microbial communities associated with anaerobic benzene degradation in a petroleum- contaminated aquifer." Appl. Environ. Microbiol. 65: 3056-3063.
! ! 101!
Sakai, N., F. Kurisu, O. Yagi, F. Nakajima and K. Yamamoto (2009). "Identification of putative benzene-degrading bacteria in methanogenic enrichment cultures." J. Biosci. Bioeng. 108: 501- 507.
Schmid, M., K. Walsh, R. Webb, W. I. Rijpstra, K. v. d. Pas-Schoonen, M. J. Verbruggen, T. Hill, B. Moffett, J. Fuerst, S. Schouten, J. S. Damsté, J. Harris, P. Shaw, M. Jetten and M. Strous (2003). "Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria." Syst. Appl. Microbiol. 26(4): 529- 538.
Sokolova, T. G., N. A. Kostrikina, N. A. Chernyh, T. V. Kolganova, T. P. Tourova and E. A. Bonch-Osmolovskaya (2005). "Thermincola carboxydiphila gen. nov., sp. nov., a novel anaerobic bacterium from a hot spring of the lake Baikal." Int. J. Syst. Evol. Microbiol. 55: 2069-2073.
Song, B., L. J. Kerkhof and M. M. Haggblom (2002). "Charecterization of bacterial consortia capable of degrading 4-chlorobenzoate and 4-bromobenzoate under denitrifying conditions." FEM Microbiol. Lett. 213: 183-188.
Tamura, K., M. Nei, J. Dudley and S. Kumar (2007). "MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0." Mol. Biol. Evol. 24(8): 1596-1599.
Ulrich, A. C. (2004). Characterization of anaerobic benzene-degrading cultures. Toronto, University of Toronto. Ph.D. thesis.
Ulrich, A. C., H. R. Beller and E. A. Edwards (2005). "Metabolites Detected during 13 Biodegradation of C6-Benzene in Nitrate-Reducing and Methanogenic Enrichment Cultures." Environ. Sci. Technol 39: 6681-6691.
Ulrich, A. C. and E. A. Edwards (2003). "Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures." Environ. Microbiol. 5(2): 92-102.
Vogt, C., S. Gödeke, H. C. Treutler, H. Weiß, M. Schirmer and H. H. Richnow (2007). "Benzene oxidation under sulfate-reducing conditions in columns simulating in situ conditions." Biodegradation 18: 625-636.
Weisburg, W. G., S. M. Barns, D. A. Pelletier and D. J. Lane (1991). "16S Ribosomal DNA amplification for phylogenetic study." J. Bacteriol. 173: 697-703.
Whang, C. Y. (2005). Gene expression analysis of an anaerobic benzene-degrading consortium using differential diasplay. Toronto, University of Toronto. M.A.Sc thesis.
You, J., A. Das, E. M. Dolan and Z. Hu (2009). "Ammonia-oxidizing archaea involved in nitrogen removal." Water Res. 43(7): 1801-1809.
! ! 102!
Zavarzina, D. G., T. G. Sokolova, T. P. Tourova, N. A. Chernyh, N. A. Kostrikina and E. A. Bonch-Osmolovskaya (2007). "Thermincola ferriacetica sp. nov., a new anaerobic, thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction." Extremophiles 11: 1-7.
! ! !
Chapter 5. Attempts at Isolating Pure Cultures of Benzene- Degrading Nitrate-reducing Microbes
! 103! ! 104! ! 5.1. Introduction
One of the objectives of this study was to isolate and characterize pure cultures of benzene- degrading nitrate-reducing microbes in order to provide a connection between benzene decomposition and microorganisms that mediate it. Obtaining an isolate is also valuable to elucidate the benzene degradation pathway. In mixed cultures, it is unclear whether the compounds are formed directly from benzene catabolism by a single microorganism or as a result of several sequential metabolic steps involving different microbes. In contrast in a pure culture, all enzymes and intermediates produced during decomposition of benzene would be from one microorganism rather than a mixed community, making it possible to identify the degradation pathway. To date, only a few denitrifying benzene-degrading bacteria related to the genus Dechloromonas and Azoarcus in the beta-subclass of Proteobacteria have been isolated (Coates et al. 2001; Chakraborty and Coates 2004; Kasai et al. 2006; Kasai et al. 2007). One of these bacteria named as Dechloromonas strain JJ is no longer able to degrade benzene. A recent study of the other Dechloromonas strain (RCB) indicated the absence of anaerobic monoaromatic degradation genes from the genome of this bacterium (Salinero et al. 2009). It was suggested that Dechloromonas strain RCB might activate benzene utilizing oxygen produced intracellularly (Salinero et al. 2009). There is only one pure culture of an obligate anaerobic benzene-degrader called Ferroglobus placidus (Holmes et al. 2011). This microorganism grows at 85°C and couples benzene degradation to reduction of FeIII. Since the growth temperature for Ferroglobus placidus is 85°C, it would not be a member of anaerobic benzene degrading communities found in contaminated ground water.
5.2. Materials and Methods 5.2.1. Isolation procedure
Four enrichment cultures, Cartwright pw1, Cartwright 1b, Cartwright Consolidated, and Swamp Consolidated were selected for isolation purposes. The method employed here to isolate benzene-mineralizing microorganisms was serial dilution followed by subsequent plating. Eight 10-fold dilutions of enrichment cultures were prepared in defined anaerobic medium (Burland and Edwards 1999). Based on the results presented in Chapter 3 to decrease the lag time observed at the onset of culture establishment, FeS and resazurin were omitted from the medium. Dilution cultures were incubated anaerobically at 25°C after addition of 2 mM nitrate and 192
! ! 105! ! µM benzene. Some of the dilutions showed benzene degradation activity within a few weeks. At this time, they were streaked on agar plates. The agar plates used in this study contained similar medium to that of dilutions except that we added 1.5% agar, 5 mM filter-sterilized acetate as a carbon source and electron donor, and 2 mM filter-sterilized nitrate as an electron acceptor.
Plates were incubated under an atmosphere of 80% N2, 10% CO2 and 10% H2 at 25°C. After fourteen days of incubation, small white colonies appeared on the plates. These colonies were removed from the agar plates, transferred into fresh medium, and supplied with 64 µM benzene and 2 mM nitrate to determine their ability to degrade benzene.
5.2.2. Identification of isolated colonies
To identify the isolated colonies and to ensure their purity, 16S rRNA gene fragments were amplified by polymerase chain reaction (PCR) directly from each colony using the universal bacterial primers 27f (5’ AGAGTTTGATCCTGGCTCAG 3’) and 1492r (5’ GGTTACCTTGTTACGACTT 3’) (Edwards et al. 1989; Weisburg et al. 1991). Each 50 !l PCR reaction contained 1! ThermoPol PCR buffer (New England Biolabs, Mississauga, Ontario, Canada), 0.4 µM of each primer, 0.3 mM of deoxynucleoside triphosphates, 1.5 U of Taq DNA polymerase (New Englands Biolabs), and the colony of interest. PCR amplification conditions were as follows: initial denaturation at 94°C for 10 minutes, then 30 cycles of denaturation at 94°C for 1 minute, primer annealing at 52°C for 1 minute, and chain extension at 72°C for 1.45 minutes, followed by final chain extension at 72°C for 10 minutes. A PTC-200 Peltier Thermal Cycler (MJ Research, Inc., Waltham, Massachusetts, USA) was used to perform PCR. The PCR products were purified using GenEluteTM PCR Clean-up Kit (Sigma-Aldrich, St. Louis, MO, USA). The amplified 16S rRNA gene fragments were then directly sequenced by the University Health Network Research DNA Sequencing Facility (Toronto, Ontario, Canada) using a Beckman Coulter CEQ 2000 automatic Sequencer. The sequencing primers were 27f and 1492r. Approximately 600 nucleotides of the front end and the back end of the sequence were determined using these primers. To obtain the complete 16S rRNA gene sequences of isolated colonies, internal sequencing primers were designed using the PRIMER3 program (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). The internal primer used for Dechlorosoma-like sequences (Dsoma) was AZ 545f (5’ AGGCGGTTTCGTAAGACAGA 3’) and the primer used for Dechloromonas-like sequences (DCh) was DC 453f (5’
! ! 106! ! ATACCCAGTGTGGATGACGG 3’). The 16S rRNA gene sequences of isolated bacteria are provided in Appendix F.
5.2.3. Analysis of 16S rRNA gene sequences
The NCBI Genbank blastn tool (Altschul et al. 1990) and the Ribosomal Database project II, Seqmatch tool, were employed to determine phylogenetic similarity between sequences obtained in this study and those from known organisms. Sequences were aligned by ClustalW (Larkin et al. 2007). Phylogenetic tree was generated using the Neighbor-Joining method in MEGA 4 with 1000 bootstraps (Tamura et al. 2007). Agrobacterium tumefaciens was used to root the phylogenetic tree.
5.2.4. Fluorescence microscopy
Fluorescence microscopy was employed to check the purity of serially diluted cultures. In preparation for fluorescence microscopy, samples were centrifuged at 12000 ! g in an Eppendorf Model 5415 D centrifuge for 10 minutes. The pellet of cells was re-suspended in 4% paraformaldehyde (PFA) in phosphate buffer. Cells were fixed in this solution for 30 minutes. Then, DAPI was added to the samples at a final concentration of 1 µg/ml. Cells were stained with DAPI for 30 minutes and visualized under a microscope.
5.3. Results 5.3.1. Characterization of dilution cultures
Isolation experiments were conducted with four microbial consortia: Cartwright pw1, Cartwright 1b, Cartwright Consolidated, and Swamp Consolidated. Eight serial dilutions (from 10-1 to 10-8) were prepared from each of the microcosms, and were supplied with benzene and nitrate. Benzene degradation activity was observed in some of the bottles. A list of dilutions that mineralized benzene is given in Table 5.1. The rate of benzene degradation in these cultures was 6.0 (1.2)-9.1 (3.7) µM/day. The measured nitrate to benzene consumption ratio was in the range 10.6 (2.3)-14.7 (1.7) moles of nitrate reduced per moles of benzene oxidized (Table 5.1). This ratio is consistent with the range 13.1-14.3 for the oxidation of benzene linked to the incomplete Figure 5. Phylogenetic tree showing reduction of nitrate to nitrite (refer to section 2.1.1 of Chapter 2 and Table 2.3 for details). Nitrite the position of DCh and Dsoma relative to known microorganisms !-Proteobacteria ! ! 107! ! accumulated in all of the bottles providing further evidence that benzene mineralization is coupled to the reduction of nitrate to nitrite and not to its reduction to the nitrogen gas.
Table 5.1. List of dilution cultures showed benzene degradation activity.
Average rate of Ratio of nitrate benzene Culturea Dilution reduced/benzene nd degradation oxidizedc (µM/day)b 10-1 6.0 (1.2) 13.3 (2.9) 11 Cartwright 1b 10-2 8.2 (1.3) 11.9 (2.5) 6 10-1 8.7 (2.6) 13.7 (1.6) 11 Cartwright pw1 10-2 8.3 (2.9) 12.3 (1.6) 9 10-5 7.9 (2.4) 12.3 (2.8) 7 10-1 9.1 (2.8) 13.2 (2.1) 10 Cartwright 10-2 9.1 (3.3) 11.9 (3.0) 12 Consolidated 10-3 8.5 (3.5) 10.6 (2.3) 6 10-4 8.6 (1.8) 13.4 (2.6) 8 10-1 7.9 (2.0) 14.7 (1.7) 7 Swamp 10-2 9.1 (3.7) 12.7 (1.8) 8 Consolidated 10-5 8.8 (3.7) 13.2 (2.5) 8 a Cartwright 1b is a parent culture; Cartwright Consolidated and Swamp Consolidated are established by addition of several cultures at different levels of enrichment; Cartwright pw1 is more enriched than other cultures (8th generation). b Rate of benzene degradation for each culture bottle was calculated by taking an average of benzene degradation rates observed for “n” rounds of benzene mineralization process. c For benzene oxidation coupled to the reduction of nitrate to nitrite, the theoretical number of moles of nitrate consumed per moles of benzene mineralized is 7.7-15; This ratio is 2.6-6 for the mineralization of benzene coupled to reduction of nitrate to the nitrogen gas (Appendix A). d Numbers in the brackets are the standard deviations of “n” values corresponding to “n” rounds of benzene degradation.
! ! 108! ! To examine the purity of dilutions, fluorescence microscopy was performed on the most diluted cultures capable of degrading benzene including 10-2 dilution of Cartwright 1b, 10-5 dilution of Cartwright pw1, 10-4 dilution of Cartwright Consolidated, and 10-5 dilution of Swamp Consolidated. As an example, a fluorescent image of the 10-5 dilution of Swamp Consolidated is shown in Figure 5.1 (Appendix G provides the microscopic images for the rest of cultures). The results of microscopy indicated that the cultures were not pure and at least two types of cells, i.e. short rod and long rod bacterial cells, were present in all of the samples. qPCR was also employed to determine the presence of specific species, i.e. Azoarcus, Peptococcaceae, Dechloromonas, Chlorobi, and Anammox, in the bottles (refer to section 4.3.2. of Chapter 4 for details). The majority of dilutions contained Azoarcus, Peptococcaceae, Dechloromonas, and Chlorobi. This observation also indicated that the serially diluted microbial cultures were not pure.
Figure 5.1. Fluorescence microscopy image of 10-5 dilution of Swamp Consolidated culture at a 40X magnification.
! ! 109! ! 5.3.2. Phylogenetic relationship between isolated bacteria and known microbes
To isolate bacteria responsible for benzene degradation, several dilution cultures were streaked on agar plates containing acetate and nitrate. Small white colonies appeared on the plates after two weeks. To identify these colonies, the PCR amplification of 16S rRNA gene was carried out followed by sequencing of amplified fragments.
Analysis of 16S rRNA gene sequences of isolated colonies revealed the presence of two phylotypes nicknamed DCh and Dsoma. The closest Genbank match to DCh is Dechloromonas sp. JJ, which is an isolated bacterium known to degrade benzene under nitrate-reducing conditions (Table 5.2) (Coates et al. 2001). The nearest Genbank match to Dsoma is Dechlorosoma sp. Iso1, which couples acetate oxidation to the reduction of perchlorate (Coates et al. 1999). This strain can also use nitrate as electron acceptor.
The phylogenetic tree in Figure 5.2 shows the evolutionary relationships between isolated bacteria and other known microorganisms.
Table 5.2. Close Genbank matches to colonies isolated from serially diluted cultures.
Group Sequence length Close matches NCBI %Similarity accession number DCh 1373 Dechloromonas sp. JJ AY032611 97 Dsoma 1388 Dechlorosoma sp. Iso1 "#$%&'(& 99
5.3.3. Catabolic study
To investigate the ability of isolated bacteria to degrade benzene, they were transferred into fresh medium and supplemented with benzene and nitrate. No benzene mineralization was observed in the culture bottles. The results indicated that the isolated bacteria could not oxidize benzene under nitrate-reducing conditions.
! ! 110! !
DCh"! *
DCC1! (Ulrich ! (Ulrich 2004)![benzene, 2004) toluene, [benzene, and benzoate] toluene, {nitrate and and benzoate] oxygen}! {nitrate and oxygen} [benzene, benzoate, toluene, ethylbenzene, Xylene, 4-chlorobenzoate, and simple organic acids] {chlorate, perchlorate, nitrate, and oxygen}
[benzene, toluene, benzoate, and simple organic acids] {nitrate, and oxygen} [hydrogen and acetate] {chlorate, perchlorate, nitrate, and oxygen} [acetate and lactate] {chlorate, perchlorate, oxygen, and nitrate}
Dsoma"! * "-Proteobacteria [acetate, simple volatile fatty acids, and simple organic acids] {perchlorate, oxygen and nitrate} [acetate, simple volatile fatty acids, and simple organic acids] {perchlorate, [simple organic acids] {oxygen} oxygen, and nitrate} ACC1! (Ulrich ! (Ulrich 2004)![benzene, 2004) toluene, [benzene, and benzoate] toluene, {nitrate} and! benzoate] {nitrate} [phenol, benzoate, 4-hydroxybenzoate, and p-cresol] {nitrate and oxygen} [ethylbenzene, toluene, benzoate and acetate ] {nitrate and oxygen} [benzoate, 3-hydroxybenzoate, and acetate] {nitrate and oxygen} (L33694) [toluene, benzoate, simple organic acids and fatty acids] {nitrate and oxygen } [benzene, toluene, and m-xylene] {nitrate and oxygen} [benzene, toluene, m-xylene, and benzoate] {nitrate and oxygen} (Out-group) !- Proteobacteria
Figure 5.2. Phylogenetic tree showing relationship between bacteria isolated from our benzene-degrading nitrate- reducing enrichment cultures and other classified microorganisms. * represents bacteria isolated in this study; ! represents DCC1 and ACC1 which are identified as active benzene-degrading Dechloromonas and Azoarcus species in two co-cultures
established from one of our microbial consortia by Ulrich (2004). The substrates and electron donors that can be used by each bacterium are given within []. The electron acceptors utilized by each species are shown within {}. Numbers at branching points are bootstrap values. Scale bar represents 2% nucleotide substitution. Numbers in parenthesis are NCBI accession
numbers.
! ! 111! ! 5.4. Discussion 5.4.1. Phylogenetic and physiological characterization of isolated bacteria
The results of 16S rRNA gene sequencing of isolated bacteria from benzene-degrading nitrate-reducing cultures indicates that they belong to two groups: DCh and Dsoma. DCh is closely related to the members of Dechloromonas genus and Dsoma is affiliated with the members of Azospira genus in the beta subclass of Proteobacteria (Figure 5.2). The Dechloromonas species together with the closely related Dechlorosoma bacteria represent the predominant perchlorate-reducing microbes in the environment (Chakraborty and Coates 2004).
One of the close Genbank matches to DCh is Dechloromonas sp. JJ. Strain JJ was enriched with a humic-substance (2,6-anthrahydroquinone disulphonate) as the electron donor, nitrate as the electron acceptor, and acetate as the carbon source. Dechloromonas sp. JJ completely oxidized benzene in the absence of oxygen and coupled benzene oxidation to nitrate reduction (Coates et al. 2001). Another member of this genus, Dechloromonas strain RCB that was isolated as a hydrocarbon-oxidizing chlorate reducer, could also mineralize benzene using nitrate as the electron acceptor (Coates et al. 2001). Prior work with Cartwright benzene-degrading nitrate-reducing enrichment cultures in our laboratory by Ulrich (2004) resulted in two co- cultures with active benzene-oxidizing Dechloromonas (DCC1) and Azoarcus (ACC1) species. As it is shown in Figure 5.2 DCh is phylogenetically similar to DCC1 (98% similarity). Close relationship between DCh and other benzene-degrading denitrifying Dechloromonas species suggests that DCh may also be involved in mineralization of benzene under nitrate-reducing conditions.
Three close matches to Dsoma are Dechlorosoma sp. Iso 1, Dechlorosoma suillum, and Dechlorosoma sp. KJ (Figure 5.2). These bacteria oxidize acetate while reducing perchlorate (Coates et al. 1999; Logan et al. 2001). In addition to acetate, they use short chain fatty acids and simple dicarboxylic acids (Coates et al. 1999; Logan et al. 2001). None of them can oxidize benzoate (Coates et al. 1999; Logan et al. 2001). Chlorate, oxygen, and nitrate are utilized by these species as alternative electron acceptors for their growth. This discussion indicates that Dsoma is not a benzene degrader and that growth of this bacterium on agar plates is due to presence of acetate and nitrate.
! ! 112! ! 5.4.2. Metabolic characteristics
The ability of isolated colonies, i.e. DCh and Dsoma, to degrade benzene using nitrate as an electron acceptor was evaluated by re-suspending colonies into the medium containing benzene and nitrate. None of the isolated bacteria showed benzene mineralization activity. Plausible explanations for this lack of activity are as follows: (i) Instead of benzene, acetate was used as a carbon source and electron donor during isolation process and this may have caused the bacteria to lose their ability to oxidize benzene. It is likely that genes required for the mineralization of benzene are located on a plasmid. Plasmids are extrachromosomal circular double-stranded DNA, distinct from the normal bacterial genome, and can both enter and leave cells. In the absence of selective pressure (in this case, the presence of benzene), microorganisms would not need the corresponding benzene oxidation genes on the plasmid and therefore, the plasmid could exit the cell. (ii) The isolated colonies lack benzene degradation capability. Instead of benzene, they just use acetate as a source of energy and as electron donor and nitrate as electron acceptor for their growth. This view is supported by the hypothesis that anaerobic degradation of benzene in enrichment cultures is carried out by syntrophic relationship between different bacterial species (refer to section 4.4.2 of Chapter 4 for more details). As it is explained in Chapter 4, in this syntrophic association, the primary benzene degrader (Peptococcaceae) initially attacks benzene and converts it to hydrogen, acetate, or other low molecular weight fermentation products. Then, secondary bacterial partners oxidize the low molecular weight by-products of benzene oxidation using nitrate as the electron acceptor. The lack of ability to mineralize benzene in isolated colonies strongly suggests that these colonies are not the primary consumers of benzene and act as secondary bacterial partners that grow on acetate or other low molecular weight compounds produced by primary benzene degraders.
The key to successfully isolating syntrophic microorganisms is either cultivating them in co- cultures with hydrogen consuming organisms or isolating them using substrates that allow their growth in pure cultures (Kamagata and Tamaki 2005). In the former approach, pure cultures of known hydrogen consuming microorganisms such as methanogens or sulfate-reducing bacteria are pre-grown and mixed with inoculum of consortia so that the syntrophs grow and form colonies on a “lawn” of hydrogen consumers in solidified medium. In the latter approach, a suitable substrate that can support the growth of syntrophs in pure culture is found by trial and
! ! 113! ! error. Another mean of isolating and enriching of Peptococcaceae bacteria (gram-positive) in our cultures would be to inhibit the growth of other species present in microcosms using antibiotics. For instance aminoglycoside antibiotics are active against most gram-negative bacteria while lack activity against gram-positive microorganisms (Beers et al. 2003). These antibiotics have affinity for negatively charged residues in outer membrane of gram-negative bacteria and disrupt the integrity of bacterial cell membrane, resulting in leakage of intracellular contents (Shakil et al. 2008). They also bind to the 30S ribosomal subunit and inhibit protein synthesis in gram- negative bacteria. Therefore, they could be employed for inhibiting growth of gram-negative microorganisms.
5.5. Conclusions
Two groups of bacteria were isolated from benzene-degrading nitrate-reducing enrichment cultures by employing serial dilution followed by subsequent plating on agar plates containing acetate and nitrate. One group was closely related to Dechloromonas species and the other to Dehlorosoma species. None of the isolated colonies had the ability to mineralize benzene under nitrate-reducing conditions. Based on the results presented above, it is concluded that the isolated bacteria are not responsible for the degradation of benzene. Their presence and viability in the enriched cultures is due to their ability to consume acetate and other by-products resulted from degradation of benzene by benzene-degrading bacteria.
5.6. Recommendations for further isolation trials
If mineralization of benzene in the enrichment cultures is mediated by syntrophic association between different genera of bacteria, isolation of a single bacterium that could degrade benzene in a pure culture will not be possible. However, the sequential serial dilution of microbial consortia using benzene, as the sole source of carbon and energy, is a promising technique to establish highly enriched cultures of benzene-degrading microbes and their syntrophic partners. Acetate should not be employed as a replacement for benzene during enrichment and isolation process because this may result in loss of benzene degradation ability or in enrichment and isolation of acetate-oxidizing bacteria rather than primary benzene degraders.
The results presented in Chapter 4 suggest that Peptococcaceae-related bacteria are responsible for initial attack on benzene. Previous studies have shown that some members of
! ! 114! ! Peptococcaceae family have the ability to form spores (Imachi et al. 2002; de Bok et al. 2005; Imachi et al. 2006; Qiu et al. 2006; Zavarzina et al. 2007). Hence, a possible means of isolating and obtaining pure culture of Peptococcaceae from the enriched microbial consortia is to treat the culture with heat (heat the culture at 80 °C for 10 minutes) or heat and ethanol to eliminate vegetative cells. The spore forming bacteria will survive this process and can then be isolated from the culture. Isolated bacteria can be grown on benzene in co-culture with hydrogen and acetate scavengers. Crotonate has been successfully used to establish pure cultures of bacteria that grew only as obligate syntrophs in coculture with hydrogen-utilizing microbes (Beaty and McInerney 1987; Jackson et al. 1999). This compound can potentially be utilized as a substrate for enriching or obtaining an isolated benzene degrader from the enrichment cultures.
! !
5.7. References Chapter 5
Altschul, S. F., W. Gish, W. Miller, E. W. Myers and D. J. Lipman (1990). "Basic local alignment search tool." J. Mol. Biol. 215: 403-410.
Beaty, P. S. and M. J. McInerney (1987). "Growth of Syntrophomonas wolfei in pure culture on crotonate." Arch. Microbiol. 147: 389-393.
Beers, M. H., A. J. Fletcher, T. V. Jones, R. Porter, M. Berkwits, J. L. Kaplan (2003). "The merck manual of medical information." New Jersey, Merck Research Laboratories.
Burland, S. M. and E. A. Edwards (1999). "Anaerobic benzene biodegradation linked to nitrate reduction." Appl. Environ. Microbiol. 65(2): 529-533.
Chakraborty, R. and J. D. Coates (2004). "Anaerobic degradation of monoaromatic hydrocarbons." Appl. Microbiol. Biotechnol. 64: 437-446.
Coates, J. D., R. Chakraborty, J. G. Lack, S. M. O’Connor, K. A. Cole, K. S. Bender and L. A. Achenbach (2001). "Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas." Nature 411: 1039-1043.
Coates, J. D., U. Michaelidou, R. A. Bruce, S. M. O'Connor, J. N. Crespi and L. A. Achenbach (1999). "Ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria." Appl. Environ. Microbiol. 65(12): 5234-5240. de Bok, F. A. M., H. J. M. Harmsen, C. M. Plugge, M. C. d. Vries, A. D. L. Akkermans, W. M. d. Vos and A. J. M. Stams (2005). "The first true obligately syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii Sp. nov., co-cultured with Methanospirillum hungatei, and emended description of the genus Pelotomaculum " Int. J. Syst. Evol. Microbiol. 55: 1697-1703.
Edwards, U., T. Rogall, H. Blocker, M. Emde and E. C. Bottger (1989). "Isolation and direct complete nucleotide determination of entire Genes. Characterization of a gene coding for 16S ribosomal RNA " Nucleic Acids Res 17: 7843-7853.
Holmes, D. E., C. Risso, J. A. Smith and D. R. Loveley (2011). "Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus." Appl. Environ. Microbiol. 77(17): 5926-5933.
Imachi, H., Y. Sekiguchi, Y. Kamagata, S. Hanada, A. Ohashi and H. Harada (2002). "Pelotomaculum thermopropionicum gen. nov., sp. nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium." Int. J. Syst. Evol. Microbiol. 52: 1729-1735.
Imachi, H., Y. Sekiguchi, Y. Kamagata, A. Loy, Y. L. Qiu, P. Hugenholtz, N. Kimura, M. Wanger, A. Ohashi and H. Harada (2006). "Non-sulfate-reducing, syntrophic bacteria affiliated with Desulfotomaculum Cluster I are widely distributed in methanogenic environments." Appl. and Environ. Microbiol. 72(3): 2080-2091.
! 115! ! 116!
Jackson, B. E., V. K. Bhupathiraju, R. S. Tanner, C. R. Woese and M. J. McInerney (1999). "Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms." Arch. Microbiol. 171(2): 107-114.
Kamagata, Y. and H. Tamaki (2005). "Cultivation of uncultured fastidious microbes." Microbes Environ. 20(2): 85-91.
Kasai, Y., Y. Kodama, Y. Takahata, T. Hoaki and K. Watanabe (2007). "Degradative capacities and bioaugmentation potential of anaerobic benzene-degrading bacterium strain DN11." Environ. Sci. Technol. 41: 6222-6227.
Kasai, Y., M. Manefield and K. Watanabe (2006). "RNA-based stable isotope probing and isolation of anaerobic benzene-degrading bacteria from gasoline-contaminated groundwater." Appl. Environ. Microbiol. 72 (5): 3586-3592.
Larkin, M. A., G. Blackshields, N. P. Brown3, R. Chenna, P. A. McGettigan1, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson and D. G. Higgins (2007). "Clustal W and Clustal X version 2." Bioinformatics 23(1): 2947-2948.
Logan, B. E., H. ZHANG, P. MULVANEY, M. G. MILNER, I. M. HEAD and R. F. UNZ (2001). "Kinetics of perchlorate- and chlorate-respiring bacteria " Appl. and Environ. Microbiol. 67: 2499-2506.
Qiu, Y. L., Y. Sekiguchi, S. Hanada, H. Imachi, I. Tseng, S. S. Cheng, A. Ohashi, H. Harada and Y. Kamagata (2006). "Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens." Arch. Microbiol. 185: 172-182.
Salinero, K. K., K. Keller, W. S. Feil, H. Feil, S. Trong, G. D. Bartolo and A. Lapidus (2009). "Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation " BMC Genomics 10: 351-74.
Shakil, S., R. Khan, R. Zarrilli, A. U. Khan (2008). "Aminoglycosides versus bacteria- adescription of action, resistance mechanism and nosocomial battleground." J. Biomed. Sci. 15: 5-14.
Tamura, K., M. Nei, J. Dudley and S. Kumar (2007). "MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0." Mol. Biol. Evol. 24(8): 1596-1599.
Ulrich, A. C. (2004). Characterization of anaerobic benzene-degrading cultures. Toronto, University of Toronto. Ph.D. thesis.
Weisburg, W. G., S. M. Barns, D. A. Pelletier and D. J. Lane (1991). "16S Ribosomal DNA amplification for phylogenetic study." J. Bacteriol. 173: 697-703.
! ! 117!
Zavarzina, D. G., T. G. Sokolova, T. P. Tourova, N. A. Chernyh, N. A. Kostrikina and E. A. Bonch-Osmolovskaya (2007). "Thermincola ferriacetica sp. nov., a new anaerobic, thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction." Extremophiles 11: 1-7.
! !
Chapter 6: Metatranscriptome Analysis of a Benzene-degrading Nitrate-Reducing Culture
Prepared for eventual publication as:
Metatranscriptome Analysis of a Benzene-Degrading Nitrate-Reducing Culture Roya Gitiafroz, Fei Luo, Yunchen Gong, Lutgarde Raskin, and Elizabeth A. Edwards. 2011.
! 118! ! 119!
6.1. Introduction Mineralization of benzene by indigenous microorganisms has attracted attention as a potentially efficient and inexpensive means of remediation of polluted sites. Early studies conducted by Vogel and Grbi!-Gali! (Vogel and Grbi!-Gali! 1986; Grbi!-Gali! and Vogel 1987) on methanogenic enrichment cultures indicated the possibility of benzene mineralization under anaerobic conditions. Ever since, it has been shown that benzene is susceptible to biodegradation under a variety of terminal electron-accepting conditions including nitrate- reducing, sulfate-reducing, iron-reducing, and methanogenic. Although anaerobic benzene degradation is important for removing this compound from polluted sites, the process has remained poorly understood owing to scarcity of pure and enriched benzene-degrading cultures. The metabolic pathway of anaerobic benzene degradation is largely unknown. Based on metabolites detected during mineralization of benzene, three mechanisms including hydroxylation to phenol, methylation to toluene, and carboxylation to benzoate have been proposed as the initial steps in this process (Figure 6.1). Phenol, toluene, and benzoate are then converted into benzoyl-coA through different peripheral pathways.
There are several reports that exclude benzene hydroxylation and methylation as the activation mechanism of benzene ring due to inability of anaerobic pure and enriched cultures to consume phenol and toluene (Kasai et al. 2007; Musat and Widdel 2008; Abu Laban et al. 2009). Kunapuli et al. (2008) and Abu Laban et al. (2009) reported the abiotic formation of phenol from benzene due to the exposure of culture samples to air. Therefore, caution should be exercised in interpreting phenol as a metabolite of benzene degradation (Abu Laban et al. 2010). Recently, the genome of facultative anaerobe, Dechloromonas aromatica strain RCB, which can degrade benzene and its derivatives in the absence of oxygen, was sequenced (Salinero et al. 2009). None of the known key enzymes for anaerobic catabolism of monoaromatic hydrocarbons, including those involved in central benzoyl-CoA pathway as well as enzymes facilitating toluene and phenol degradation, were present in the genome of this bacterium. Therefore, the mechanisms and genes employed by Dechloromonas aromatica strain RCB to mineralize benzene and other aromatic compounds remained unidentified and uncharacterized.
! ! 120!
OH COO- COSCoA ATP AMP + P - ATP+CoA AMP+PP i OH + CO2 Pi i
Phenylphosphate Phenylphosphate 4-hydroxybenzoate synthase carboxylase CoA ligase Phenol O P O- OH OH 4-hydroxybenzoyl-CoA OH! 4-hydroxybenzoate + 2H +2Fdred H+ Phenylphosphate 4-hydroxybenzoyl- (1) CoA reductase H2O+2Fdox H2O/OH• COO- COSCoA ATP+CoA AMP+PPi CO2 3 Acetyl-CoA + CO2 (2) Benzoate-CoA ligase Benzene Benzoate Benzoyl-CoA Succinyl-CoA Benzoylsuccinyl-CoA thiolase CoA CH3-X X=S-adenosyl-methionine, Tetrahydrofolate, COSCoA or Cobalamin O (3) ! - COO Benzoylsuccinyl-CoA X
2-[hydroxy (phenyl)methyl]- 2H succinyl-CoA dehydrogenase - COO COSCoA COSCoA COSCoA CH COO- HO 3 - - - - COO COO Succinyl-CoA COO- 2H COO COO H2O
Benzylsuccinate Succinyl-CoA: (R)- (R)-benzylsuccinyl- Phenylitaconyl- synthase benzylsuccinate CoA dehydrogenase CoA hydratase CoA transferae
Toluene Benzylsuccinate Benzylsuccinyl- Phenylitaconyl 2-[hydroxyl(phenyl) CoA -CoA methyl]-succinyl-CoA
Figure 6.1. Benzene hydroxylation to phenol (1), carboxylation to benzoate (2) and methylation to toluene (3) as proposed activation mechanisms of benzene ring (Coates et al. 2002). Phenol, benzoate, and toluene are further metabolized to benzoyl-CoA through peripheral pathways (Carmona et al. 2009). The name of enzymes involved in each step appear in bold and
below or next to each arrow.
In the present study, an RNA-centered meta-transcriptomic approach was applied to attempt to elucidate the anaerobic benzene degradation pathway and the responsible genes in an enriched benzene-degrading nitrate-reducing culture called Cartwright Consolidated. The mRNA
! ! 121! sequences from benzene-grown cells were compared with those of benzoate-grown cells. This study identified carboxylase genes that were specifically transcribed in the presence of benzene. These genes may encode for subunits of a carboxylase enzyme that is involved in initial attack on benzene ring, as proposed by Abu Laban (2010). These results provide compelling evidence for carboxylation as the initial reaction in benzene degradation and further our understanding of this process. The knowledge of the genes involved in the degradation of benzene will be useful for creating molecular tools to assess biodegradation capacity and monitor bioremediation in contaminated sites.
6.2. Materials and Methods 6.2.1. Nitrate-reducing enrichment cultures
Nitrate-reducing microcosms were originally prepared with soil and groundwater from a decommissioned gasoline station on Cartwright Avenue in Toronto, Ontario (Nales et al. 1998; Burland and Edwards 1999). Over the past 16 years, these cultures have been repeatedly transferred into defined mineral media (Burland and Edwards 1999; Ulrich and Edwards 2003) and supplied with benzene at concentrations ranging from 190 to 256 µM (from a neat stock) and 2-5 mM sodium nitrate (from a 500 mM anaerobic stock). The microbial consortia have been incubated statically and in the dark inside a Coy anaerobic chamber (Coy Laboratory Products,
Madison, WI) under an atmosphere of 80% N2, 10% H2 and 10% CO2. The nitrate-reducing enrichment culture used in this study is called Cartwright Consolidated.
6.2.2. Substrate utilization experiments
In these experiments, the Cartwright Consolidated enrichment culture was tested for its ability to degrade benzoate, a possible metabolite of benzene degradation. Six identical subcultures were prepared by transferring 30 ml of the original culture into 60 ml glass bottles sealed with screw cap Mininert valves (Supelco Inc.). Three of the bottles were supplied with 128 µM benzene and 2 mM nitrate while the other three were fed with 128 µM benzoate and 2 mM sodium nitrate. Sterile controls were made with 30 ml of medium (no culture added), 128 µM of either benzene or benzoate, and 2 mM sodium nitrate. Subsequently, a second experiment was conducted to study the impact of benzoate on benzene mineralization. In this experiment, nine subcultures were prepared from the Cartwright Consolidated. Three of these subcultures
! ! 122! were fed only with benzene (positive controls), three were simultaneously fed with benzene and benzoate, and the last three were fed first with benzoate and then with benzene (after consumption of benzoate). Nitrate was added to all of the bottles. Negative controls were prepared by adding benzene, benzoate, and nitrate to fresh medium that did not contain the culture (three replicates). The feeding concentration of benzene, benzoate, and nitrate were as above.
6.2.3. Differential transcription experiments
A differential expression study was performed with the goal of identifying genes specific to the anaerobic benzene degradation pathway. In this experiment, 200 ml of the Cartwright Consolidated culture was transferred into a bottle and supplied with benzoate at a concentration of 128 µM and 2 mM sodium nitrate. Total RNA was extracted from 50 ml of culture during mineralization of benzoate (in this case when !25% of benzoate was degraded). After complete benzoate oxidation, 128 µM of benzene and 2 mM sodium nitrate were added to the same bottle. Finally, when !65% of benzene was degraded, another 50 ml of culture was removed for RNA extraction. In this way, a set of paired RNA samples was obtained: one RNA sample from benzoate-amended culture and one from the same culture subsequently amended with benzene. If benzoate is considered a metabolite of benzene mineralization, then mRNA sequences corresponding to the genes involved in degradation of benzoate through benzoyl-CoA pathway should be present in both RNA samples. The RNA from benzene-grown cells should also contain gene transcripts for enzymes involved in benzene transformation to benzoate.
We attempted to extract RNA at the onset of benzoate and benzene degradation. However, this proved to be challenging due to the presence of lag periods prior to mineralization of benzene. Therefore, at the time of RNA extraction, 25% of benzoate was degraded while 65% of benzene was mineralized. In addition to the above pair of RNA, three other pairs of RNA were extracted in a similar manner.
6.2.4. RNA extraction and Pyrosequencing
RNA extraction was performed according to the protocol explained by Waller et al. (2005) with some modifications. First, 50 ml of culture was dispensed into an anaerobic centrifuge tube on ice inside the anaerobic chamber (Coy Laboratory Products, Madison, WI). Cells were
! ! 123! collected by centrifugation at 9900 ! g for 20 minutes at 4°C. The cell pellet was resuspended in 250 µl of ice-cold extraction buffer comprised of 1.4 M NaCl, 22 mM EDTA, and 35 mM sodium dodecyl sulfate. 900 µl of ice-cold acid-phenol-chloroform-isoamyl alcohol (125:24:1, pH 4.5; Ambion, Austin, TX) was added to the mixture of cells and the extraction buffer. This mixture was transferred into a 1.5 ml screw-cap microcentrifuge tube containing 100 µl of Zirconia/silica beads (0.5 mm; BioSpec Products Inc., Bartlesville, OK). The tube was agitated horizontally in a mini bead-beater-96 (Biospec products Inc., Bartlesville, OK) for 4 minutes and then centrifuged at 14000 ! g for 15 minutes at 4°C. The aqueous phase was transfered into a new 1.5 ml microcentrifuge tube, which contained 900 µl of ice-cold acid-phenol-chloroform- isoamyl alcohol, and then centrifuged at 14000 ! g for 15 minutes at 4°C (this step was repeated twice). The supernatant was moved to a new tube. RNA was precipitated by adding 0.1 volume of ammonium acetate (7.5 M; Sigma-Aldrich) and 1.1 volume of isopropanol and storing the tube at -20°C overnight. The RNA pellet was collected by centrifugation for 15 minutes at 4°C and resuspended in RNase/DNase free water (Sigma-Aldrich). The contaminating DNA was removed using Turbo DNA-free Kit (Ambion, Austin, Texas). The resulting RNA samples were sequenced by the Center for Applied Genomics (The Hospital for Sick Children, MaRS center, Toronto, Ontario) using a Roche GS FLX Titanium sequencer (Roche Applied Sciences/454 Life sciences, Branford, CT). Sequences were assembled using Newbler (refer to Appendix H).
The above method was employed to extract 8 high quality RNA samples from Cartwright Consolidated culture during its growth on either benzoate or benzene (refer to Appendix H for more details, Figures H.1 and H.2 and Table H.1). One pair of RNA was sequenced using pyrosequencing and a second pair was sequenced using Illumina sequencing technology. The results obtained from pyrosequencing were the main focus of this study. The Illumina sequences are being analyzed by another student (Fei Luo), owing to the complexity and time consuming nature of the analyses.
6.2.5. Community profiling and functional analysis of mRNA
Direct pyrosequencing of RNA samples from cells grown with benzoate alone and with benzene alone produced reads corresponding to ribosomal RNA as well as messenger RNA. Analysis of rRNA and mRNA sequences provided information regarding the community
! ! 124! structure of the samples and the genes that were transcribed during degradation of each compound, respectively.
Figure 6.2 shows the schematic of the procedure used in this study for identifying rRNA and mRNA sequences. In the first step, all RNA reads were compared to a small subunit and a large subunit rRNA reference database (SSUrdb and LSUrdb) using the NCBI Genbank blastall implementation of BLASTN. The SSUrdb and LSUrdb contained sequences from all three domains of life and were constructed from publicly available rRNA databases by Urich et al. (2008). The BLAST output file was subsequently analyzed using MEGAN software version 4 (Huson et al. 2007; Huson et al. 2011). MEGAN uses the results of a BLAST comparison as an input and places each read on a node in the NCBI taxonomy. The outcome of this analysis can be depicted as a tree representing the NCBI taxonomy of sample. MEGAN also allows the comparative taxonomic analysis of multiple data sets. This function of MEGAN was employed to compare the community structures of benzoate-fed and benzene-fed cells. RNA sequences that had no BLAST matches or were not assigned to any taxonomy by MEGAN were considered as non-ribosomal RNA. All of the unassigned sequences were assembled using Newbler software (454 life sciences, Roche, Branford, Connecticut) and further translated in six reading frames and compared to Genbank non-redundant protein database using BLASTX. Manual inspection of the output files allowed identifying several genes that play important roles in benzoate and benzene degradation process within the benzene-degrading nitrate-reducing enrichment culture.
6.2.6. Analytical methods
The concentration of benzene was determined by injecting 300 µl samples from headspace of culture bottles into a Hewlett-Packard 6890 series gas chromatograph equipped with a HP-5, 30 m ! 0.32 mm I. D. column and a flame ionization detector. The injector, the FID detector, and the oven temperatures were at 200°C, 250°C, and 85°C, respectively. The carrier gas was nitrogen at a flow rate of 3 ml/min.
Benzoate concentration was measured by injecting 10 µl liquid samples withdrawn from the cultures into a Hewlett-Packard 1090 series II high performance liquid chromatograph (HPLC). This HPLC was equipped with a Hypersil BDS-C18 (5 µm particle size, 250 mm length ! 2 mm I. D.) column and a UV detector. The mobile phase contained 25% Acetonitrile and 75%
! ! 125!
KH2PO4 at a concentration of 50 mM (adjusted to pH=3 using phosphoric acid). The flow rate of mobile phase was 0.3 ml/min. The UV detection wavelength and the retention time for benzoate were 230 nm and 2.8 minutes, respectively.
Total RNA sequences
BlastN against Community profile Small and Large subunit MEGAN based on Small and rRNA reference data bases Large subunit rRNA
Non-ribosomal RNA
Assembly using Newbler
Translation in all six reading frames and aligned to NCBI non-redundant protein data base using BLASTX
Putative mRNA
Manual inspection to identify genes involved in metabolism of aromatic hydrocarbons
Figure 6.2. Schematic of different steps taken for analysis and identification of rRNA and mRNA sequences in the pool of total RNA. The programs used are highlighted in bold.
! ! 126!
6.3. Results and discussion 6.3.1. Metabolism of benzoate and benzene by Cartwright Consolidated enrichment culture
Previous studies conducted on nitrate-reducing cultures in our laboratory by Ulrich et al. 13 13 (2005) using C6-benzene resulted in the detection of C-labeled benzoate as a metabolite of benzene oxidation. If benzoate is an intermediate of benzene degradation, microbial consortia should be able to utilize this compound. An experiment was carried out to investigate whether the Cartwright Consolidated enrichment culture, which had been maintained on benzene as the sole source of carbon and energy, could also mineralize benzoate. This experiment was performed in triplicate. Figure 6.3 shows the benzoate and benzene degradation curves. Benzoate was consumed rapidly within 18 hours (Figure 6.3a). This result is consistent with the hypothesis that benzoate is an intermediate of benzene decay. Although mineralization of benzoate occurred rapidly and without a delay, a lag time of 14 to 34 days was evident prior to the onset of benzene degradation (Figure 3.6b). A possible explanation for this lag time could be the challenge microorganisms should overcome to activate the non-substituted benzene ring, which has the highest C-H bond dissociation energy among all hydrocarbons (Carmona et al. 2009). No decrease in benzoate and benzene concentrations was observed in sterile controls.
200
150 (a) 100
Benzoate 50
concentration (µM) 0 0 5 10 15 20 Hours
150 (b)
100
Benzene 50
concentration (µM) 0 0 10 20 30 40 50 Days Figure 6.3. Degradation of benzoate (a) and benzene (b) by benzene- degrading nitrate-reducing Cartwright Consolidated culture. This experiment performed in three replicates. Closed symbols represent positive treatments and open symbols represent sterile controls.
! ! 127!
The effect of benzoate on degradation of benzene was also studied. In this experiment, identical subcultures of Cartwright Consolidated were either first supplied with benzoate and then with benzene in a sequential order, or simultaneously exposed to both compounds. Three replicates were set for each treatment. Incubations that only contained benzene served as positive controls. In all of the bottles, benzoate was mineralized fast within 18 hours. However, benzene degradation was accompanied with a delay in every treatment as well as in the positive control. Table 6.1 shows lag times observed in the cultures. Bottles that were first exposed to benzoate and then fed with benzene had a much longer lag time (42 days) than the positive controls, whereas cultures that were fed with these substrates concurrently showed almost the same lag times as the positive controls (23 days). Based on these results, it appears that absence of benzene in the culture during benzoate degradation adversely influences the ability of the culture to mineralize benzene afterwards. Several studies have shown that when microorganisms are supplied with mixtures of benzene, toluene, xylenes, and ethylbenzene, bacteria use these substrates in a sequential order. Toluene is usually degraded most rapidly, followed by isomers of xylene, benzene, and ethylbenzene (Evans et al. 1991a; Evans et al. 1991b; Beller et al. 1992; Edwards and Grbi!-Gali! 1992; Edwards et al. 1992; Gulensoy and Alvarez 1999; Meckenstock et al. 2004; Siddique et al. 2007). This agrees with our observation that benzoate degrades preferentially and ahead of benzene. There are reports that show mineralization of benzene in benzene-degrading sulfate-reducing, nitrate-reducing, and methanogenic cultures as well as bioaugmented methanogenic aquifer columns was impeded or totally inhibited in the presence of more readily utilized substrates such as toluene (Edwards and Grbi!-Gali! 1992; Nales et al. 1998; Phelps and Young 1999; Da Silva and Alvarez 2004). Similarly, in a nitrate-reducing culture fed with benzene, toluene, and benzoate, degradation of benzene lagged until toluene and benzoate were completely consumed by the culture (Ulrich et al. 2005). Inhibition of benzene degradation might be due to a temporary repression of anaerobic benzene degradation genes in the presence of more favorable compounds such as toluene and benzoate. In the case of our mixed culture, it is also possible that the presence of benzoate and the absence of benzene provide an opportunity for benzoate-degrading bacteria to grow. Growth of these bacteria may in turn have a negative impact on the primary benzene-degrader in the culture, resulting in the observed longer lag times.
! ! 128!
Table 6.1. Comparison between the lag times observed prior to degradation of benzene for culture bottles amended first with benzoate and after its consumption with the benzene, supplied simultaneously with benzoate and benzene, and positive controls that were fed only with benzene. This experiment was performed in triplicate. The standard deviations are given for the average of three replicates.
Treatment Average lag time (day) Benzoate then benzene 42±11 Benzoate and benzene simultaneously 23±4 Benzene only (positive control) 23±10
The nitrate to benzoate and nitrate to benzene consumption ratios were 10.3 ± 0.4 (moles of nitrate reduced/mole of benzoate oxidized) and 14.3 ± 0.7 (moles of nitrate reduced/mole of benzoate oxidized) (n=6, 6 bottles, one measurement per bottle), respectively. Nitrite accumulated in the culture as nitrate was reduced during mineralization of benzoate and benzene. It appears that degradation of benzoate and benzene was coupled to reduction of nitrate to nitrite than to reduction of nitrate to nitrogen gas. The analysis of mRNA sequences from Cartwright Consolidated culture grown on benzoate indicated that all of the genes encoding the enzymes of denitrification pathway i.e. nitrate reductase (narGHI), nitrite reductase (nirS), nitric oxide reductase (norCB), and nitrous oxide reductase (nosZ) were transcribed (Table H.2, Appendix H). Transcripts of nitrite reductase (nirS), nitric oxide reductase (norCB), and nitrous oxide reductase (nosZ) were also detected in the culture supplied with benzene (Table H.2, Appendix H). While the genes necessary for complete reduction of nitrate to nitrogen gas were transcribed, the culture only partially reduced nitrate to nitrite during mineralization of benzoate and benzene. Therefore, nitrite accumulated in the culture. These results suggest that benzene or a metabolite of benzene degradation such as benzoate may have an inhibitory impact on the synthesis (at the level of translation) or activity of enzymes of nitrate reduction pathway such as nitrite reductase.
6.3.2. Analysis of RNA sequences
Table 6.2 summarizes the results of pyrosequencing. The RNA samples extracted from cells supplied with benzoate or benzene contained 250500 and 196075 sequences, respectively. Among these sequences only 1 to 2% correspond to mRNA and the rest were identified as rRNA.
! ! 129!
Table 6.2. Total number of RNA, rRNA, and mRNA sequences for the RNA samples extracted from Cartwright Consolidated culture during its growth on benzoate and benzene.
Benzoate RNA Benzene RNA Number of Number of % Sequences % Sequences sequences sequences Total RNA 250500 100 196075 100 mRNA 4970 2 2153 1.1 rRNA 245530 98 193922 98.9
6.3.3. Comparison between community profiles of Cartwright Consolidated culture during growth on benzoate and benzene
16S rRNA gene clone libraries prepared previously from our benzene-degrading nitrate- reducing enrichment cultures indicated the presence of several genera of bacteria including Peptococcaceae, Rhodocyclaeae (Azoarcus and Dechloromonas), Planctomamycetes (Anammox), and Chlorobi (Ulrich and Edwards 2003; Nandi 2006). Recently, Burkholderiales were also identified as a member of these microbial consortia (refer to Chapter 4). In the present study, we investigated the differences in the community structure of Cartwright Consolidated caused by changing the source of carbon and energy from benzene to benzoate.
The total RNA extracted from Cartwright Consolidated during growth on benzoate and benzene contained a very large ratio of ribosomal RNA sequences compared to mRNA sequences, which could be used to identify the community structure of culture. Sequences corresponding to small subunit and large subunit rRNA were identified and taxonomically profiled using the MEGAN software and two ribosomal RNA databases compiled by Urich et al. (2008). Figure 6.4 and Figure H.3 (Appendix H) compare microbial communities of benzoate- grown cells and benzene-grown cells based on small subunit and large subunit rRNA, respectively. A major difference between these two populations is the number of sequences related to Peptococcaceae (Figure 6.4). These numbers are much higher (about 10 to 100 times more) in the culture with benzene as a substrate, in agreement with our previous observations that indicated among monitored species, only Peptococcaceae showed significant growth during mineralization of benzene (refer to Chapter 4 for details). Several other members of Firmicutes
! ! 130! phylum are also present in higher numbers in culture grown on benzene compared to the one grown on benzoate (Figure 6.4). Although, Azoarcus /Aromatoleum and Burkholderiales comprise a large portion of culture population, no significant difference is observed in their quantities in the Cartwright Consolidated grown with benzene or benzoate (Figure 6.4). These results suggest that Peptococcaceae is the primary consumer of benzene. It plays a key role in benzene degradation and other species rely on metabolites produced by Peptococcaceae for their growth and survival. Recently, Kleinsteuber et al. (2008) reported a significant increase in the population of Cryptanaerobacter/Pelotomaculum-related microorganisms within Peptococcaceae in a benzene-degrading sulfidogenic consortium after repeated supplementation with benzene. They hypothesized that Peptococcaceae was responsible for the initial attack on benzene and producing intermediates that are used by different bacteria present in the culture. However, during cultivation on toluene, benzoate, and phenol, Peptococcaceae was outcompeted by other community members and its population decreased greatly. This data provides further support for our qPCR results obtained in Chapter 4, where increase in the number of Peptococcaceae was directly linked to benzene degradation.
! ! 131!
3_GAC_SSU_blastn 4_GAC_SSU_blastn Actinobacteria (111; 206) Bacteroidetes/Chlorobi group (1381; 783) Chlamydiae/Verrucomicrobia group (67; 43) Chloroflexi (1161; 1264) Deinococcus-Thermus (98; 192) Fibrobacteres/Acidobacteria group (116; 94) Bacilli (6; 27) Firmicutes Clostridiales Clostridiaceae (0; 8) Clostridia Peptococcaceae (27; 3849) Thermoanaerobacterales (0; 14) Gemmatimonadetes (18; 22) Nitrospirae (32; 32) Planctomycetes (39; 69)
Bacteria Alphaproteobacteria (190; 207) cellular organisms Burkholderiales (6078; 5137) Gallionellales (6; 0) root Hydrogenophilales (15; 31) Neisseriales (5; 0) Betaproteobacteria Nitrosomonadales (12; 0) Proteobacteria Aromatoleum (469; 428) Azoarcus (25676; 24894) Rhodocyclaceae Azospira (98; 176) environmental samples (101; 93) Thauera (30; 48) Zoogloea (23; 38) delta/epsilon subdivisions (49; 90) Gammaproteobacteria (61; 169) Tenericutes (34; 18) unclassified Bacteria (5; 24) Eukaryota (1902; 49) Not assigned (343; 400) No hits
Figure 6.4. The MEGAN tree showing comparison between community compositions of Cartwright Consolidated culture during growth on benzoate (red colors) versus growth on benzene (blue colors). This community profiling is based on taxonomic affiliation of the ribosomal RNAs obtained in this study to the small subunit rRNA reference database compiled by Urich et al. (2008). The size of each circle at the tree nodes is scaled by the number of reads assigned to the corresponding taxon. The first number and second number inside each prentices represents the total number of hits to that node plus the number of hits in the subtree rooted at that node for benzoate supplied cells and benzene supplied cells, respectively. For comparison purpose, we normalized the number of counts such that each data set contains 100,000 reads. 6.3.4. Genes transcribed in the presence of benzoate
! ! 132!
The anaerobic mineralization of benzoate has been studied in denitrifying facultative anaerobes such as Thauera aromatica (Breese et al. 1998; Harwood et al. 1999), Magnetospirilum spp. (López Barragán et al. 2004b; Shinoda et al. 2005), and Azoarcus spp. (López Barragán et al. 2004a; Rabus et al. 2005), in the strictly anaerobic iron-reducing Geobacter spp. (Wischgoll et al. 2005; Kung et al. 2009; Aklujkar et al. 2010), in fermentative Syntrophus aciditrophicus (McInerney et al. 2007), and in the photosynthetic Rhodopseudomonas palustris (Egland et al. 1997; Larimer et al. 2003). Figure 6.5 shows the steps and enzymes employed by different bacterial species for degradation of this compound. The initial step is activation of benzoate to benzoyl-CoA by benzoate-CoA ligase, which is coupled to hydrolysis of ATP to AMP and diphosphate. This is a general feature shared by all of the above bacteria (Fuchs 2008; Carmona et al. 2009). Benzoyl-CoA is then reduced and dearomatized to Cyclohex-1,5-diene-1-carbonyl-CoA. In aromatic-degrading facultative anaerobes and phototrophs described so far, this reaction is catalyzed by benzoyl-CoA reductase (BCR), which is an ATP-dependent oxygen sensitive enzyme consisting of four subunits (!"#$) (Boll and Fuchs 1995; Boll et al. 1997). In obligate anaerobes and fermenting bacteria, an ATP- independent membrane protein complex containing eight components BamBCDEFGHI (BamB- I) is proposed to be involved in reduction of benzoyl-CoA (Figure 6.5) (Wischgoll et al. 2005; McInerney et al. 2007; Heintz et al. 2009). Although, facultative, obligate and fermenting bacteria all use the product of benzoyl-CoA reductase as the substrate and generate 3-hydroxy- pimelyl-CoA as the final product via a modified "-oxidation pathway in a similar manner, phototrophic bacteria employ a different pathway (Peters et al. 2007; Kuntze et al. 2008; Carmona et al. 2009). In this pathway the cyclic monoenoyl-CoA produced from reduction of benzoyl-CoA is used as the substrate resulting in the formation of pimelyl-CoA (Egland et al. 1997; Pelletier and Harwood 1998; Harwood et al. 1999; Pelletier and Harwood 2000). Finally degradation of 3-hydroxypimelyl-CoA and pimelyl-CoA via "-oxidation and decarboxylation steps yields 3 molecules of acetyl-CoA, which is channeled into the cell’s central metabolism (López Barragán et al. 2004a).
! ! 133!
- COO COSCoA CoA, ATP AMP, PPi
Benzoate-CoA ligase 1. BzdA, 2. BclA, 3. BamY, Benzoate 4. BamY, 5. BadA Benzoyl-CoA Facultative & Obligate anaerobes & BadDEFG 3. BcrCBAD 2. BzdNOPQ 1. reductase Benzoyl Phototrophic bacteria fermenting bacteria
4. BamB 4. 3. 2ATP, 2H
BamB Dearomatization Benzene ring
2H - CoA CoA 2ADP, 2Pi
- - I? I? Phototrophic bacteria
COSCoA COSCoA COSCoA 2H
Cyclohex-1,5-diene-1-carbonyl-CoA Cyclohex-1-ene-1-carbonyl-CoA 5.
BadK H2O Cyclohexadienoyl-CoA hydratase H2O
1. BzdW, 2. Dch, 3. BamR, 4. BamR, COSCoA COSCoA HO HO
Modified 2-Hydroxycyclohexane- 6-Hydroxycyclohex-1-ene-1-carbonyl-CoA NAD+ 1-carbonyl-CoA Hydroxyenoyl-CoA dehydrogenase 5. +
Bad NAD 1. BzdX, 2. Had, 3. BamQ, 4. BamQ NADH+H+
H +
! NADH+H
- oxidation COSCoA O COSCoA O
6-Ketocxycyclohex-1-ene-1-carbonyl-CoA 2-Ketocyclohexane-1- 2H O carbonyl-CoA Oxoenoyl-CoA hydrolase 2 5.
1.BzdY, 2. Oah, 3. BamA, Bad H2O 4. BamA I
COSCoA COSCoA HO COO- COO-
3-Hydroxypimelyl-CoA Pimelyl-CoA
Lower pathway 3 Acetyl-CoA + CO2 Figure 6.5. Anaerobic pathway of benzoate degradation (Carmona et al, 2009). The name of enzymes catalyzing each step is given in bold below or beside each arrow. The genes encoding the corresponding enzymes for various bacteria are provided either next to or below the arrows in the figure. The numbers next to the name of each gene represent the gene identified in Azoarcus spp. (1), Magnetospirillum spp. and T. aromatica (2), Gobacter spp. (3), S. aciditrophicus (4), and R. plustris ! (5), respectively. ! 134!
The analysis of mRNA sequences from Cartwright Consolidated culture supplied with benzoate showed that all of the genes encoding the necessary enzymes for conversion of benzoate to 3-hydroxypimelyl-CoA through benzoyl-CoA pathway were transcribed (Appendix H provides the gene sequences obtained in this study). List of these genes and those present in other anaerobes are provided in Table 6.3. The sequences corresponding to benzoate-CoA ligase, benzoyl-CoA reductase, enoyl-CoA hydratase, hydroxyenoyl-CoA dehydrogenase, and oxoenoyl-CoA hydrolase in Cartwright Consolidate were more closely related to those found in denitrifying Azoarcus spp. than other bacteria (Table H.3, Appendix H). Bam or bad-like genes of strictly anaerobic Geobacter spp., fermenting Synrophus aciditrophicus, and phototrophic Rhodopseudomonas palustris were not identified in the culture. Transcripts of several other genes that are important in the uptake and mineralization of benzoate were also present in our benzoate-grown cells (Table H.3). Table 6.4 lists them and their possible functions. One of these genes annotated as bzdB encodes for an ABC-type transporter system. In Azoarcus spp., bzdB is located in a cluster of genes for anaerobic benzoate degradation and therefore, it is proposed to be involved in the uptake of benzoate (Rabus et al. 2005; Carmona et al. 2009). We also identified transcript of a ferredoxin gene (fdx/bzdM). Ferredoxin has been considered as the primary electron donor of benzoyl-CoA reductase (López Barragán et al. 2004a; Rabus et al. 2005). Upon exposure to benzoate, cells in Cartwright Consolidated transcribed KorABC and bzdV-like genes for a three subunit 2-oxoglutarate:ferredoxin oxidoreductase and a NADPH:acceptor (ferredoxin) oxidoreductase, respectively. In Azoarcus evansii, Azoarcus strain CIB, and Azoarcus EbN1 (Aromatoleum aromaticum EbN1) these two enzymes have been shown to belong to a system that regenerates ferredoxin (fdx/bzdM) (Ebenau-Jehle et al. 2003; López Barragán et al. 2004a; Rabus et al. 2005). Ferredoxin is reduced by the combined action of these two enzymes. A gene similar to the bzdR of Azoarcus strain CIB was identified in our culture. López Barragán et al. (2004a) showed that the product of bzdR negatively regulates the expression of the catabolic operon of benzoate degradation (bzd operon). It represses the PN promoter that controls the bzd operon in response to the presence of first intermediate of benzoate mineralization i.e. benzoyl-CoA (Carmona et al. 2009). Other mRNA sequences that were transcribed in Cartwright Consolidated were related to bzdU, bzdS, bzdT, and bzdZ of Azoarcus microorganisms. Although they are identified as part of benzoate
! ! 135!
degradation gene cluster, no specific functions have been assigned to them so far (López Barragán et al. 2004a).
Table 6.3. List of the genes encoding the enzymes necessary for degradation of benzoate to 3-hydroxypimelyl-CoA/Pimelyl-CoA that were expressed in Cartwright Consolidated culture and those identified in other benzoate-degrading anaerobes. Gene name Strict anaerobes/ Phototrophic Our Culture Denitrifying bacteria fermenting Enzyme name bacteria bacteria
Cartwright Azoarcus Thauera aromatica/ Rhodopseudomonas Geobacter spp./ Consolidated spp.a Magnetospirillum palustris Syntrophus spp. aciditrophicus Benzoate-CoA ligase BzdA/BclA BzdA/BclA BclA BadA BadA Benzoyl-CoA reductase !-subunit BzdQ/BcrA BzdQ/BcrA BcrA BadF "-subunit BzdO/BcrB BzdO/BcrB BcrB BadE BamBCDEFGHI #-subunit BzdN/BcrC BzdN/BcrC BcrC BadD $-subunit BzdP/BcrD BzdP/BcrD BcrD BadB
Enoyl-CoA hydratase BzdW/Dch BzdW/Dch Dch BadK BamR Hdroxyenoyl-CoA dehydrogenase BzdX/Had BzdX/Had Had BadH BamQ Oxoenoyl-CoA hydrolase BzdY/Oah BzdY/Oah Oah BadI BamA a Rabus et al. (2005) anotated the genes for benzoate degradtion pathway of Azoarcus EbN1 (Aromatoleum aromaticum EbN1) the same as those of Thauera aromatica. However, these genes are actually more similar to the genes from other Azoarcus species than to the ones for Thauera aromatica.
! ! 136!
Table 6.4. List of several other genes that were expressed in Cartwright Consolidated culture during growth on benzoate and their possible functions in benzoate mineralization.
Gene Name Gene Product Function BzdB Putative ABC transporter subunit Benzoate uptake
Fdx/BzdM Ferredoxin Primary electron donor of benzoyl-CoA reductase KorA 2-oxoglutarate:ferredoxin oxidoreductase, !-subunit Providing electrons for ferredoxin
KorB 2-oxoglutarate:ferredoxin oxidoreductase, "-subunit Providing electrons for ferredoxin KorC 2-oxoglutarate:ferredoxin oxidoreductase, #-subunit Providing electrons for ferredoxin
BzdV NADPH:acceptor (ferredoxin) oxidoreductase Providing electrons for ferredoxin BzdR Anaerobic benzoate transcriptional regulator Controlling the inducible expression of the bzd catabolic operon involved in anaerobic benzoate degradation BzdS Hypothetical protein Unknown BzdT Transcriptional regulator Unknown BzdU Hypothetical protein Unknown BzdZ Putative dehydrogenase Unknown
! ! 137!
The transcription of anaerobic benzoate degradation genes similar to those of nitrate- reducing Azoarcus species suggests that the Cartwright Consolidated culture uses a pathway for the activation, dearomatization, and modified !-oxidation of benzoate, analogous to the pathway employed by denitrifying bacteria.
Table H.4 of Appendix H provides a through analysis of all other mRNA sequences identified in RNA sample extracted from the cells grown on benzoate.
6.3.5. Genes transcribed during growth of culture on benzene
Analysis of the mRNA data from the Benzene-amended culture indicated the presence of several transcripts that may play important roles in the oxidation of benzene (refer to Appendix H for corresponding nucleotide sequences). Table 6.5 contains the list of these sequences and their closest NCBI relatives based on BLASTX results. Two of the transcribed genes were analogous to AbcA and AbcD, two subunits of a putative benzene carboxylase enzyme called AbcDA, which was recently identified in a strictly anaerobic benzene-degrading iron-reducing enrichment culture BF by Abu Laban et al. (2010) as well as in a pure culture of Ferroglobus placidus (Holmes et al. 2011). The protein products of AbcA and AbcD have 43% and 37% sequence identity to " and # subunits of a phenylphosphate carboxylase (ppcA and ppcD), respectively (Abu Laban et al. 2010). Phenylphosphate carboxylase, which catalyzes the carboxylation of phenylphosphate to 4-hydroxybenzoate, is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds. This enzyme uses CO2 as a substrate, requires K+ and a divalent metal cation (Mg2+ or Mn+2) for its activity, does not contain biotin or thiamine diphosphate, and is inhibited by oxygen (Schühle and Fuchs 2004). Phenylphosphate carboxylase consists of four subunits (ppcABCD). PpcA and ppcB, the " and ! subunits of enzyme, are similar to UbiD aryl decarboxylase which is involved in decarboxylation of 3-octaprenyl-4-hydroxybenzoate to 3-octaprenylphenol during ubiquinone biosynthesis (Schühle and Fuchs 2004). PpcC ($ subunit) belongs to phosphatase protein family. PpcD (# subunit) is a hypothetical protein. The role of $ subunit of phenylphosphate carboxylase is dephosphorylation of phenylphosphate and at the same time ensuring trapping of the released phenolate in a reactive form bound to the core carboxylase enzyme (Schühle and Fuchs 2004). The ", !, and # subunits form the core carboxylase enzyme and catalyze the reversible
! ! 138! carboxylation of the phenolate intermediate. The proteomics experiments conducted by Abu Laban and colleagues showed the specific expression of AbcA and AbcD in benzene-grown cells versus phenol- and benzoate-grown cells. Hence, it was suggested that the AbcDA was responsible for the initial activation and direct carboxylation of benzene to benzoate (Abu Laban et al. 2010). We also found two other transcribed sequences in our culture that displayed 100% and 94% identity to the gene products of ORFs 126 and 133 of the aforementioned iron-reducing consortium, respectively (Table 6.5). These two ORFs were specifically expressed during anaerobic benzene degradation in culture BF and were hypothesized to encode for other potential subunits of benzene carboxylase. An UbiD/UbiX-like carboxylase (3-octaprenyl-4- hydroxybenzoate carboxylase) gene similar to that encoded by ORF124 of culture BF was transcribed in the Cartwright Consolidated culture (the preliminary analysis of Illumina sequences of another RNA sample from Cartwright Consolidated). It should be noted here that all of these carboxylase genes were transcribed only in cells grown with benzene and not in those fed with benzoate. Therefore, these results provide further support for the carboxylation of benzene as the initial step in benzene mineralization. This reaction is probably catalyzed by a multi-subunit carboxylase enzyme, which is encoded by the genes analogous to AbcA, AbcD, ORF126, ORF 133 and ORF 124 of culture BF.
In addition to the sequences encoding for a putative carboxylase enzyme, several genes corresponding to the enzymes of the benzoyl-CoA pathway were identified in the benzene- amended Cartwright Consolidated culture (Table 6.5). Two of the transcribed sequences were similar to BamD and BamE, which are the two subunits of a putative benzene ring dearomatizing enzyme in strictly anaerobic and fermenting bacteria (Figure 6.5). BamB another subunit of this enzyme as well as mRNA sequences encoding benzoyl-CoA reductase enzyme of dentitrifying Azoarcus species were detected in the Illumina data. As shown in Table 6.5, another gene that was transcribed during growth of cells on benzene was a dienoyl-CoA hydratase (Dch/BzdW), similar to that of Azoarcus spp. BzdX and BzdY-like sequences encoding for hydroxyacyl-CoA dehydrogenase and ring-opening hydrolase of Azoarcus bacteria were present in Illumina data. Based on the results of this study, after benzene is converted to benzoate, further metabolism of benzoate proceeds via the benzoyl-CoA pathway.
! ! 139!
Table 6.5. Aromatic compound degradation genes that were specifically expressed in Cartwright Consolidated culture in the presence of benzene as the sole electron donor and carbon source. ! NCBI closest gene products (Results of BlastX) Sequence ID Size Gene name Product Function Organism NCBI Accession# %Identity E-value (bp) GMRHGY404IQFR7 511 AbcA Putative anaerobic benzene Direct Clostridia gb|ADJ94002.1| 99 6.00E-88 carboxylase carboxylation of bacterium benzene to enrichment culture benzoate clone BF GMRHGY404JC5OM 355 AbcA Putative anaerobic benzene Direct Clostridia gb|ADJ94002.1| 96 3.00E-60 carboxylase carboxylation of bacterium benzene to enrichment culture benzoate clone BF GMRHGY404IXLZN 557 AbcD Putative anaerobic benzene Direct Clostridia gb|ADJ94001.1| 93 3.00E-63 carboxylase carboxylation of bacterium benzene to enrichment culture benzoate clone BF GMRHGY404IQ0MN 483 AbcD Putative anaerobic benzene Direct Clostridia gb|ADJ94001.1| 96 4.00E-65 carboxylase carboxylation of bacterium benzene to enrichment culture benzoate clone BF GMRHGY404I47SF 296 - Hypothetical protein Direct Clostridia gb|ADJ93997.1| 94 1.00E-22 carboxylation of bacterium benzene to enrichment culture benzoate clone BF Isotig00071 780 - Hypothetical protein Direct Clostridia gb|ADJ93990.1| 100 E-150 carboxylation of bacterium benzene to enrichment culture benzoate clone BF GMRHGY404IZ92L 573 BamD Putative benzoate-degrading A subunit of a Clostridia gb|ADJ94008.1| 85 4.00E-68 protein putative benzoyl- bacterium CoA reductase enrichment culture clone BF GMRHGY404IBD8N 524 BamE Putative benzoate-degrading A subunit of a Clostridia gb|ADJ93918.1| 74 5.00E-68 protein putative benzoyl- bacterium CoA reductase enrichment culture clone BF GMRHGY404I8T7K 499 Dch/BzdW Dienoyl-CoA hydratase Acyl-CoA Aromatoleum ref|YP_160034.1| 67 2.00E-52 GMRHGY404HZ0K7 hydratase aromaticum EbN1 (Azoarcus EbN1)
! ! 140!
One of the proposed mechanisms for the activation of benzene under anaerobic conditions is direct hydroxylation of benzene to phenol (Vogel and Grbi!-Gali! 1986; Weiner and Lovley 1998; Caldwell and Suflita 2000; Chakraborty and Coates 2005). Phenol is then converted to benzoyl-CoA via several reactions catalyzed by phenylphosphate synthase, phenylphosphate carboxylase, 4-hydroxybenzoate-CoA ligase and 4-hydroxybenzoyl-CoA reductase (Figure 6.1) (Breinig et al. 2000; Rabus et al. 2005; Schleinitz et al. 2008). If the initial step of benzene mineralization in our culture were a hydroxylation process resulting in phenol formation, we would have expected to observe the transcription of the genes involved in phenol degradation. However, none of the genes responsible for the metabolism of phenol (such as phenylphosphate synthase, 4-hydroxybenzoate-CoA ligase and 4-hydroxybenzoyl-CoA reductase) were transcribed in the cells grown with benzene. This result suggests that benzene activation does not occur through a hydroxylation step in our culture.
! !Methylation of benzene to toluene is another process that has been hypothesized as the first step in benzene mineralization (Coates et al. 2002; Ulrich et al. 2005)"!Subsequent degradation of toluene to benzoyl-CoA proceeds via addition of the methyl group of toluene onto fumarate catalyzed by benzylsuccinate synthase followed by !-oxidation pathway!(Biegert et al. 1996; Beller and Spormann 1997a; Beller and Spormann 1997b; Coschigano et al. 1998; Leuthner et al. 1998; Leuthner and Heider 2000; Achong et al. 2001; Leutwein and Heider 2001; Leutwein and Heider 2002; Chakraborty and Coates 2004; Rabus et al. 2005; Selmer et al. 2005)"!The enzymes involved in catalyzing different steps of the !-oxidation pathway are succinyl- CoA:benzylsuccinate-CoA transferase, (R)-benzylsuccinyl-CoA dehydrogenase, phenyl- itaconyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, and benzoylsuccinyl-CoA thiolase (Figure 6.1). None of the mRNA sequences obtained from the Cartwright consolidated culture during growth on benzene encodes the enzymes for the toluene degradation pathway. This eliminates the possibility of benzene activation through a methylation process in this culture.
Finally, several studies propose carboxylation of benzene to benzoate as the activation mechanism of benzene ring in the absence of oxygen (Caldwell and Suflita 2000; Phelps et al. 2001; Kunapuli et al. 2008; Musat and Widdel 2008; Abu Laban et al. 2009; Abu Laban et al. 2010). Benzoate-CoA ligase further converts benzoate to benzoyl-CoA that is in turn mineralized by the well-established benzoyl-CoA pathway (Figure 6.1). For carboxylation to be the first
! ! 141! reaction in anaerobic oxidation of benzene, one would expect the transcription of benzoyl-CoA pathway genes as well as genes encoding for a carboxylase enzyme. The caboxylase enzyme should be transcribed or upregulated in the presence of benzene. We identified several genes that encode for different subunits of a carboxylase enzyme specifically transcribed in cells supplied with benzene. Therefore, it is proposed that direct carboxylation of benzene to benzoate is the initial step in anaerobic degradation of benzene in Cartwright Consolidated culture.
A complete analysis of all of the other mRNA sequences obtained from the benzene-supplied cells is provided in Table H.5 of Appendix H.
6.3.6. A complex interaction between different bacteria in Cartwright Consolidated culture facilitates benzene degradation
As it was explained earlier, transcripts of carboxylase-related genes were detected in Cartwright Consolidated culture during its growth on benzene (Figure 6.6). These genes encoded a putative benzene carboxylase, which is proposed to mediate carboxylation of benzene and is analogous to that identified in a strict iron-reducing culture BF. In BF culture, the carboxylase enzyme was from a Gram-positive Peptococcaceae-related microorganism (Abu Laban et al. 2010). Cartwright Consolidated also contains Peptococcaceae as one of its community members. The analysis of rRNA data showed that the Peptococcaceae sequences increased 10 to 100 times by changing the substrate from benzoate to benzene (Table 6.6). These results collectively suggest that Peptococcaceae-related bacteria rely on benzene as the source of energy and are responsible for the initial attack on benzene and its activation to benzoate by the putative benzene carboxylase. While direct carboxylation of benzene to benzoate may occur, it is also possible that several transient intermediates are formed prior to formation of benzoate. Benzoate is further mineralized through benzoyl-CoA degradation pathway. Several mRNA sequences were identified in Cartwright Consolidated corresponding to benzoyl-CoA pathway enzymes. The genes encoding for benzene ring dearomatization enzyme that were transcribed in our culture had similarity either to those identified in iron-reducing culture BF (Bam-like) or to benzoyl-CoA reductase genes of Azoarcus species (Bcr/Bzd-like) (Figure 6.6). The transcribed sequences involved in modified !-oxidation of the benzoyl-CoA pathway were related to those of denitrifying Azoarcus bacteria (BzdW, BzdX, and BzdY). According to these findings, a syntrophic association is proposed in Cartwright Consolidated culture for mineralization of
! ! 142! benzene. In this syntrophic relationship, the Peptococcaceae-related microorganisms activate benzene using the carboxylase enzyme and convert it to one of the benzoyl-CoA pathway products (Cyclohex-1,5-diene-1carbonyl-CoA), which is further metabolized to 3- Hydroxypimelyl-CoA by nitrate-reducing Azoarcus spp. It is also possible that Peptococcaceae releases some of the benzoate obtained from benzene into the culture medium, which can then be utilized by nitrate-reducing bacteria. This will explain the presence of benzoyl-CoA reductase gene transcripts related to those of Azoarcus spp. when cells grew on benzene. One more possibility to consider is that the Peptococcaceae activates benzene and converts it to low molecular fermentation products that can be used by other members of the culture. However, the transcribed genes encoding for enzymes catalyzing conversion of Cyclohex-1,5-diene-1carbonyl- CoA to 3-hydroxy-pimelyl-CoA were similar to those of denitrifying Azoarcus microorganisms. Peptococcaceae might have acquired these genes from Azoarcus through horizontal gene transfer during several years of culture enrichment.
During growth of cells on benzoate all of the gene transcripts involved in benzoate degradation were from nitrate-reducing Azoarcus spp. It is possible that Azoarcus bacteria outcompete the Peptococcaceae-related microorganisms using benzoate as the growth substrate. Another explanation would be that Peptococcaceae bacteria might lack the enzymes required for active intake of benzoate; therefore the pathway for benzoate degradation in these microorganisms would not be stimulated in the presence of benzoate.
! ! 143!
COO-
CO2
Putative benzene carboxylase ABcDA Benzene Benzoate
Benzoate-CoA ligase BzdA/BclA
COSCoA
Benzoyl-CoA
BamB BamD BzdQ/BcrA Benzoyl-CoA reductase BzdO/BcrB BamE BzdN/BcrC BzdO/BcrB BzdP/BcrD BzdN/BcrC BzdP/BcrD
Cyclohex-1,5-diene-1-carbonyl-CoA
Cyclohexadienoyl-CoA hydratase BzdW/Dch BzdW/Dch
6-Hydroxycyclohex-1-ene-1-carbonyl-CoA
Hydroxyenoyl-CoA dehydrogenase BzdX/Had BzdX/Had
6-Ketocyclohex-1ene-1-carbonyl-CoA
Oxoenoyl-CoA hydrolase BzdY/Oah BzdY/Oah
3-Hydroxy pimelyl-CoA Figure 6.6. Genes that were transcribed in the cells grown on benzoate (red) and the ones grown on benzene (blue).
Table 6.6. Peptococcaceae number of small and large subunit ribosomal RNA sequences in the cells amended with benzene and the ones supplied with benzoate. ! Substrate Number of small subunit Number of large subunit ribosomal RNA sequences ribosomal RNA sequences Benzene 3849 418 Benzoate 27 60
! ! 144!
6.4. Conclusions
In summary, analysis of transcripts from cells grown on benzene or benzoate resulted in the identification of carboxylase genes that were specifically transcribed in the presence of benzene. These genes are subunits of a putative benzene carboxylase enzyme that activates benzene through a carboxylation reaction producing benzoate. It is also plausible that several transient intermediates are formed prior to formation of benzoate. Benzoate is then mineralized via benzoyl-CoA biodegradation pathway. In the Cartwright Consolidated culture, initial attack on benzene was carried out by members of Peptococcaceae family. These microorganisms degraded benzene to benzoate or to one of the benzoyl-CoA pathway metabolites, which was then consumed by other bacteria, such as Azoarcus present in the culture.
6.5. Recommendations for future work
It is essential to study the metagenome of Cartwright Consolidated culture. This will allow to map the genes that are transcribed during benzene or benzoate degradation to the genome of different bacteria and obtain further information regarding the arrangement of the corresponding genes on the genome.
Enzyme assay experiments and purification of the putative benzene carboxylase can provide us with a better understanding of the actual structure and function of this enzyme. Although this enzyme might be involved in direct carboxylation of benzene to benzoate, one should also consider the possibility of formation of several transient intermediates prior to benzoate formation.
Benzene-degrading nitrate-reducing enriched cultures should be tested for their ability to degrade other potential metabolites of benzene degradation such as toluene and phenol. A similar meta-transcriptomic approach should be employed to identify the genes that are differentially expressed when culture is supplied with benzene, toluene or phenol.
Another approach for identifying the genes involve in mineralization of benzene would be to compare the proteomes of benzene-, phenol-, and benzoate-grown cells.
! !
6.6. References Chapter 6
Abu Laban, N., D. Selesi, C. Jobelius and R. U. Meckenstock (2009). "Anaerobic benzene degradation by gram-positive sulfate-reducing bacteria." FEMS Microbiol. Ecol. 68: 300-311.
Abu Laban, N., D. Selesi, T. Rattei, P. Tischler and R. U. Meckenstock (2010). "Identification of enzymes involved in anaerobic benzene degradation by strictly anaerobic iron-reducing enrichment culture." Environ. Microbiol. 12(10): 2783-2796.
Achong, G. R., A. M. Rodriguez and A. M. Spormann (2001). "Benzylsuccinate synthase of Azoarcus sp. strain T: cloning, sequencing, transcriptional organization, and its role in anaerobic toluene and m-xylene mineralization." J. Bacteriol. 183: 6763-6770.
Aklujkar, M., N. D. Young, D. Holmes, M. Chavan, C. Risso, H. E. Kiss, C. S. Han, M. L. Land and D. R. Lovley (2010). "The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments." BMC Genomics 11: 490.
Beller, H. R., D. Grbi!-Gali! and M. Reinhard (1992). "Microbial degradation of toluene under sulfate-reducing conditions and the influence of iron on the process." Appl. Environ. Microbiol. 58: 786-793.
Beller, H. R. and A. M. Spormann (1997a). "Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T." J. Bacteriol. 179: 670-676.
Beller, H. R. and A. M. Spormann (1997b). "Benzyl-succinate formation as a means of anaerobic toluene activation by sulfate-reducing strain PRTOL1." Appl. Environ. Microbiol. 63: 3729- 3731.
Biegert, T., G. Fuchs and J. Heider (1996). "Evidence that anaerobic oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate." Eur. J. Biochem 238: 661-668.
Boll, M., S. S. Albracht and G. Fuchs (1997). "Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. A study of adenosinetriphosphatase activity, ATP stoichiometry of the reaction and EPR properties of the enzyme." Eur. J. Biochem 244(3): 840- 851.
Boll, M. and G. Fuchs (1995). "Benzoyl-coenzyme A reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172." Eur. J. Biochem 234: 921-933.
Breese, K., M. Boll, J. Alt-Mörbe, H. Schägger and G. Fuchs (1998). "Genes coding for the benzoyl-CoA pathway of anaerobic aromatic metabolism in the bacterium Thauera aromatica." Eur. J. Biochem 256(1): 148-154.
! 145! ! ! 146!
Breinig, S., E. Schiltz and G. Fughs (2000). "Genes involved in anaerobic metabolism of phenol in the bacterium Thauera aromatica." J. Bacteriol. 182: 5849-5863.
Burland, S. M. and E. A. Edwards (1999). "Anaerobic benzene biodegradation linked to nitrate reduction." Appl. Environ. Microbiol. 65(2): 529-533.
Caldwell, M. E. and J. M. Suflita (2000). "Detection of phenol and benzoate as intermediates of anaerobic benzene biodegradation under different terminal electron accepting condition." Environ. Sci. Technol. 34: 1216-1220.
Carmona, M., M. T. Zamarro, B. Blázquez, G. Durante-Rodríguez, J. F. Juárez, J. A. Valderrama, M. J. Barragán, J. L. García and E. Díaz (2009). "Anaerobic catabolism of aromatic compounds: a genetic and genomic view." Microbiol. Mol. Biol. Rev. 73(1).
Chakraborty, R. and J. D. Coates (2004). "Anaerobic degradation of monoaromatic hydrocarbons." Appl. Microbiol. Biotechnol. 64: 437-446.
Chakraborty, R. and J. D. Coates (2005). "Hydroxylation and carboxylation-two crucial steps of anaerobic benzene degradation by Dechloromonas Strain RCB." Appl. Environ. Microbiol. 71(9): 5427-5432.
Coates, J. D., R. Chakraborty and M. J. McInerney (2002). "Anaerobic benzene biodegradation-a new era." Res. Microbio. 153: 621-628.
Coschigano, P. W., T. S. Wehrman and L. Y. Young (1998). "Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: Putative role of a glycine free radical." Appl. Environ. Microbiol. 64: 1650-1656.
Da Silva, M. L. B. and P. J. J. Alvarez (2004). "Enhanced Anaerobic biodegradation of benzene-, toluene-, ethylbenzene-, xylene-ethanol mixtures in bioaugmented aquifer columns." Appl. Environ Microbiol 70: 4720-4726.
Ebenau-Jehle, C., M. Boll and G. Fuch (2003). "2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction." J. Bacteriol. 185: 6119-6129.
Edwards, E. A. and D. Grbi!-Gali! (1992). "Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions." Appl. Environ. Microbiol. 58: 2663-2666.
Edwards, E. A., L. E. Wills, M. Reinhard and D. Grbi!-Gali! (1992). "Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions." Appl. Environ. Microbiol. 58(794-800).
Egland, P. G., D. A. Pelletier, M. Dispensa, J. Gibson and C. S. Harwood (1997). "A cluster of bacterial genes for anaerobic benzene ring biodegradation." Proc. Natl. Acad. Sci. USA.
! ! ! 147!
Evans, P. J., D. T. Mang, K. S. Kim and L. Y. Young (1991a). "Anaerobic degradation of toluene by denitrifying bacterium." Appl. Environ. Microbiol. 57: 1139-1145.
Evans, P. J., D. T. Mang and L. Y. Young (1991b). "Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures." Appl. Environ. Microbiol. 57: 450-454.
Fuchs, G. (2008). "Anaerobic metabolism of anaerobic compounds." Ann. N. Y. Acad. Sci. 1125: 82-99.
Grbi!-Gali!, D. and T. M. Vogel (1987). "Transformation of toluene and benzene by mixed methanogenic cultures." Appl. Environ. Microbiol. 53: 254-260.
Gulensoy, N. and P. J. J. Alvarez (1999). "Diversity and correlation of specific aromatic hydrocarbon biodegradation capabilities." Biodegradation 10: 331-340.
Harwood, C. S., G. Burchhardt, H. Herrmann and G. Fuchs (1999). "Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway." FEMS Microbiol. Rev. 22: 439-458.
Heintz, D., S. Gallien, S. Wischgoll, A. K. Ullmann, C. Schaeffer, A. K. Kretzschmar, A. V. Dorsselaer and M. Boll (2009). "Differential membrane proteome analysis reveals novel proteins involved in the degradation of aromatic compounds in Geobacter metallireducens." Mol. Cell Proteomics 8(9): 2159-2169.
Holmes, D. E., C. Risso, J. A. Smith and D. R. Loveley (2011). "Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus." Appl. Environ. Microbiol. 77(17): 5926-5933.
Huson, D. H., A. Auch, J. Qi and S. C. Schuster (2007). "MEGAN analysis of metagenome data " Genome Research 17: 377-386.
Huson, D. H., S. Mitra, H. J. Ruscheweyh, N. Weber and S. C. Schuster (2011). "Integrative analysis of environmental sequences using MEGAN 4." submitted.
Kasai, Y., Y. Kodama, Y. Takahata, T. Hoaki and K. Watanabe (2007). "Degradative capacities and bioaugmentation potential of anaerobic benzene-degrading bacterium strain DN11." Environ. Sci. Technol. 41: 6222-6227.
Kleinsteuber, S., K. M. Schleinitz, J. Breitfeld, H. Harms, H. H. Richnow and C. Vogt (2008). "Molecular charecterization of bacterial communities mineralizing benzene under sulfate- reducing conditions." FEMS Microbiol. Ecol. 66: 143-157.
Kunapuli, U., C. Griebler, H. Beller and R. U. Meckenstock (2008). "Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture." Environ. Microbiol. 10: 1703-1712.
! ! ! 148!
Kung, J. W., C. Löffler, K. Dörner, D. Heintz, A. V. D. S. Galliend, T. Friedrich and M. Boll (2009). "Identification and characterization of the tungsten-containing class of benzoyl- coenzyme A reductases." Proc. Natl. Acad. Sci. USA 106(42): 17687-17692.
Kuntze, K., Y. Shinoda, H. Moutakki, M. J. McInerney, C. Vogt, H. H. Richnow and M. Boll (2008). "6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes." Environ. Microbiol. 10(6): 1547-1556.
Larimer, W. F., P. Chain, L. Hauser, J. Lamerdin, S. Malfatti, L. Do, M. L. Land, D. A. Pelletier, J. T. Beatty, A. S. Lang, F. R. Tabita, J. L. Gibson, T. E. Hanson, C. Bobst, J. L. T. y. Torres, C. Peres, F. H. Harrison, J. Gibson and C. S. Harwood (2003). "Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris." Nature Biotechnology 22: 55-61.
Leuthner, B. and J. Heider (2000). "Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of betaoxidation of the intermediate benzyl suucinate." J. Bacteriol. 182: 272-277.
Leuthner, B., C. Leutwein, H. Schulz, P. Horth, W. Haehnel, E. Schiltz, H. Schagger and J. Heider (1998). "Biochemical and genetic charecterization of benzylsuccinatesynthasefrom Thauera aromatica: a new glycyl radical enzyme catalyzng the first step in anaerobic toluene metabolism." Mol. Microbiol. 28: 615-628.
Leutwein, C. and J. Heider (2001). "Succinyl-CoA:(R)-benzylsuccinate CoA trasferase: an enzyme of the anaerobic toluene catabolic pathway in denitrifying bacteria." J. Bacteriol. 183: 4288-4295.
Leutwein, C. and J. Heider (2002). "(R)-Benzylsuccinyl-CoA dehydrogenase of Thauera aromatica, an enzyme of the anaerobic toluene catabolic pathway." Arch. Microbiol. 178: 517- 524.
López Barragán, M. J., M. Carmona, M. T. Zamarro, B. Thiele, M. Boll, G. Fuchs, J. L. García and E. Díaz (2004a). "The bzd gene cluster, coding for anaerobic benzoate catabolism, in Azoarcus sp. Strain CIB." J. of Bacteriol. 186: 5762-5774.
López Barragán, M. J., E. Díaz, J. L. García and M. Carmona (2004b). "Genetic clues on the evolution of anaerobic catabolism of aromatic compounds." Microbiol. 150: 2018-2021.
McInerney, M. J., L. Rohlin, H. Mouttaki, U. Kim, R. S. Krupp, L. Rios-Hernandez, J. Sieber, C. G. Struchtemeyer, A. Bhattacharyya, J. W. Campbell and R. P. Gunsalus (2007). "The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth." Proc. Natl. Acad. Sci. USA 104: 7600-7605.
! ! ! 149!
Meckenstock, R. U., R. I. Warthmann and W. Schafer (2004). "Inhibition of anaerobic microbial o-xylene degradation by toluene in sulfidogenic sediment columns and pure cultures." FEMS Microbiol. Ecol. 47: 381-386.
Musat, F. and F. Widdel (2008). "Anaerobic degradation of benzene by a marine sulfate- reducing enrichment culture, and cell hybridization of the dominant phylotype." Environ. Microbiol. 10(1): 10-19.
Nales, M., B. J. Butler and E. Edwards (1998). "Anaerobic benzene biodegradation: a microcosm survey." Bioremediat. J. 2: 125-144.
Nandi, M. (2006). Biochemical and molecular charecterization of anaerobic benzene-degrdading cultures. Toronto, University of Toronto. M.A.Sc thesis.
Pelletier, D. A. and C. H. Harwood (2000). "2-Hydroxycyclohexanecarbonyl coenzyme A dehydrogenase, an enzyme charecteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris." J. Bacteriol. 182: 2753-2760.
Pelletier, D. A. and C. S. Harwood (1998). "2-ketocyclohexanecarbonyl coenzyme A hydrolase, the ring cleavage enzyme required for anaerobic benzoate degradation by Rhodopseudomonas palutris." J. Bacteriol. 180: 2330-2336.
Peters, F., Y. Shinoda, M. J. McInerney and M. Boll (2007). "Cyclohexa-1,5-Diene-1-Carbonyl- Coenzyme A (CoA) Hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a Common Benzoyl-CoA degradation pathway in facultative and strict anaerobes." J. Bacteriol. 189(3): 1055-1060.
Phelps, C. D. and L. Y. Young (1999). "Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments." Biodegradation 10: 15-25.
Phelps, C. D., X. Zhang and L. Y. Young (2001). "Use of stable isotopes to identify benzoate as a metabolite of benzene degradation in a sulphidogenic consortium." Environ. Microbiol. 3: 600- 603.
Rabus, R., M. Kube, J. Heider, A. Beck, K. Heitmann, F. Widdel and R. Reinhardt (2005). "The genome sequence of an anaerobic-degrading denitrifying bacterium, strain EbN1." Arch. Microbiol. 183: 27-36.
Salinero, K. K., K. Keller, W. S. Feil, H. Feil, S. Trong, G. D. Bartolo and A. Lapidus (2009). "Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation " BMC Genomics 10: 351-74.
Schleinitz, K. M., S. Schmeling, N. Jehmlich, M. v. Bergen, H. Harms, S. Kleinsteuber, C. Vogt and G. Fuchs (2008). "Phenol degradation in the strictly anaerobic iron-reducing bacterium Geobacter metallireducens GS-15." Appl. Environ. Microbiol. 75: 3912-3919.
! ! ! 150!
Sch"hle, K., Fuchs, G. (2004). "Phenylphosphate carboxylase: a new C-C lyase involved in anaerobic phenol metabolism in Thauera aromatica." J. Bacteriol. 186(14): 4556-4567.
Selmer, T., A. J. Pierik and J. Heider (2005). "New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria." Biol. Chem. 386: 981-988.
Shinoda, Y., J. Akagi, Y. Uchihashi, A. Hiraishi, H. Yukawa, H. Yurimoto, Y. Sakai and N. Kato (2005). "Anaerobic degradation of aromatic compounds by Magnetospirillum strains: Isolation and degradation genes." Biotechnol. Biochem. 69: 1483-1491.
Siddique, T., P. M. Fedorak, M. MacKinnom and J. M. Foght (2007). "Metabolism of BTEX and naphta compounds to methane in oil sands tailings." Environ. Sci. Technol. 41: 2350-2356.
Ulrich, A. C., H. R. Beller and E. A. Edwards (2005). "Metabolites detected during 13 biodegradation of C6-benzene in nitrate-reducing and methanogenic enrichment cultures." Environ. Sci. Technol 39: 6681-6691.
Ulrich, A. C. and E. A. Edwards (2003). "Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures." Environ. Microbiol. 5(2): 92-102.
Urich, T., A. Lanze´n, J. Qi, D. H. Huson, C. Schleper and S. C. Schuster (2008). "Simultaneous assessment of soil microbial community structure and function through analysis of the meta- transcriptome " PLoS One 3(6): e2527.
Vogel, T. M. and D. Grbi!-Gali! (1986). "Incorporation of Oxygen from Water into Toluene and Benzene During Anaerobic Fermentative Transformation." Appl. Environ. Microbiol. 52: 200- 202.
Waller, A. S., R. Krajmalnik-Brown, F. E. Lo¨ffler and E. A. Edwards (2005). "Multiple reductive-dehalogenase-homologous genes are simultaneously transcribed during dechlorination by Dehalococcoides-containing cultures." Appl. Environ. Microbiol. 71(12): 8257-8264.
Weiner, J. M. and D. R. Lovley (1998). "Rapid Benzene Degradation in Methanogenic Sediments from a Petroleum-Contaminated Aquifer." Appl. Environ. Microbiol. 64: 1937-1939.
Wischgoll, S., D. Heintz, F. Peters, A. Erxleben, E. Sarnighausen, R. Reski, A. V. Dorsselaer and M. Boll (2005). "Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens." Mol. Microbiol. 58(5): 1238-1252.
! !
Chapter 7: General Discussion and Synthesis
! 151! ! 152!
7.1. Optimizing growth of benzene-degrading nitrate-reducing cultures
Similar to many other aromatic hydrocarbons, benzene is a common environmental contaminant and its chronic exposure negatively impacts human and ecosystem health. Among existing methods for the treatment of contaminated sites, in situ bioremediation that involves transforming hazardous materials into benign products by microorganisms is a potentially efficient and cost-effective approach. A variety of parameters such as concentration and bioavailability of contaminants or other essential nutrients, pH, redox, presence/absence of key co-substrates, and presence of toxins can affect microbial growth and the efficiency of bioremediation (Atlas 1981; Chapelle 1999). Identification of factors that hinder or facilitate degradation of benzene and microbial growth is of great importance to bioremediation of contaminated sites. One of the objectives of the current study was to identify these factors.
7.1.1. Inhibitory impact of nitrite on mineralization of benzene
Benzene-degrading nitrate-reducing cultures maintained in our laboratory coupled benzene mineralization to incomplete reduction of nitrate to nitrite. As a result, nitrite accumulated either transiently or permanently in the cultures. Accumulation of this compound had a negative impact on mineralization of benzene. A possible explanation for the loss of benzene degradation capacity is toxicity of nitrite to the primary benzene degrader (Peptococcaceae) or to its syntrophic partners such as denitrifying Azoarcus and Dechloromonas species. Nitrite has an inhibitory effect on the growth and activity of sulfate-reducing, denitrifying, and syntrophic bacteria, and methanogens (Williams et al. 1978; Klüber and Conrad 1998a; Klüber and Conrad - 1998b; O'Reilly and Colleran 2005). Nitrite (HNO2, the undissociated form of NO2 ) dismutates to reactive intermediates such as nitric oxide (NO) and nitroxyl anion (NO-) (Baumann et al. 1997). These compounds react with the metal centers of iron-sulfur proteins such as ferredoxin (an enzyme necessary for energy production) and form iron-nitrosyl complexes (Reddy et al. 1983). This in turn causes the destruction of the iron-sulfur cluster and results in growth inhibition. Several studies indicate that nitrite impedes a variety of biological functions including active transport and oxidative phosphorylation, consistent with inhibition of electron transport processes by destruction of iron-sulfur centers (Williams et al. 1978; Rowe et al. 1979; Reddy et al. 1983). Since iron-sulfur proteins play important roles in electron transport and synthesis of ATP in microorganisms, their inactivation by formation of iron-nitrosyl complexes would
! ! ! 153! prevent growth (Reddy et al. 1983). This provides an explanation for the loss of benzene degradation ability in the cultures in the presence of nitrite. It is also possible that nitrite accumulation inhibits the synthesis or activity of enzymes of the nitrate reduction pathway and therefore, inhibits growth of nitrate-reducing bacteria present in the cultures. As mentioned in Chapters 4 and 6, nitrate-reducing bacteria play an essential role in mineralization of benzene by removing the metabolites produced by the primary benzene degrader and by pulling the benzene degradation process toward completion. Inactivation of these microorganisms by nitrite causes intermediates of benzene mineralization to build up. Build-up of these compounds consequently stops benzene mineralization. Nitrite accumulated both during growth of cultures on benzene and on benzoate. It is likely that benzene or more likely a metabolite of benzene degradation inhibits activity or synthesis of nitrite-reductase (the enzyme involved in reduction of nitrite to nitric oxide) resulting in accumulation of nitrite in the cultures.
Except for Cartwright Consolidated, all of the cultures maintained in our laboratory accumulate nitrite. The molecular investigation of this culture by qPCR indicated that it contained a large population of Anammox bacteria (refer to Chapter 4). The role proposed for these microorganisms in the cultures was to remove and recycle nitrite (refer to Chapter 4). Hence, a strategy for preventing accumulation of nitrite in the microcosms and counteracting the inhibitory impact of this compound on the mineralization of benzene would be to enhance the growth of Anammox microorganisms in the cultures. Another approach for removing nitrite would be to simulate growth of denitrifying bacteria present in the culture by adding acetate as a substrate. Oxidation of acetate coupled to reduction of nitrite to nitrogen gas could possibly prevent nitrite accumulation. However, caution should be taken using acetate. This compound may result in growth of undesired bacteria, which negatively impact the growth of benzene- degrader. Microorganisms present in the culture could couple oxidation of acetate to reduction of nitrate and therefore, consume the nitrate available for degradation of benzene. Furthermore, since acetate is a possible intermediate of benzene degradation, its addition to the culture might inhibit the mineralization of benzene. Hence, acetate should be added to the cultures in just sufficient amounts to remove nitrite.
! ! ! 154!
7.1.2. Effect of omission of FeS and resazurin and addition of autoclaved culture on the lag time
Cultures in our laboratory experience significant lag times after transferring them into fresh medium. The possible explanations for this observation is the presence of components in the medium that pose inhibitory effects on degradation of benzene or the lack of some mineralization-mediating compounds from the medium. Experiments conducted in this study showed that simultaneous omission of resazurin and FeS from the medium shortens lag periods. Consequently, to decrease the lag time observed at the onset of culture establishment, dilution cultures prepared from the original microcosms and subcultures were maintained in FeS- and resazurin-free medium. While this helped to reduce the lag time, it caused extinction of Anammox bacteria in dilution cultures. qPCR performed on these cultures indicated that Anammox microorganisms were not present in the bottles (refer to Chapter 4). Anammox bacteria require reduced conditions (strict anaerobic conditions) for their growth. Removal of FeS (reducing agent) from the medium may adversely impact the growth of these species. As mentioned earlier, Anammox organisms are responsible for removal of nitrite from the microbial consortia. The absence of these bacteria from dilution cultures resulted in more nitrite accumulation in the bottles and more frequent need for its removal by centrifugation.
It was discussed in Chapter 3 that transfers into autoclaved culture had shorter lag times than those into regular medium. Addition of autoclaved culture reduced the lag periods most likely by providing components such as cofactors or minerals missing from the medium. In addition, autoclaving the culture destructed the cells and components of the cells which could be used as a carbon or nutrient source by benzene-degrading bacteria to promote their growth. Further analysis of the autoclaved culture using inductively coupled plasma mass spectrometry (ICP- MS) may shed light on its composition.
While long lag periods were observed during growth of cultures on benzene, benzoate mineralization occurred rapidly and without a delay (refer to Chapter 6). This suggests that the observed lags are related to initial activation of the benzene ring. Maybe some components that are essential for activation of benzene are missing from the medium. These compounds might need to be synthesized by microorganisms before benzene mineralization can start. The results of meta-transcriptomic study provided strong evidence for carboxylation as the initial reaction in
! ! ! 155! benzene degradation in our microbial consortia. The missing components of the medium could be minerals or cofactors such as biotin (a cofactor responsible for carbon dioxide transfer in several carboxylase enzymes) that are involved in this carboxylation reaction. Previously, it was shown that addition of acetate to benzene-degrading nitrate-reducing cultures significantly reduced the observed lag time upon transfer of culture into fresh medium (Nandi 2006). Acetate may be involved in the activation of benzene ring. It may also be used as a carbon source by benzene degrading bacteria increasing their initial cell numbers and therefore, reducing the lag time. The Addition of biotin and acetate to the medium and assays of the carboxylase enzyme would provide further clues on the role of these chemicals in the benzene degradation process.
7.2. Exploring microbial interactions in benzene-degrading nitrate-reducing cultures
Anaerobic benzene degrading cultures derived from contaminated sites represent complex communities. In these microbial consortia the interactions of microorganisms can affect the benzene mineralization capacity and long-term reliability of the cultures. Therefore, an understanding of how the community members interact and in particular identifying members that are responsible for initial attack on benzene can lead to development of cultures with high degradation capacity and tolerance to a broad range of chemical, physical, and environmental stresses that can be applied to the contaminated sites.
As discussed in Chapter 4 and 6 of this thesis, the results of qPCR and meta-transcriptomic experiments collectively indicate that Peptococcaceae relies on benzene as the growth substrate. Peptococcaceae degrades benzene in syntrophic association with other bacteria present in the culture. In this interaction, Peptococcaceae converts benzene to fermentation products such as acetate, hydrogen, and benzoate. Other members of community such as Azoarcus and Dechloromonas metabolize the fermentation products using nitrate as an electron acceptor. Consumption of fermentation products by these microorganisms will reduce the partial pressure of hydrogen and therefore pull the benzene degradation process toward completion. Chlorobi could also consume hydrogen during its photosynthetic growth. Chlorobi may alternatively grow phototrophically using sulfide present in culture medium as the electron donor. Anammox bacteria perform an important role in the global nitrogen cycle by coupling oxidation of ammonium to reduction of nitrite and production of nitrogen gas (Moore et al. 2011). In our cultures, Anammox organisms grow because of the simultaneous presence of ammonium and
! ! ! 156! nitrite in these cultures, and their presence fortuitously removes nitrite and recycles nitrite to nitrate. This nitrate can in turn drive more benzene degradation, explaining why sometimes benzene consumption is more than that expected for the amount of nitrate fed to the culture.
From this work it can be concluded that the optimal strategy to maintain cultures that degrade benzene through a syntrophic association is to supply the cultures with substrates and compounds that promote the growth of the benzene degrader as well as its syntrophic partners. For example, adding acetate and biotin to the culture may promote the growth of the primary benzene degrader. Using a reducing agent to provide strict anaerobic conditions helps the growth of Anammox bacteria. Removal of nitrite by Anammox bacteria promotes the growth of both Peptococcaceae and nitrate-reducing bacteria.
7.3. Carboxylation as the initial step in degradation of benzene
Elucidating key metabolic steps, genes and enzymes in the anaerobic benzene degradation pathway is an equally important task to the identification of microorganisms. Detection of intermediates that are produced during benzene mineralization and genes/enzymes that are involved in this process would provide the most conclusive evidence of benzene decomposition by microbial communities in contaminated sites. In addition, anaerobic activation of benzene ring requires unique reactions that are of importance for their potential applications in the field of green chemistry and biotechnology.
As it was explained in Chapter 6, analysis of transcripts from cells grown on benzene provides compelling evidence for carboxylation as the activation mechanism of benzene ring. The results obtained here as well as recent proteomic and transcriptomic studies of a benzene- degrading iron-reducing enrichment culture (Abu Laban et al. 2010) and a pure culture of hyperthermophilic archaen Ferroglobus placidus (Holmes et al. 2011), collectively indicate transcription, expression, and up-regulation of carboxylase genes in benzene grown-cells versus benzoate-grown cells. These genes probably encode for different subunits of a putative benzene carboxylase enzyme involved in initial attack on benzene and its carboxylation. Although these findings expand our knowledge of anaerobic benzene degradation pathway, direct association of the carboxylase genes to different subunits and functions of the enzyme, the mechanism of carboxylation reaction, and the substrate of the carboxylase enzyme (source of carboxyl group)
! ! ! 157! are still unknown. In addition, there is a need to study the catalytic activity of this enzyme and its role in metabolism of benzene. Enzyme assay experiments and purification of putative benzene carboxylase can provide clues on the structure and function of this enzyme. Biochemical assay of enzymes usually require a large amount of biomass and proteins, which is a challenge in anaerobic benzene-degrading cultures due to their low yields. Therefore, a prerequisite for enzyme assay experiments would be to generate a large amount of biomass of benzene-grown cultures. Another approach for elucidating the role of this enzyme would be to express it in another host. Some of the issues that may be encountered during heterologous protein expression are low protein yield, formation of insoluble aggregates, improper folding of protein, and difficulty in isolating and purifying a desired protein. Nevertheless, anaerobic carboxylation of benzene is a reaction that generates novel carbon bonds, which are always desired in synthesis of chemicals. It also has the potential to be used as a strategy for capturing and sequestering atmospheric carbon dioxide.
Recently, Ettwig et al. (2010) described the discovery of a new “intra-aerobic” pathway for reduction of nitrite, which results in production of oxygen. In this pathway nitrite is reduced to nitric oxide. Two molecules of nitric oxide are then converted into O2 and N2 (Ettwig et al. 2010). This newly discovered mechanism might play an important role in degradation of recalcitrant substrates such as benzene under nitrate-reducing conditions. Oxygen generated from nitrite could be used by microorganisms to aerobically degrade these compounds. In our culture, genes encoding for oxygenase enzymes and required for aerobic mineralization of benzene were not transcribed in the cells grown on benzene. Therefore for our cultures, we can rule out the possible role of oxygen generated from nitrite in benzene degradation.
! ! !
7.4. References Chapter 7
Abu Laban, N., D. Selesi, T. Rattei, P. Tischler and R. U. Meckenstock (2010). "Identification of enzymes involved in anaerobic benzene degradation by strictly anaerobic iron-reducing enrichment culture." Environ. Microbiol. 12(10): 2783-2796.
Atlas, R. M. (1981). "Microbial degradation of petroleum hydrocarbons: An environmental perspective." Microbiol. Rev. 45: 180-209.
Baumann, B., J. R. v. d. Meer, M. S. M and A. J. Zehnder (1997). "Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH." Antonie Van Leeuwenhoek 72(3): 183-189.
Chapelle, F. H. (1999). "Bioremediation of petroleum hydrocarbon-contaminated ground water: The perspectives of history and hydrology." Ground Water 37: 122-132.
Ettwig, K. F., M. K. Butler, D. L. Paslier, E. Pelletier, S. Mangenot, M. M. M. Kuypers, F. Schreiber, B. E. Dutilh, J. Zedelius, D. d. Beer, J. Gloerich, H. J. C. T. Wessels, T. v. Alen, F. Luesken, M. L. Wu, K. T. v. d. Pas-Schoonen, H. J. M. O. d. Camp, E. M. Janssen-Megens, K. J. Francoijs, H. Stunnenberg, J. Weissenbach, M. S. M. Jetten and M. Strous (2010). "Nitrite- driven anaerobic methane oxidation by oxygenic bacteria." Nature 464: 543-550.
Holmes, D. E., C. Risso, J. A. Smith and D. R. Loveley (2011). "Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus." Appl. Environ. Microbiol. 77(17): 5926-5933.
Klüber, H. D. and R. Conrad (1998a). "Effects of nitrate, nitrite, NO and N2O on methanogenesis and other redox processes in anoxic rice field soil." FEMS Microbiol. Ecol. 25(3): 301-318.
Klüber, H. D. and R. Conrad (1998b). "Inhibitory effects of nitrate, nitrite, NO and N2O on methanogenesis by Methanosarcina barkeri and Methanobacterium bryantii." FEMS Microbiol. Ecol. 25(4): 331-339.
Moore, T. A., Y. Xing, B. Lazenby, M. D. Lynch, S. Schiff, W. D. Robertson, R. Timlin, S. Lanza, M. C. Ryan, R. Aravena, D. Fortin, I. D. Clark and J. D. Neufeld (2011). "Prevalence of anaerobic ammonium-oxidizing bacteria in contaminated groundwater." Environ. Sci. Technol. 45(17): 7217-7225.
Nandi, M. (2006). Biochemical and molecular charecterization of anaerobic benzene-degrdading cultures. Toronto, University of Toronto. M. A. Sc. Thesis.
O'Reilly, C. and E. Colleran (2005). "Toxicity of nitrite toward mesophilic and thermophilic sulphate-reducing, methanogenic and syntrophic populations in anaerobic sludge." J. Ind. Microbiol. Biotechnol. 32(2): 46-52.
! ! 158! ! 159!
Reddy, D., J. R. Lancaster and D. P. Cornforth (1983). "Nitrite inhibition of Clostridium botulinum: electron spin resonance detection of iron-nitric oxide complexes." Science 221(4612): 769-770.
Rowe, J. J., J. M. Yarbrough, J. B. Rake and R. G. Eagon (1979). "Nitrite inhibition of aerobic bacteria." Curr. Microbiol. 2(1): 51-54.
Williams, D. R., J. J. Rowe, P. Romero and R. G. Eagon (1978). "Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport." Appl. Environ. Microbiol. 36(2): 257-263.
! ! !
Chapter 8: Conclusions and Engineering Significance
! 160! ! ! 161!
8.1. Summary
Benzene is a highly toxic (carcinogenic) industrial compound that is found in many petroleum products and utilized for the production of various chemicals, rubbers, lubricants, dyes, detergents, fuel oils, and drugs. Benzene is among the most prevalent contaminants of groundwater and soil. Bioremediation, which involves utility of biological processes, is a preferred approach compared to the more invasive and expensive technologies such as “pump and treat” for remediating polluted fields. Because hydrocarbon-contaminated sites usually contain extensive anoxic zones due to depletion of oxygen by aerobic hydrocarbon utilizing microorganisms, anaerobic mineralization processes are very important in these fields. Therefore, a fundamental understanding of anaerobic benzene biodegradation is essential for design and implementation of effective management and remediation strategies in polluted sites.
In this thesis the biochemical and molecular characterization of benzene-degrading nitrate- reducing microbial consortia was described. The first objective was to understand conditions that promote or inhibit degradation of benzene in order to improve the growth of microorganisms in the cultures. The results suggest that nitrite inhibits mineralization of benzene. Concomitant removal of FeS (a solid) and resazurin (a redox indicator) from the medium of enrichment cultures decreases the lag time observed upon transfer of cultures into fresh medium. Addition of autoclaved supernatant from an actively benzene-degrading microcosm to a stalled culture proved to be useful in reviving benzene degradation capacity of culture. These results implicate a complex interplay between solid and dissolved components in the medium, affecting bioavailability of key nutrients.
The second objective was to identify community members of the cultures and determine their functional roles in order to identify those initiating attack on benzene. The results indicate a syntrophic interaction between different microorganisms in the culture to mineralize benzene. Members of Peptococcaceae family are primary benzene degraders and are involved in initial attack on benzene. They convert benzene to benzoate or low molecular weight fermentation products, which are further metabolized by other bacteria present in the culture such as Azoarcus, Dechloromonas, and Chlorobi. Molecular investigations also revealed the presence of Anammox bacteria in the culture. They facilitate removal of nitrite produced during denitrification process and stabilize the culture.
! ! ! 162!
The third objective was to establish a pure culture of a benzene-degrading nitrate-reducing microbe. The attempts to obtain a pure culture resulted in the isolation of Dechloromonas- and Dechlorosoma-like microorganisms from the enrichment cultures. These bacteria were dependent on acetate and nitrate for growth and could not degrade benzene.
The final objective was to elucidate anaerobic benzene degradation pathway and responsible genes. Putative carboxylase genes were identified that were specifically transcribed in the presence of benzene. These genes encode a putative benzene carboxylase enzyme. Several genes corresponding to the enzymes of benzoyl-CoA pathway were also transcribed in the benzene- grown cells. These results together with other recently published data provide compelling evidence for carboxylation as the activation mechanism of benzene ring. Discovery of these genes is a stepping stone in the field of anaerobic benzene biodegradation.
8.2. Conclusions
1. All of the enrichment cultures accumulate nitrite during mineralization of benzene coupled to nitrate reduction with the exception of Cartwright Consolidated, which only transiently builds up nitrite. This is likely because Cartwright Consolidated contains Anammox species that mediate nitrite removal.
2. Simultaneous omission of FeS and resazurin from the medium and addition of autoclaved culture significantly reduces the lag time observed upon transfer of culture into fresh medium.
3. The Swamp Consolidated culture contains five major OTUs. These OTUs are phylogenetically most closely related to Peptococcaceae (Swampcons-N1), Rhodocyclaceae (Swampcons-N2), Chlorobi (Swampcons-N3 and Swampcons-N4), and Burkholderia (Swampcons-N5). Peptococcaceae is the most dominant bacterium in the clone library, representing 30% of the analyzed clones.
4. The number of 16S rRNA gene copies of Peptococcaceae increases significantly during mineralization of benzene. This indicates that growth of Peptococcaceae in the culture is directly correlated with benzene degradation. None of the other monitored bacteria, i.e. Azoarcus, Dechloromonas, Chlorobi, and Anammox show this characteristic.
5. Benzene in the cultures is degraded by syntrophic association between different genera of
! ! ! 163! bacteria. In this association, Peptococcaceae degrades benzene to hydrogen, acetate, or low molecular weight fermentation products that can be used by its syntrophic partners. Chlorobi possibly acts as a hydrogen scavenger. The function of Azoarcus and Dechloromonas within the flow of carbon and electrons would be oxidizing acetate or other low molecular weight products as well as hydrogen, using nitrate as an electron acceptor.
6. The role of Anammox microorganisms in the culture is nitrite removal. This process detoxifies and stabilizes the culture.
7. The Cartwright Consolidated culture can consume benzoate, a possible metabolite of benzene degradation. Absence of benzene in the culture during benzoate degradation has a negative impact on the ability of the culture to mineralize benzene afterwards. It is possible that presence of benzoate and absence of benzene provides an opportunity for growth of benzoate-degrading bacteria. Growth of these bacteria may in turn adversely affect the primary benzene-degrader in the culture.
8. mRNA sequences corresponding to carboxylase genes were identified that were specifically transcribed in the presence of benzene. These genes probably encode for different subunits of a putative benzene carboxylase enzyme which is involved in carboxylation of benzene to benzoate. Furthermore, transcripts for the genes that encode different enzymes of benzoyl-CoA degradation pathway were present in the culture. These findings suggest that benzene is activated to benzoate by the putative benzene carboxylase. Benzoate is further metabolized through a pathway for biodegradation of benzoyl-CoA.
8.3. Engineering significance
To demonstrate that bioremediation takes place in a contaminated site, at least four lines of evidence are required: 1. loss of contaminants from the site, 2. laboratory data indicating the presence of microbial populations required for degradation of compounds of interest, 3. evidence showing actual in situ microbial activity, e.g. production of predicted metabolites, depletion of terminal electron acceptor or accumulation of reduced terminal electron acceptor products, and detection of enzymes of the pathway, and 4. rate at which contaminants are removed (Smets and Pritchard 2003; Foght 2008). Therefore, it is important to both identify the microorganisms that are involved in anaerobic benzene degradation and elucidate the corresponding pathway,
! ! ! 164! metabolites, genes and enzymes. Metabolites, genes and enzymes can be used as biomarkers for biodegradation of contaminants. Furthermore, study of the population dynamics of mixed communities during mineralization process will allow scientists as well as practitioners to understand the community structure, interactions between different species, and degradation potential of the consortium. This in turn will enable them to produce stable consortium for efficient degradation of contaminants in the fields.
Mineralization of contaminants in polluted fields through biological activity of microorganisms can be enhanced either by addition of limiting nutrients (biostimulation) or addition of microbes with the ability to degrade compounds of interest (bioaugmentation). Identifying microorganisms that mediate degradation process and enhancing our knowledge of their growth requirements will enable better design of biostimulation and bioaugmentation processes for remediation of contaminated sites. In other words, continued investigation of methods for optimizing in situ bioremediation enables us to identify fast, economical, and efficient means for removal of recalcitrant compounds such as benzene from contaminated fields (Wilson and Bouwer 1997).
There is a renewed interest in understanding the mechanism of activation of the benzene ring under anaerobic conditions. Due to the low chemical reactivity of benzene ring, its anaerobic biodegradation requires unusual biochemical reactions that are of great importance for their potential applications in the field of biotechnology (Carmona et al. 2009). For instance, these mechanisms could provide us with the clues on how to decompose tougher compounds such as poly aromatic hydrocarbons (PAHs).
Therefore, the focus of this thesis was on identifying microorganisms that facilitate decay of benzene in the enriched cultures and its anaerobic mineralization pathway. Peptococacceae was found as the active benzene degrader in the enrichment cultures. Presence or growth of this bacterium in contaminated sites will provide evidence for occurrence of benzene biodegradation. A carboxylase enzyme was specifically transcribed in the presence of benzene in nitrate-reducing culture. The genes corresponding to this enzyme can potentially be employed as biomarkers for biodegradation activity in the field. In addition, carboxylation as activation mechanism of benzene ring is a reaction that generates novel C-C bonds. These types of reactions are always desired in chemical synthesis. Moreover, carboxylation of benzene is a form of carbon fixation,
! ! ! 165! and as such is important.
The research conducted in this thesis enhanced our knowledge of benzene mineralization under nitrate-reducing conditions both in terms of microorganisms involved in this process and the metabolic pathway.
8.4. Recommendations for future work
1. Further growth optimization trials are needed, as the culture still does not grow as well as theoretical yields would predict. Other variables to check include benzene concentration, addition of selenium and molybdenum (or tungstate) to the cultures, addition of acetate and biotin, use of solid supports and Amberlite-XAD7 ion exchange resin, and concentration of trace elements such as iron. These parameters have been shown to influence the growth of other benzene-degrading cultures.
2. It is recommend wrapping the cultures in aluminum foil to prevent growth of phototrophic bacteria such as Chlorobi. This will allow investigating whether Chlorobi is a critical member of the culture and its presence is required for maintaining the degradation capacity.
3. To identify bacteria that benefit from benzoate, qPCR experiments should be conducted to follow the abundance of different phylotypes in the culture during benzoate mineralization process.
4. The impact of addition of hydrogen on degradation of benzene should be studied. Based on thermodynamic calculations if benzene is degraded via a syntrophic interaction with hydrogen as a central intermediate, then addition of hydrogen at partial pressures higher than 0.24 atm inhibits mineralization of benzene.
5. Since some members of Peptococcaceae family have the ability to form spores, treating the culture with heat (heat the culture at 80 °C for 10 minutes) or heat and ethanol to eliminate vegetative cells, may be a possible means of isolating Peptococcaceae from the cultures. The isolated Peptococcaceae can then be grown on benzene in co-culture with a methanogenic microorganism.
! ! ! 166!
6. Crotonate could be used to obtain pure cultures of Peptococcaceae. This compound has been previously utilized to establish pure cultures of bacteria that grew only as obligate syntrophs in coculture with hydrogen-utilizing microbes.
7. It is suggested to study the metagenome of Cartwright Consolidated culture. This will enhance current state of knowledge regarding the putative benzene carboxylase enzyme and probably lead to its complete sequence. It will also yield information about the arrangement of genes on the genome of benzene degrading bacteria.
8. Determination of the genes involved in the anaerobic benzene pathway can be accomplished by comparing the proteome of benzene-, phenol-, and toluene-grown cells.
9. Enzyme assay experiments and purification of the putative benzene carboxylase should be employed to better understand the actual structure and function of this enzyme.
10. It is possible that more than one pathway is employed for benzene mineralization in the microbial consortia. Therefore, it is important to study the consumption of other possible metabolites of benzene degradation, i.e. toluene and phenol, by benzene-degrading cultures. A meta-transcriptomic approach similar to that employed in this study should be used to determine the genes that are differentially expressed in the presence of benzene, toluene, or phenol.
! ! !
8.5. References Chapter 8
Carmona, M., M. T. Zamarro, B. Blázquez, G. Durante-Rodríguez, J. F. Juárez, J. A. Valderrama, M. J. Barragán, J. L. García and E. Díaz (2009). "Anaerobic catabolism of aromatic compounds: a genetic and genomic view." Microbiol. Mol. Biol. Rev. 73(1).
Foght, J. (2008). "Anaerobic biodegradation of aromatic hydrocarbons: Pathways and Prospects." J Mol Microbiol Biotechnol 15: 93-120.
Smets, B. F. and P. H. Pritchard (2003). "Elucidating the microbial component of natural attenuation." Curr. Opin. Biotechnol. 14: 283-288.
Wilson, L. P. and E. J. Bouwer (1997). "Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review." J. Ind. Microbiol. Biotechnol. 18: 116-130.
! 167! ! !
Appendix A: Bacterial Energetics and Stoichiometric Calculations
! 168! ! 169!
Calculation of free energy and overall energy reactions
The Gibb’s standard free energy of reactions (!G°") for the redox couples in section 2.1 of Chapter 2 are calculated using the standard free energy of half-reactions provided in Rittmann and McCarty (2001). Table A.1 shows these half-reactions and their standard free energies, which are calculated by subtracting the sum of free energy of formation of products from the sum of free energy of formation of reactants and considering stoichiometric coefficients (Rittmann and McCarty 2001). The free energy of formation for individual compounds is given in Table A.2. Based on convention, the half-reactions are written as reduction reactions and are balanced to a single electron. Therefore, the unit for free energy is kJ per electron equivalent (Table A.1).
Table A.1. Half-reactions and their Gibb's standard free energy at pH=7.0
Reduced/Oxidized !G°" Reduction half-reaction compounds (kJ/e-eq)
1 " + " 1 " 1 Nitrite/Nitrate NO3 +H +e = NO2 + H2 O -41.65 2 2 2
1 " 6 + " 1 3 Nitrogen/Nitrate NO3 + H +e = N2 + H2 O -72.20 5 5 10 5 ! 1 " 6 + " 1 3 Benzene/Carbon dioxide HCO3 + H +e = C6 H6 + H2 O 27.37 5 5 30 5 ! 1 1 " + " 1 " 13 Benzoate/Carbon dioxide CO2 + HCO3 +H +e = C6 H5 COO + H2 O 27.34 5 30 30 30 ! 1 1 " + " 1 " 3 Acetate/Carbon dioxide CO2 + HCO3 +H +e = CH3 COO + H2 O 27.40 8 8 8 8 ! !
!
! ! !
! ! 170!
Table A.2. Free energy of formation of chemical species at 25°C
Substance Form kJ/mola
Carbon dioxide (CO2) aq -386.02
Water (H2O) l -237.178 Hydrogen ion (H+) aq -39.87 - Bicarbonate (HCO3 ) aq -586.85 - Nitrate (NO3 ) aq -111.34 - Nitrite (NO2 ) aq -37.2
Benzene (C6H6) aq 133.9 - Benzoic (C6H5COO ) aq -245.6 - Acetate (CH3COO ) aq -369.41
a Free energy of formation were taken from Rittmann and McCarty (2001).
As an example, the free energy of half-reaction for the reduction of nitrate to nitrite is calculated below:
1 " + " 1 " 1 NO3 +H +e = NO2 + H2 O (A.1) 2 2 2 In the first step, the free energy of formation of each compound that is provided in Table A.2 is multiplied by its stoichiometric coefficient: ! 1 1 1 ("111.34),1("39.87),0, ("37.2), ("237.178) 2 2 2 The free energy of half-reaction is then calculated as follows:
, 1 1 1 - ! "G° = (#37.2)+ (#237.178)#(( (#111.34)+1(#39.87)+0)) = #41.65 KJ/e eq (A.2) 2 2 2 Coupling of the half-reactions in Table A.1 allows to construct the energy reactions in Table 2.1 of Chapter 2. It is assumed that free energy of reaction is used only for energy production and ! not for cell synthesis. As examples, overall energy reactions for oxidation of benzene coupled to complete reduction of nitrate to the nitrogen gas and its incomplete reduction to nitrite are given bellow.
A) Oxidation of benzene coupled to the reduction of nitrate to the nitrogen gas:
! ! 171!
The initial step in constructing the overall energy reaction is to write down the electron donor and the electron acceptor half reactions:
1 " 6 + " 1 3 - Donor half-reaction Rd: HCO3 + H +e = C6 H6 + H2 O !Gd°"= 27.37 kJ/e eq (A.3) 5 5 30 5
1 " 6 + " 1 3 - Acceptor half-reaction Ra: NO3 + H +e = N2 + H2 O !Ga°"= -72.20 kJ/e eq (A.4) 5 5 10 5 !
The overall energy yielding reaction (Re) then can be written as: ! " " R e=R a"Rd : C6H6 + 6NO 3 # 6HCO3 + 3N2 (A.5)
The free energy of oxidation-reduction reaction (!Gr°") is calculated by subtracting the free energy of electron donor half reaction from that of electron acceptor: ! - !Gr°"=!Ga°"- !Gd°"= -72.20 - 27.37 = -99.57 kJ/e eq (A.6) The unit of free energy of formation can be converted from kJ/ e- eq to kJ/mole of benzene oxidized by multiplying the free energy of formation by stoichiometric coefficient of benzene, which is 30.
!Gr°"= -2987 kJ/mol of benzene oxidized B) Oxidation of benzene coupled to the reduction of nitrate to nitrite:
1 " 6 + " 1 3 - Donor half-reaction Rd: HCO3 + H +e = C6 H6 + H2 O !Gd°"= 27.37 kJ/e eq (A.7) 5 5 30 5 1 " 6 + " 1 " 1 - Acceptor half-reaction Ra: NO3 + H +e = NO2 + H2 O !Ga°"= -41.65 kJ/e eq (A.8) 2 5 2 2 The overall energy yielding reaction R =R -R : ! e a d " " " + C 6 H6 + 15 NO3 +3H2 O # 6 HCO3 + 15 NO2 + 6 H (A.9) ! - The overall free energy of reaction: !Gr°"=!Ga°"- !Gd°"= -41.65 - 27.37 = -69.02 kJ/e eq= -2070 kJ/mole of benzene oxidized (A.10) ! Calculation of fe, fs and overall stoichiometric equations
Growth of bacteria involves two basic reactions, one for production of energy and the other for cell synthesis. Therefore, a portion of electrons transferred from electron donor to electron
acceptor (fe) is used for energy production and the other portion (fs) for growth and cell synthesis. This section describes the method developed by Rittmann and McCarty (2001) to
estimate fe and fs parameters and elaborate on the calculation of the stoichiometric equations in Table 2.2 of Chapter 2.
! ! 172!
Employing the method of Rittmann and McCarty (2001), the fraction of electron donor used
for energy (fe) and the fraction of donor used for synthesis of cells (fs) can be calculated. If one considers that “A” equivalents of electron donor is required for production of energy per equivalent of cells formed, “A” can be calculated according to the following equation:
"GP "Gpc n + A = # # (A.11) #"Gr The parameters in this equation are described below.
!Gp is the energy required to convert the carbon source to the common organic intermediates ! such as pyruvate used by cells for synthesizing their macromolecules. !Gp is computed as the difference between the free energy of the pyruvate half-reaction and that of carbon source. For
example for benzene as a carbon source, !Gp is calculated according to the following equation:
!Gp= !G°"pyruvate - !G°"benzene (A.12)
1 1 " + " 1 " 2 Pyruvate half-reaction: CO2 + HCO3 +H +e = CH3 COCOO + H2 O 5 10 10 5 - !G°"pyruvate= 35.09 kJ/e eq (A.13)
1 " 6 + " 1 3 Benzene half-!reaction: HCO3 + H +e = C6 H6 + H2 O 5 5 30 5 - !G°" benzene = 27.37 kJ/e eq (A.14)
Therefore, !Gp is: ! - !Gp= 35.09 - 27.37 = 7.72 kJ/e eq (A.15)
!Gpc is the energy needed to convert pyruvate carbon to cellular carbon. The estimated value of
!Gpc is 3.33 kJ per gram of cells (McCarty 1971). To estimate “A”, we need to convert the unit
of !Gpc from kJ per gram of cells to kJ per electron. For this purpose, it is essential to know the nitrogen source. If ammonium is the nitrogen source, then an electron equivalent of cells will be - 113/20=5.65 grams (McCarty 1971). Thus, !Gpc is 18.8 kJ/e eq.
!Gr is the free energy released per equivalent of donor oxidized for energy production (see
previous section for detail calculations of !Gr ). # is the energy-transfer efficiency. For anaerobic heterotrophic growth, it is within the range 40- 70% . An average of 60% is usually used in calculations (McCarty 1971).
n is equal to +1 for !Gp> 0 and -1 for !Gp< 0.
! ! 173!
- Substituting values of !Gp (A.15), !Gpc=18.8 kJ/e eq, !Gr (A.4), "=0.6, and n=1 into equation A.11, one can estimate “A” for the degradation of benzene coupled to the reduction of nitrate to the nitrogen gas as given below: 7.72 18.8 1 + A= 0.6 0.6 = 0.74 (A.16) 0.6"# 99.57 Value of “A” for the oxidation of benzene linked to incomplete reduction of nitrate to nitrite can
also be calculated by using !Gr from (A.10): ! 7.72 18.8 1 + A= 0.6 0.6 = 0.99 (A.17) 0.6"# 69.02
Since “A” equivalents of donor is used for energy and 1 equivalent is used for cell synthesis, ! the total amount of donor consumed is 1 + A. Therefore, fe and fs can be computed from “A” using the following equations: A f e= (A.18) 1+ A 1 f s= (A.19) 1+ A ! fe and fs for the mineralization of benzene coupled to the reduction of nitrate to the nitrogen gas or reduction of nitrate to nitrite are calculated by substituting “A” from equations A.16 and ! A.17 into equations A.18 and A.19, respectively. Estimated values of fe and fs are 0.43 and 0.57 for oxidation of benzene linked to reduction of nitrate to nitrogen and 0.52 and 0.48 for benzene oxidation associated with reduction of nitrate to nitrite. Calculating the fractions of electrons consumed for energy production and cell synthesis, now one can approximate the overall stoichiometric reactions, which include cell synthesis, according to the following equation:
Overall reaction R= fe (Ra-Rd) + fs (Rc-Rd) (A.20)
, where Ra is the electron acceptor half-reaction, Rd is the electron donor half-reaction, and Rc is the cell synthesis half-reaction.
When ammonium is the nitrogen source for cell synthesis, Rc can be written as follows (Rittmann and McCarty 2001):
1 1 " 1 + + " 1 9 CO2 + HCO3 + NH4 +H +e = C5 H7 O2 N+ H2 O (A.21) 5 20 20 20 20
! ! ! 174!
Based on equation A.20, The overall stoichiometric reaction for degradation of benzene coupled to the reduction of nitrate to nitrogen or reduction of nitrate to nitrite are provided below:
" + " + C 6 H6 +2.6NO3 + 0.9NH4 +3.4CO2 +2.6H2 O#1.3N2 +5.1HCO3 +0.9C5 H7 O2 N+3.4H (A.22)
" + " " + C H +7.7NO3 + 0.7NH4 +2.9CO +3.7H O#7.7NO2 +5.3HCO3 +0.7C H O N+6H ! 6 6 2 2 5 7 2 (A.23) If the free energy of oxidation-reduction reaction is only used for energy production ! purposes, the stoichiometric ratio of nitrate to benzene for oxidation of benzene coupled to the complete reduction of nitrate to the nitrogen gas or its incomplete reduction to nitrite will be 6.0 and 15, respectively (Equations A.5 and A.9). However in practice, some of this free energy is used for cell synthesis. In this case the stoichiometric ratio of nitrate to benzene will be 2.6 and 7.7 for the mineralization of benzene linked to the reduction of nitrate to nitrogen gas or reduction of nitrate to nitrite (Equations A.22 and A.23). The stoichiometric equations for all of the redox couples listed in Table 2.1 and 2.2 were calculated using approach explained in this Appendix.
Calculation of fe, fs based on observed yields
Ulrich and Edwards (2003) showed that the observed yields (8.6-22 g cells/mole benzene) for our nitrate-reducing cultures were well below the theoretical yields obtained based on !=0.6
(Chapter 2). This indicates that the fs value for our microbial consortia is not as high as the value
predicted assuming !=0.6. The observed yields were used to calculate fs and fe and to form the stoichiometric equations for mineralization of benzene linked to reduction of nitrate to nitrite or nitrogen gas. This section explains the steps taken.
In the first step fs is calculated using the following equation:
g cells e" eqcells 113gcells mole cells 30e"eqcells Y( ) =fs ( " ) # # " # (A.24) mole donor e eqdonor mole cells 20e eqcells mole donor In this equation Y is the observed yield. Substituting the observed yield values of 8.6 or 22 into
equation A.24, one can calculate fs: !
! ! 175!
fs=0.05 (for Y=8.6)
fs=0.13 (for Y=22)
All of the remaining steps for deriving overall stoichiometric reactions are explained in the previous section.
" + " + C6 H6 +5.7NO3 + 0.08NH4 +0.3 CO2 +0.23H2 O#2.9N2 +5.9HCO3 +0.08C5 H7 O2 N+0.3H (A.25) " + " " + C H +14.3 NO3 + 0.08 NH4 +0.3 CO +3.1 H O#14.3NO2 +5.9HCO3 +0.08C H O N+6H 6 6 2 2 5 7 2 (A.26) ! Calculation of yield from the change in number of 16S rRNA gene copies per mole of ! benzene
In Chapter 4, the yield for Peptococcaceae and Azoarcus was calculated in terms of grams of cells per mole of benzene from the change in number of 16S rRNA gene copies per mole of benzene. This section explains the steps that were taken. First the size of Peptococcaceae and Azoarcus were determined by taking an average of the cells size of several known Peptococcaceae (Imachi et al. 2002; Imachi et al. 2006; Qiu et al. 2006) and Azoarcus species (Reinhold-Hurek et al. 1993; Rabus and Widdel 1995). These values are provided below:
Peptococcaceae cell size: 0.87 µm (width) ! 2.8 µm (length) (rod shaped) Azoarcus cell size: 0.7 µm (width) ! 2.5 µm (length) (rod shaped)
Using the cell sizes, the volume of cell can be calculated as follows:
2 0.87 2 3 VPeptococcaceae = " r h = 3.14159 #( ) # 2.8 =1.66µm 2 3 VAzoarcus= 0.96 µm Since 80% of a cell consists of water, it is assumed that cell density is equal to that of water. This ! assumption allows to calculate the wet mass of cells.
g µm3 Wet mass of Peptococcaceae cell = " # V =1.03 ( ) #1.66µm3 #10$12( ) =1.71#10$12 g ml ml Wet mass of Azoarcus cell= 9.91! 10-13 g
! ! ! 176!
Only 20% of mass of cell is dry. Multiplying the mass of wet cell by 0.2 will give us the dry mass of cell:
Dry mass of Peptococcaceae cell= 0.2 ! 1.71! 10-12 = 3.43! 10-13 g Dry mass of Azoarcus cell= 1.98! 10-13 g
Based on the average 16S rRNA copies found in the genome of cultured bacteria (Pelotomaculum thermopropionicum and Azoarcus spp.) (Klappenbach et al. 2000), the average 16S rRNA copies per genome of Peptococcaceae and Azoarcus were assumed to be 2 and 4 copies, respectively. The yields of Peptococcaceae and Azoarcus were 4.41 ± (2.07) ! 1013 and 2.39 (± 0.9) ! 1013 16S rRNA gene copies/mol of benzene degraded (refer to Chapter 4). These values can now be expressed in terms of g cells per mol benzene according to the following equation:
16S rRNA gene copies 1 Y = Y( )" " Dry mass of cell(g) = Peptococcaceae mole of benzene 16S rRNA gene copies/cell
1 g cell 4.41"1013 " "3.43"10#13 = 7.56 2 mol benzene
YAzoarcus= 1.18 g cell/mol benzene ! Calculation of yield based on ratio of nitrate to benzene
In our cultures, 12 moles of nitrate is consumed per each mole of degraded benzene. This ratio was used to calculate the yield as follows:
mole nitrate 1mole benzene 2e#eq nitrate fe =12 " # " = 0.8 mole benzene 30e eq 1mole nitrate
fs=1-fe=0.2 ! e"eq cell 1mole cell 113gcell 30e"eqdonor gcell Y = 0.2 " # " # # = 33.9 e eq donor 20e eq 1molecell 1moledonor mole benzene
!
! ! 177!
Calculation of reaction free energy for nonstandard conditions
In this section, the steps should be taken for calculation of energy of reaction for nonstandard conditions is described. The following equation can be employed to adjust the reaction free energy for nonstandard conditions for a reaction that involves n different constituents (Rittmann and McCarty 2001):
n ! "G = "G '+R T#v i lna i (A.27) i=1
Here, vi represents the stoichiometric coefficient for constituent Ai in the reaction which is
! negative if Ai appears on the left side of reaction and positive if it appears on the right side, ai is
the activity of constituent Ai, T is absolute temperature (K), R is the ideal gas law constant (8.314 J/mol K) (Rittmann and McCarty 2001). In the example that follows, the free energy of fermentation of benzene to acetate and hydrogen for nonstandard concentrations of reactants and products is calculated.
# + C 6H6 + 6H2O " 3CH3COO + 3H2 + 3H (A.28)
In the first step, the standard free energy of reaction is calculated. The free energy of formation ! of benzene, water, acetate, hydrogen, and hydrogen ion are 133.9, -237.178, -369.41, 0, -39.87 kJ/mol, respectively. Therefore, !G°" can be written as follows:
! " G '= [3# $369.41+ 3# 0 $ 3# 39.87]$[133.9 $ 6 # 237.178] = 61.3 kJ /mol (A.29)
For the above energy reaction vi is -1, -6, 3, and 3 for benzene, water, acetate, and hydrogen, ! respectively. Substituting this information into equation A.27 gives:
[CH COO# ]3 [H ]3 "G = "G!'+R T ln 3 2 (A.30) [C H ][H O]6 6 6 2 It is assumed that the concentration of acetate in aqueous solution is 0.00005 M, the hydrogen
! partial pressure is 0.001 atm, the benzene concentration is 0.0001M, the activity of water is close to one and temperature is 25°C. It is also assumed that the activities of the constituents of reaction are equal to their molar concentrations or partial pressures.
! ! 178!
[0.00005]3 [0.001]3 kJ "G = 61.3+ 0.008314 # 298.15# ln 6 = $40.8 (A.31) [0.0001][1] mol
For the hydrogen partial pressure of 10-4 and 10-6 atm the free energy of reaction will be -60 and
! -90 kJ/mol, respectively. The free energy of reaction becomes positive for hydrogen partial pressures above 0.2 atm.
! !
Appendix B: Anaerobic Medium Composition
! 179! ! 180!
Anaerobic medium used for maintaining our cultures
The medium used for maintaining our anaerobic benzene-degrading nitrate-reducing cultures contains various components such as phosphate buffer, salts, magnesium chloride, sodium bicarbonate, redox indicator, trace minerals, iron sulfide, and vitamins. Table B.1 provides the list of these compounds and their concentrations in the medium.
Table B.1. Components of our anaerobic medium and their corresponding concentrations
Component Composition Concentration (mg/l)
KH2PO4 209.6 Phosphate buffer K2HPO4 428.5
NH4Cl 535
Salts CaCl2.6H2O 70
FeCl2.4H2O 20
. Magnesium chloride MgCl2 6H2O 101.6
Sodium bicarbonate NaHCO3 69
Redox indicator Resazurin 1
H3BO3 0.6
ZnCl2 0.2 Na2MoO4.2H2O 0.2
NiCl2.6H2O 1.5
Trace Minerals MnCl2.4H2O 2
CuCl2.2H2O 0.2
CoCl2.6H2O 3
Na2SeO3 0.04
Al2(SO4)3.18H2O 0.2
Iron sulfide FeS 20 Biotin 0.002 Folic acid 0.002 Pyridoxine HCl 0.01 Riboflavin 0.005 Thiamine 0.005 Vitamins Nicotinic acid 0.005 Pantothenic acid 0.005 PABA 0.005 Cyanocobalamin (vitamin B12) 0.005 Thioctic (lipoic) acid 0.005
Coenzyme M 0.1
! !
Appendix C: Effect of Addition of Autoclaved Culture on the Observed Lag Times
! 181! ! 182!
Benzene degradation curves for Cartwright pw1 10-5 and Cartwright Consolidated 10-3 and for their transfers into an autoclaved culture
160
(a) Without autoclaved culture With autoclaved culture 120
80
40
Benzene concentration (µM) 0 0 20 40 60 80 100 120 140 160
(b) Without autoclaved culture 120 With autoclaved culture
80
40
Benzene concentration (µM) 0 0 20 40 60 80 100 120 140 160 Time (day)
Figure C.1. Anaerobic benzene mineralization in dilution cultures, a) Cartwright pw1 10-5 and b) Cartwright Consolidated 10-3, both with and without autoclaved culture. Arrows point to the time at which benzene is supplied to the culture.
! !
Appendix D: Partial 16S rRNA Gene Sequences Obtained from Swamp Consolidated Enriched Culture
! 183! ! 184!
Swampcons-N1 TTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACG GAGTTAATTAGGAAGCTTGCTTTTTAATTAACTTAGTGGCGGACGGGTGAGTAACGC GTGGGCAATCTGCCCGTAAGAGGGGGATAACACCTAGAAATGGGTGCTAATACCGC ATAACATCGTGGTGTTGCATGATACTGCGATCAAAGGAGCAATCCGCTTACGGATGA GCCCGCGTCTGATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCAGT AGCCGGCCTGAGAGGGTGACCGGCCACACTGGAACTGAGACACGGTCCAGACTCCT GCGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAAC GCCGCGTGAGTGATGAAGGCCTTCGGGTTGTAAAACTCTGTCTTCAGGGAAGAAAC AAATGACGGTACCTGAGGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA ATACGTAGGTGGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGC
Swampcons-N2 ATGCTTTACACATGCAAGTCGAACGGCAGCACGGGGGCAACCCTGGTGGCGAGTGG CGGACGGGTGAGTAATGCATCGGAACGCGTCCTGTAATGGGGGATAACCTAGCGAA AGTTAGGCTAATACCGCATACGTCCTGAGGGAGAGAGCGGGGGATCGTAAGACCTC GTGTTATAGGAGCGGCCGATGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCA AGGCGACGATCCGTAGCGGGTCTGAGAGGACGGCCCGCCACACTGGGACTGAGACA CGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACC CTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTCGG CCGGGAAGAAATCGTGCGGGCTAATACCCTGTATGGACGACGGTACCGGAAGAAGA AGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAA TCGGAATTACTGGGCGTAAAGCGT
Swampcons-N3 GTGCTTAATACATGCAAGTCAACTGAAACAGTTGTAGCAATACGGCTGTGGAGGTG GCGCACGGGTGAGTAACACGTAGGTAATCTGCCTTCAGGACTGACACAACTCCGAG AGATCGGAGCTAATATCAGATAATGCAGCGGCTTGACATCAAGACAGTTGTTAAAG CTTCGGTGCCTGGAGATGAGCCTGCGCCCCATTAGGTAGTTGGCGGAGTAACAGCCC ACCAAGCCTGCGATGGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGA GACACGGTCCAGACTCCTACGGGAGGCAGCAGTAAGGAATATTGCTCAATGGCCGA AAGGCTGAAGCAGCAACGCCGCGTGAGGGATGAAGACCTTATGGTTGTAAACCTCT GTAGATGGGGAGAAATTTTCCCGTTTCGGGAATTTGATAGTACCCATAAAGTAAGCC CCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGG ATTTACTGGGTGTAAAGGGT
! ! 185!
Swamcons-N4 GTGCTTAATACATGCAAGTCTACGATTTTTAAGGTAGCAATATTTTAAAGAGAGTGG CGCACGGGTGAGTAACACGTAGGTAATCTGCCTTTAGGTCTGACATAACTCGTCGAA AGACGGACTAATATCAGATAATGCAGCGATCCGGCATCGGATTGTTGTCAAAGCTTC GGCGCCCAAAGATGAGCCTGCGGTCCATTAGGTAGTTGGCGGAGTAACGGCCCACC AAGCCAACGATGGATAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGAC ACGGTCCAGACTCCTACGGGAGGCAGCAGTAAGGAATATTGCTCAATGGCCGAAAG GCTGAAGCAGCAACGCCGCGTGAAGGATGAAGGGTCTTTGGCTTGTAAACTTCTGTA AAAGGGGAAAAATAATCCCGCATTGCGGGACTTGATTGTACCCTTAAAGTAAGCCC CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGA TTTACTGGGTGTAAAGGGT
Swamcons-N5 ATGCTTTACACATGCAAGTCGAACGGCAGCGCGGGGGCAACCCTGGCGGCGAGTGG CGAACGGGTGAGTAATACATCGGAACGTACCTGGTAGTGGGGGATAGCTCGGCGAA AGCCGGATTAATACCGCATACGCACCATGGTGGAAAGCGGGGGATCGCAAGACCTC GCGCTATCGGAGCGGCCGATGTCGGATTAGCTTGTTGGTGGGGTAATGGCCTACCAA GGCTACGATCCGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACAC GGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCC TGATCCAGCAATGCCGCGTGTGCGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTC CGGAAAGAAATCCGTCTGGCTAATACCCGGATGGGATGACGGTACCCGGAAGAATA AGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAA TCGGAATTACTGGGCGTAAAGCGT
! !
Appendix E: Quantitative PCR (qPCR) Calculations
! 186! ! 187!
qPCR experiments calculations
In this section a sample calculation for quantification of 16S rRNA gene copies/ml of culture is described. The following sample calculation is for the enumeration of Azoarcus species in the DNA sample.
Several steps were taken to build a qPCR calibration curve for Azoarcus. In the first step the Azoarcus 16S rRNA gene-containing plasmid insert was amplified using PCR with T7 and M13 reverse primers. The size of this amplicon was calculated by adding the size of Azoarcus plasmid insert (1469 bp) to the size of flanking sequences from TOPO plasmid (179 bp).
Azoarcus amplicon size = 1469 bp + 179 bp = 1648 bp/16S rRNA copy (E.1) This amplicon was cleaned using GenEluteTM PCR Clean-up Kit (Sigma-Aldrich, St. Louis, MO, USA) and its concentration was quantified by Nanodrop spectrophotometer. Concentration of Azoarcus amplicon = 134.8 ng/µl (E.2) Using Azoarcus amplicon size and concentration, the Avogadro number (6.02E23 ) and the average molecular weight of a base pair in double stranded DNA (660 g/bp), the number of 16S rRNA gene copies/µl was calculated according to equation E.3: 6.02 "1023 (bp/mol bp) " Concentration (ng/µl) Quantity (copies/µl) = (E.3) Amplicon Size (bp/copy) " 660 (g/mol bp) "109 (ng/g) 6.02 "1023 "134.8 = = 7.46#10 copies/µl 1648 " 660 "109 ! The resulting amplicon was serially diluted from 10-1 to 10-8 in DNase-, RNase-, and ! Protease-free water (Sigma-Aldrich, St. Louis, MO, USA) and used to create the qPCR standard curve that is shown in Figure E.1. In this figure, the Y-axis represents the logarithm of Azoarcus 16S rRNA gene copies/µl, which is calculated for individual dilutions by multiplying equation E.3 by a dilution factor (10-1 to 10-8). The X-axis is the PCR cycle at which the fluorescence surpasses the threshold fluorescence C(T). The equation at the top left corner of the figure is the equation of standard curve. It should be noted that only dilutions that fall within the linear range are considered for constructing the standard curve. In this case, 10-1 dilution (the first point in Figure E.1) is excluded from the standard curve because its value was outside the linear range. If a dilution had a C(T) cycle higher than blank, then it would also be excluded from the standard
! ! 188!
curve.
Figure E.1. Calibration curve for Azoarcus qPCR. The first point is excluded from the standard curve because its value falls outside the linear range.
The number of 16S rRNA gene copies/µl for each sample can be calculated based on its C(T) value (X in the standard equation) using the standard curve equation. For example, a sample with C(T) value of 17.71 has 9.35E4 16S rRNA gene copies/µl. This value is converted into 16S rRNA gene copies/ml of culture by taking into account the volume of culture from which DNA was extracted, the volume of extracted DNA, and dilution factor as follows: Quantitity (copies/µl) " volume of DNA extract (µl) Quantity (copies/ml culture) = " Dilution factor volume of culture (ml)
If one considers that for the above sample, 50 µl of DNA was extracted from 50 ml of ! culture, and it was subsequently diluted by a factor of 10, this sample would contain 9.35E5
! ! 189!
Azoarcus 16S rRNA gene copies/ml of culture.
! !
Appendix F: 16S rRNA Gene Sequences of Isolated Colonies from Benzene-Degrading Nitrate-Reducing Enrichment Cultures
! 190! ! 191!
Dechloromonas DCh (1373 bp) ACTTCGGTCCGCCGGCGAGTGGCGAACGGGTGANTNATATATCGGAACGTACCTTTC NGNGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCNTATTCTGTGAGCAGGAA AGCAGGGGATCGCAAGACCTTGCGCTGATTGAGCGGCCGATATCAGATTAGCTAGT TGGNGAGGTAAAGGCTCNCCAAGGCGACGATCTGTAGNGGGTCTGANAGGATGANC CNCCACACTGGAACTGAGACACGGTCCANACTCCTACGGGAGGCAGCAGNGGGGA NTTTTGGACANNGGGGGCNACCCTGATCCNGCCATGCCGCGTGAGTGAAGAAGGCC TTCGGGTTGTAAAGCTCTTTCGGCCGGNAANAAATCGCATTGGTTAATACCCAGTGT GGATGACGGTACCGGAATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTA ATACGTAGGGTGCGAGCGTTAATCGGAATTACTNGGGCGTAAAGCGTGCGCAGGCG GTTTTGTAAGACAGGCGTGAAATCCCCGGGCTCAACCTGGGAACTGCGTTTGTGACT GCAAGGCTAGAGTATGGCAGAGGGGGGTGGAATTCCACGTGTAGCAGTGAAATGCG TAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGC TCATGCACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCC TAAACGATGTCAACTAGGTGTTGGGTGGGTAAAACCATTTAGTACCGGAGCTAACG CGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGA CGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAAC CTTACCTACCCTTGACATGTCCAGAAGCTCTTAGAGATTTGAGTGTGCCCGAAAGGG AGCTGGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTGTCGTTAATTGCCATCATTTAGTTGGGCACTT TAACGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCAT GGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTTGCCAA GCCGCGAGGTGGAGCCAATCCCAGAAAGCCGATCGTAGTCCGGATCGTAGGCTGCA ACTCGCCTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGGTGA ATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTTCCGCCA GAAGTAGGTAGCCTAACCGCAA
! ! 192!
Dechlorosoma Dsoma (1388 bp) CGGCAGCACGGGAGCTTGCTCCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATC GGAACGTACCCAGGAGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCNTAT TCTGTGAGCAGGAAAGCGGGGGATCGCAAGACCTCGCGCTCTTGGAGCGGCCGATG TCGGATTAGCTAGTTGGTGAGGTAAAAGCTCACCANGGCGACGATCCGTAGCAGGT CTGANAGGATGATCTGCCACACTGGGACTGAGACACGGCCCANACTCCTACGGGAG GCAGCAGNGGGGANTTTTGGACAATGGGGGCAACCCTGATCCNGCCATGCCGCGTG AGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTCGGCGGGNAANAAATGGCAACGG CTAATATCCGTTGTTGATGACGGTACCCGCATAAGAAGCACCGGCTAACTACGTGCC AGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAG CGTGCGCAGGCGGTTTCGTAAGACAGACGTGAAATCCCCGGGCTCAACCTGGGAAC TGCGTTTGTGACTGCGAGGCTAGAGTACGGCAGAGGGGGGTAGAATTCCACGTGTA GCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGG TTAGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG GTAGTCCACGCCCTAAACGATGTCAACTAGGTGTTGGAAGGGTTAAACCTTTTAGTA CCGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACT CAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGC AACGCGAAAAACCTTACCTACCCTTGACATGCCAGGAACTTTCCAGAGATGGATTGG TGCCCGAAAGGGAGCCTGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTG AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAATTGCCATCATTC AGTTGGGCACTTTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG TCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGTACA GAGGGTTGCCAAGCCGCGAGGTGGAGCCAATCCCAGAAAGCCGATCGTAGTCCGGA TCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCA TGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGT GGGTTCTACCAGAAGTAGTTAGCCTAACCGTAAGGA
! !
Appendix G: Microscopy Images of Dilution Cultures
! 193! ! 194!
(a)
(b)
(c)
Figure G.1. Cartwright 1b 10-2, Cartwright pw1 10-5, and Cartwright Consolidated 10-4 microscopy images taken at a 40X magnification.
! !
Appendix H: Analysis of rRNA and mRNA Sequences Obtained from Cartwright Consolidated Culture During Growth on Benzene and Benzoate
! 195! ! 196!
RNA extraction from cells grown on benzoate and benzene Employing the method explained in section 6.2.4 of Chapter 6, 8 RNA samples were extracted from Cartwright Consolidated culture during its growth on benzoate and benzene. Four of these RNA samples were obtained from the cells grown on benzoate and four were obtained from the cells grown on benzene. Figure H.1 shows the corresponding benzoate and benzene degradation curves and time points at which RNA was extracted from the culture.
160 160 Sample name: M3 Sample name: N 120 120
80 80
40 40
Benzene concentration (µM) Benzoate concentration (µM) 0 0 0 2 4 6 0 5 10 15 20 25 160 160 Sample name: M10 120 120
80 80 Sample name: M14
40 40 Benzoate concentration (µM) 0 Benzene concentration (µM) 0 0 2 4 6 0 20 40 60 80 160 %'"! Sample name: Cart cons Sample name: Cart cons benzoate benzene 120 %&"!
80 $"!
40 #"! Benzoate concentration (µM) Benzene concentration (µM) 0 "! 0 5 10 15 0 5 10 15 Time (Hours) Time (Days)
! ! 197!
160 160
120 120
80 80
40 40 Sample name: Cart cons Sample name: P2 benzene second trial
0 Benzene concentration (µM) 0 Benzoate concentration (µM) 0 2 4 6 8 10 0 5 10 15 Time (Hours) Time (Days) Figure H.1. Benzoate and benzene degradation curves. Arrows indicate time points at which RNA was extracted from Cartwright Consolidated culture. ! M10 and M14 RNA samples were sequenced using Pyrosequencing and Cart cons benzoate and Cart cons benzene RNA samples were sequenced employing Illumina sequencing. In the case of M RNA samples, the culture was fed first with benzoate and then with benzene whereas for Cart cons RNA samples, culture was supplied first with benzene and then with benzoate. In both cases, the culture was starved for one week between the feedings. After extraction of RNA, Nanodrop 1000 spectrophotometer was employed to quantify the amount of RNA. Table H.1 provides a summary of Nanodrop results. Table H.1. Quantity of RNA that is present in each individual RNA sample based on Nanodrop readings.
RNA sample Total volume of ng of RNA/µl of RNA 260/280 260/230 Total amount of name RNA sample sample RNA (µl) (µg) M3 55.8 160.8 1.68 1.77 9 N 63 848.9 1.75 2.06 53.5 M10 55 627.1 1.87 2.07 34.5 M14 60 366.5 1.74 2 22 Cart cons 60 457.2 1.84 2.09 27.4 benzoate Cart cons 57 571.3 1.67 2.02 32.6 benzene P2 55 470 2.10 2.16 25.9 Cart cons benzene second 59.4 479.6 1.76 1.96 28.5 trial
! ! 198!
The Agarose gels were employed to check the quality of extracted RNA. Figure H.2 shows the gel images for extracted RNA samples prior to the removal of contaminating DNA and after its removal. For RNA samples to be considered high quality, the 23S rRNA band on Agarose gel should be brighter or at least as bright as 16S rRNA band. In addition, the contaminating DNA that usually accompanies the RNA should be removed.
(a) Benzoate M3 M10 Cart cons benzoate P2 ! ! ! DNA
23S rRNA
16S rRNA
Cart cons benzene (b) Benzene N M14 Cart cons benzene second trial ! ! DNA 23S rRNA 16S rRNA
Figure H.2. Agarose gel images of individual RNA samples. a) The gel images of RNA extracted from benzoate-grown cells and b) The gel images of RNA extracted from benzene- grown cells. For each sample the left hand side lane corresponds to RNA gel image prior to DNA digestion and the right hand side lane represents the RNA gel image after removal of contaminating DNA. ! ! 199!
Analysis of Large subunit ribosomal RNA sequences
3_GAC_LSU_blastn 4_GAC_LSU_blastn Actinobacteria (127; 188) Benzoate Bacteroidetes/Chlorobi group (619; 237) Chlamydiae/Verrucomicrobia group (35; 33) Benzene Chloroflexi (34; 44) Cyanobacteria (45; 47) Deinococcus-Thermus (56; 56) Dictyoglomi (37; 21) Fibrobacteres/Acidobacteria group (209; 167) Bacilli (270; 882) Clostridiaceae (116; 139) Clostridiales Clostridiales incertae sedis (68; 121) Clostridia Firmicutes Peptococcaceae (60; 418) Syntrophomonadaceae (8; 16) Thermoanaerobacterales (39; 1462) Erysipelotrichi (0; 4) Negativicutes (9; 13) Bacteria cellular organisms Fusobacteria (4; 0) Planctomycetes (40; 42) root Alphaproteobacteria (200; 164) Burkholderiales (5233; 3630) Hydrogenophilales (181; 129) Methylophilales (6; 0) Betaproteobacteria Neisseriales (264; 223) Proteobacteria Nitrosomonadales (33; 31)
Rhodocyclaceae Aromatoleum (54542; 52921) Azoarcus (411; 381) Dechloromonas (105; 108) delta/epsilon subdivisions (197; 154) Gammaproteobacteria (119; 190) unclassified Proteobacteria (0; 5) Spirochaetes (16; 5) Tenericutes (3; 0) Thermotogae (15; 15) Eukaryota (1617; 110) Not assigned (448; 611) No hits Figure H.3. The MEGAN tree showing comparison between taxonomic profiles of Cartwright Consolidated culture grown with benzoate (red colors) and with benzene (blue colors). This community profiling is based on taxonomic affiliation of the ribosomal RNAs obtained in this study to the large subunit rRNA reference database compiled by Urich et al. (2008). The size of the tree nodes is scaled based on the number of reads assigned to each node. The first number and second number inside each prentices corresponds to the total number of hits to that node plus the number of hits in the subtree rooted at that node for cells supplied with benzoate and the ones fed with benzene, respectively. The number of counts is normalized in a way that each data set contains 100,000 reads.
! ! 200!
Assembly of mRNA sequences
In this study Newbler was employed to assemble the metatranscriptomic sequences. In the first step, Newbler builds a contig graph. Reads corresponding to the transcript of a certain gene should assemble into a single contig. There are usually several contigs for each transcript that form a small contig graph themselves (Figure H.4). Therefore, there are several subgraphs in assembly of trascriptomic reads. Each of these subgraphs is called an isogroup and potentially represents one gene (Figure H.4). Newbler traverses the contigs of each isogroup and generates transcript variants called isotigs. These isotigs can then be analyzed. Reads that cannot be placed in any contigs are reported as singletons in the Newbler output.
Contig 2
Contig 1 Contig 3 Contig graph
Contig 1 Contig 2 Contig 3 Isotig 1 Isogroup Contig 1 Contig 3 Isotig 2
Figure H.4. Steps for assembly of a gene by Newbler. http://contig.wordpress.com/category/using-newbler
! ! 201!
Table H.2. Denitrification pathway genes transcribed in Cartwright Consolidated culture grown on benzoate and benzene.
Sequence Id Size (bp) Gene name Function Organism NCBI Accession number%identity Score E-value COG#
Cells grown on Benzoate Nitrate reductase and nitrate/nitrite transport GMRHGY403G5PS4 497 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 83 274 3.00E-72 5013 GMRHGY403GAJ72 492 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 91 320 5.00E-86 5013 GMRHGY403GF7H1 497 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 93 325 1.00E-87 5013 GMRHGY403FKP6K 531 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 78 290 4.00E-77 5013 GMRHGY403GW449 180 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 94 77 3.00E-18 5013 GMRHGY403GQEVG 518 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 93 256 5.00E-69 5013 isotig00109 gene=isogroup00035 635 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 97 226 2.00E-88 5013 isotig00203 gene=isogroup00129 513 narG nitrate reductase, alpha subunit Aromatoleum aromaticum EbN1 ref|YP_160621.1| 87 312 1.00E-83 5013 GMRHGY403HETXU 457 narH nitrate reductase, beta subunit Aromatoleum aromaticum EbN1 ref|YP_160620.1| 94 125 8.00E-44 1140 GMRHGY403F72T7 507 narH nitrate reductase, beta subunit Aromatoleum aromaticum EbN1 ref|YP_160620.1| 87 286 5.00E-76 1140 GMRHGY403GIC2U 466 narI nitrate reductase, gamma subunit Aromatoleum aromaticum EbN1 ref|YP_160618.1| 83 270 3.00E-71 2181 GMRHGY403GBUX9 515 narK1 nitrate/nitrite antiporter Aromatoleum aromaticum EbN1 ref|YP_160622.1| 82 273 4.00E-72 2223 isotig00099 gene=isogroup00025 502 narK1 nitrate/nitrite antiporter Aromatoleum aromaticum EbN1 ref|YP_160622.1| 75 209 1.00E-52 2223 GMRHGY403FVTPW 496 narK2 nitrate/proton symporter Aromatoleum aromaticum EbN1 ref|YP_160623.1| 55 99 2.00E-19 2223 isotig00195 gene=isogroup00121 1198 narK2 nitrate/proton symporter Aromatoleum aromaticum EbN1 ref|YP_160623.1| 73 593 E-167 2223
Nitrite reductase and genes involved in cofactor biosynthesis GMRHGY403G100I 98 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 ref|YP_157499.1| 100 61.2 4.00E-08 2010 GMRHGY403GV75B 508 nirS putative dissimilatory nitrite reductase uncultured bacterium gb|AAL89549.1| 93 241 3.00E-63 GMRHGY403FWF5E 512 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 ref|YP_157499.1| 95 337 3.00E-91 2010 GMRHGY403GL87V 505 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 ref|YP_157499.1| 75 221 3.00E-63 2010 GMRHGY403FVG4G 513 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 ref|YP_157499.1| 98 343 5.00E-93 2010 GMRHGY403GCKHJ 524 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 ref|YP_157499.1| 68 260 4.00E-68 2010 GMRHGY403GGX0B 502 nirH Potential regulator involved in cofactor synthesis for nitrite reductase Aromatoleum aromaticum EbN1 ref|YP_157492.1| 75 250 4.00E-65 1522
Nitritic oxide reductase and corresponding activation factor GMRHGY403FY30B 119 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 ref|YP_157126.1| 78 37.7 1.00E-05 GMRHGY403GPUQ1 495 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 ref|YP_157126.1| 96 295 1.00E-78 GMRHGY403FVV1E 494 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 ref|YP_157126.1| 79 270 5.00E-71 GMRHGY403G31L0 494 norB putative nitric-oxide reductase subunit B Azoarcus sp. BH72 ref|YP_934592.1| 83 295 1.00E-78 isotig00142 gene=isogroup00068 543 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 ref|YP_157126.1| 93 278 2.00E-89 isotig00141 gene=isogroup00067 572 norC nitric-oxide reductase subunit C Aromatoleum aromaticum EbN1 ref|YP_157125.1| 87 210 1.00E-59 2010 GMRHGY403FYLHF 480 nitric oxide reductase activation protein Aromatoleum aromaticum EbN1 ref|YP_157132.1| 83 255 1.00E-66 4548 GMRHGY403F97SK 407 nitric oxide reductase activation protein Aromatoleum aromaticum EbN1 ref|YP_157132.1| 90 101 6.00E-31 4548 GMRHGY403FPBVP 500 nitric oxide reductase activation protein Aromatoleum aromaticum EbN1 ref|YP_157132.1| 95 208 1.00E-69 4548
Nitrous oxide reductase and transcription regulator GMRHGY403F76IV 298 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 ref|YP_160614.1| 95 145 2.00E-33 4263 GMRHGY403HGMXY 188 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 ref|YP_160614.1| 84 110 6.00E-23 4263 GMRHGY403GX9RL 391 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 ref|YP_160614.1| 54 97.4 5.00E-19 4263 GMRHGY403HBBEB 493 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 ref|YP_160614.1| 66 201 3.00E-50 4263 GMRHGY403F6RHJ 439 nosZ Nitrous-oxide reductase Ferroglobus placidus DSM 10642 ref|YP_003434591.1| 59 172 2.00E-41 isotig00162 gene=isogroup00088 477 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 ref|YP_160614.1| 88 272 6.00E-75 4263 GMRHGY403GLICE 81 nosR nitrous oxide expression regulator Variovorax paradoxus S110 ref|YP_002948075.1| 82 51.2 4.00E-05 GMRHGY403GVMWB 352 nosR transcription regulator Aromatoleum aromaticum EbN1 ref|YP_160613.1| 91 129 3.00E-50 GMRHGY403HD12H 504 nosR transcription regulator Aromatoleum aromaticum EbN1 ref|YP_160613.1| 84 100 5.00E-20 GMRHGY403F66L0 485 nosR transcription regulator Aromatoleum aromaticum EbN1 ref|YP_160613.1| 89 286 5.00E-76 GMRHGY403GHBCT 490 nosR transcription regulator Aromatoleum aromaticum EbN1 ref|YP_160613.1| 94 326 7.00E-88 GMRHGY403FRU37 524 nosL putative lipoprotein involved in nitrous oxide reduction Aromatoleum aromaticum EbN1 ref|YP_160608.1| 83 189 9.00E-47 4314 GMRHGY403G2G1I 174 nosL putative lipoprotein involved in nitrous oxide reduction Aromatoleum aromaticum EbN1 ref|YP_160608.1| 84 88.6 2.00E-16 4314
Cells grown on benzene Nitrite reductase GMRHGY404I2TYD 521 nirS cytochrome cd1 nitrite reductase precursor Methylomonas sp. 16a gb|ADB24711.1| 56 207 4.00E-52 2010 GMRHGY404IL6RW 539 nirS cytochrome cd1 nitrite reductase precursor Methylomonas sp. 16a gb|ADB24711.1| 56 207 4.00E-52 2010
Nitritic oxide reductase and corresponding activation factor GMRHGY404I9DII 147 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 ref|YP_157126.1| 81 50.4 7.00E-05 GMRHGY404I3BBW 508 norQ chaperone required for maturation of nitric oxide reductase Aromatoleum aromaticum EbN1 ref|YP_157129.1| 96 338 2.00E-91
Nitrous oxide reductase GMRHGY404HZ9MI 557 nosZ nitrous-oxide reductase Hydrogenobacter thermophilus TK-6ref|YP_003431831.1| 47 129 2.00E-28
! ! 202!
Analysis of mRNA sequences of benzoate- and benzene-grown cells
Table H.3. Benzoate-related genes that were transcribed in Cartwright Consolidated culture when it was supplied with benzoate as an electron donor and carbon source. The first section of this table provides the lists of sequences that were not assembled into a contig and the second section represents sequences that were assembled into contigs.
Sequence ID Size (bp) Gene name Product Function Organism NCBI Accesion number %identity Score E-value COG# GMRHGY403HDNLB 506 BclA/BzdA benzoate-CoA ligase Activation of benzoate to benzoyl-CoA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160038.1| 86 288 2.00E-76 GMRHGY403FX443 496 BzdA benzoate-CoA ligase Activation of benzoate to benzoyl-CoA Azoarcus sp. CIB gb|AAQ08820.1| 87 216 1.00E-54
GMRHGY403GQNP9 493 BcrA putative alpha subunit of benzoyl-CoA reductase The electron activation module of benzoyl-CoA reductase uncultured bacterium dbj|BAD91562.1| 61 180 5.00E-44 GMRHGY403HDRMV 499 BzdO Benzoyl CoA reductase, !-subunit The benzoyl-CoA reduction module Azoarcus sp. CIB gb|AAQ08807.1| 94 211 7.00E-73 1775 GMRHGY403G2HMG 503 Benzoyl CoA reductase, !-subunit The benzoyl-CoA reduction module Magnetospirillum magneticum AMB-1 ref|YP_421503.1| 80 274 3.00E-72 1775 GMRHGY403GYJ3Y 510 BcrC/BzdN Benzoyl CoA reductase, "-subunit The benzoyl-CoA reduction module Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160025.1| 90 290 6.00E-77 1775
GMRHGY403GJ00N 513 Dch/BzdW Dienoyl-CoA hydratase Acyl-CoA hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160034.1| 83 238 2.00E-61 GMRHGY403HAJL4 499 Dch/BzdW Dienoyl-CoA hydratase Acyl-CoA hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160034.1| 77 166 9.00E-40 GMRHGY403GQ93M 481 BzdX 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase Dehydrogenase Azoarcus evansii emb|CAD21637.1| 97 205 1.00E-51 GMRHGY403GSP1F 493 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase Dehydrogenase Thauera sp. MZ1T ref|YP_002890998.1| 73 243 8.00E-63 GMRHGY403F9DOE 469 Had 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase Dehydrogenase Thauera aromatica emb|CAA12244.2| 71 176 3.00E-48 GMRHGY403GDGSR 520 BzdY 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase Ring-opening hydrolase Azoarcus sp. CIB gb|AAQ08817.1| 89 322 9.00E-87
GMRHGY403F9QCI 265 putative ABC transporter subunit Benzoate-uptake Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160039.1| 93 125 1.00E-27 0683 GMRHGY403HBVTC 264 putative ABC transporter subunit Benzoate-uptake Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160039.1| 93 125 1.00E-27 0683 GMRHGY403GTHEE 265 putative ABC transporter subunit Benzoate-uptake Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160039.1| 93 125 1.00E-27 0683 GMRHGY403HBUV7 128 putative ABC transporter subunit Benzoate-uptake Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160039.1| 92 54.3 5.00E-06 0683 GMRHGY403FNO3H 485 putative ABC transporter subunit Benzoate-uptake Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160040.1| 74 152 2.00E-35 0684
GMRHGY403FQZTA 465 KorA 2-ketoglutarate: NADP oxidoreductase, alpha subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159623.1| 83 264 3.00E-69 GMRHGY403FZA8J 500 KorA 2-ketoglutarate: NADP oxidoreductase, alpha subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159623.1| 84 232 1.00E-59 GMRHGY403F5W7C 506 KorA 2-ketoglutarate: NADP oxidoreductase, alpha subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159623.1| 88 217 6.00E-78 GMRHGY403GIMYE 499 KorB 2-oxoglutarate ferredoxin oxidoreductase subunit beta Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159624.1| 86 267 3.00E-70 1013 GMRHGY403HC7JI 488 KorB 2-oxoglutarate ferredoxin oxidoreductase subunit beta Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159624.1| 81 252 1.00E-65 1013 GMRHGY403FJCWF 487 KorB 2-oxoglutarate ferredoxin oxidoreductase subunit beta Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158793.1| 94 155 3.00E-48 1013 GMRHGY403GBLM7 484 KorB 2-oxoglutarate ferredoxin oxidoreductase subunit beta Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158793.1| 93 192 1.00E-47 1013 GMRHGY403FJPQN 486 KorC 2-ketoglutarate: NADP oxidoreductase gamma subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159622.1| 89 291 2.00E-77 0497 GMRHGY403FKB56 478 KorC 2-ketoglutarate: NADP oxidoreductase gamma subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159622.1| 87 288 2.00E-76 0497 GMRHGY403FMEA4 495 KorC 2-ketoglutarate: NADP oxidoreductase gamma subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159622.1| 87 291 2.00E-77 0497 GMRHGY403G9FWL 209 KorC 2-ketoglutarate: NADP oxidoreductase gamma subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159622.1| 91 115 2.00E-24 0497 GMRHGY403FWRN1 166 KorC 2-ketoglutarate: NADP oxidoreductase gamma subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159622.1| 88 56.6 1.00E-06 0497 GMRHGY403F6LH6 484 BcrV/BzdV hypothetical protein/putative dehydrogenase subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160033.1| 98 335 1.00E-90 GMRHGY403G8ETO 521 bcrV/BzdV hypothetical protein/putative dehydrogenase subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160033.1| 83 305 1.00E-81 GMRHGY403GM23D 513 bcrV/BzdV hypothetical protein/putative dehydrogenase subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160033.1| 88 303 4.00E-81
GMRHGY403HGINX 316 BzdR Anaerobic benzoate catabolism transcriptional regulator Controlling the inducible expression of genes of benzoate catabolic Operan Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160023.1| 72 84.7 4.00E-15
GMRHGY403HC3VO 501 BzdS hypothetical protein Unkown function Azoarcus sp. CIB gb|AAQ08811.1| 80 149 2.00E-34 BzdM Ferredoxin The primary electron donor of benzoyl-CoA reductase Azoarcus sp. CIB gb|AAQ08810.1| 90 81.6 3.00E-14 1142 GMRHGY403G8R6S 496 BzdU hypothetical protein Unkown function Azoarcus sp. CIB gb|AAQ08813.1| 79 207 7.00E-67 0613 GMRHGY403HDNNY 461 BzdU hypothetical protein Unkown function Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160032.1| 86 167 4.00E-40 0613
Section 2. Assembled sequences
isotig00198 gene=isogroup00124 1685 BcrA/BzdQ Benzoyl CoA reductase, #-subunit The electron activation module of benzoyl-CoA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157401.1| 99 508 e-159 1924 BcrD/BzdP Benzoyl CoA reductase, $-subunit The electron activation module of benzoyl-CoA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157402.1| 93 390 e-106 1924 isotig00092 gene=isogroup00018 761 BcrB/BzdO Benzoyl CoA reductase, !-subunit The benzoyl-CoA reduction module Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160026.1| 93 486 E-135 1775 isotig00103 gene=isogroup00029 1093 BcrC/BzdN Benzoyl CoA reductase, "-subunit The benzoyl-CoA reduction module Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160025.1| 88 640 0 1775
isotig00129 gene=isogroup00055 1173 BzdX 6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase Dehydrogenase Azoarcus sp. CIB gb|AAQ08816.1| 92 439 e-137 isotig00151 gene=isogroup00077 1832 Oah/BzdY 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase Ring-opening hydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160036.1| 91 495 e-138 BzdZ Putative dehydrogenase Unknown Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160037.1| 77 376 e-102 1028
isotig00106 gene=isogroup00032 497 BzdB putative ABC transporter subunit Benzoate-uptake Azoarcus evansii emb|CAD21641.1| 96 326 7.00E-88 0683
isotig00087 gene=isogroup00013 563 KorA 2-ketoglutarate: NADP oxidoreductase, alpha subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159623.1| 84 281 2.00E-83 isotig00140 gene=isogroup00066 589 KorB 2-oxoglutarate ferredoxin oxidoreductase subunit beta Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159624.1| 93 198 3.00E-57 1013 contig00073 gene=isogroup00008 981 bcrV/BzdV hypothetical protein/putative dehydrogenase subunit Providing electrons for ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160033.1| 84 325 7.00E-84
isotig00094 gene=isogroup00020 509 BzdR Anaerobic benzoate catabolism transcriptional regulator Controlling the inducible expression of genes of benzoate catabolic Operan Azoarcus sp. CIB gb|AAQ08805.1| 73 238 2.00E-61
isotig00127 gene=isogroup00053 1150 BzdT Transcriptional regulator Unknown Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160031.1| 81 256 5.00E-66 BzdS hypothetical protein Unkown function Azoarcus evansii emb|CAD21633.1| 75 164 3.00E-38 Fdx/BzdM Ferredoxin The primary electron donor of benzoyl-CoA reductase Azoarcus evansii emb|CAD21632.1| 84 146 6.00E-33 1142 isotig00091 gene=isogroup00017 491 BzdU hypothetical protein Unkown function Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160032.1| 82 274 3.00E-72 0613
! ! 203!
Table H.4. Genes that were transcribed in Cartwright Consolidated culture during growth on benzoate. Section 1. non-assembled (singleton) sequences NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GSAE0 98 trmD tRNA (guanine-N1-)-methyltransferase Azoarcus sp. BH72 ref|YP_934403.1| 88 51.2 4.0E-05 GMRHGY403HCGMD 252 HlyD family secretion protein Achromobacter piechaudii ATCC 43553 ref|ZP_06688916.1| 85 64.3 5.0E-14 GMRHGY403FUWHL 238 CoxG cytochrome C oxidase assembly protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159415.1| 74 108 2.0E-22 GMRHGY403GCZTN 118 band 7 protein Thioalkalivibrio sp. K90mix ref|YP_003461331.1| 91 69.7 1.0E-10 GMRHGY403FPK5E 264 glnA glutamate-ammonia ligase, Achromobacter piechaudii ATCC 43553 ref|ZP_06688008.1| 92 117 1.0E-27 GMRHGY403GLICE 81 NosR nitrous oxide expression regulator Variovorax paradoxus S110 ref|YP_002948075.1| 82 51.2 4.0E-05 GMRHGY403HGOEN 173 TraU family protein Burkholderia glumae BGR1 ref|YP_002913230.1| 53 60.5 7.0E-08 GMRHGY403GGLRB 304 HNH endonuclease Thauera sp. MZ1T ref|YP_002891024.1| 42 66.2 2.0E-12 GMRHGY403FXNAG 188 ABC transporter related Nitrobacter hamburgensis X14 ref|YP_578254.1| 67 42.7 4.0E-07 GMRHGY403FQ8WZ 247 TonB TonB-dependent receptor Rhodothermus marinus DSM 4252 ref|YP_003291824.1| 46 53.5 9.0E-06 GMRHGY403GVORZ 225 excinuclease ABC subunit A Thiobacillus denitrificans ATCC 25259 ref|YP_314194.1| 55 81.3 4.0E-14 GMRHGY403F1N21 130 MreB rod shape-determining protein Azoarcus sp. BH72 ref|YP_931679.1| 100 71.2 4.0E-11 GMRHGY403G55FO 127 acetyl-CoA synthetase Burkholderia thailandensis TXDOH ref|ZP_02370425.1| 68 52.4 2.0E-05 GMRHGY403GD5C5 160 Integrase catalytic region Acidovorax delafieldii 2AN ref|ZP_04765470.1| 67 54.7 4.0E-06 GMRHGY403F155D 187 cheV1 chemotaxis protein CheV-like Azoarcus sp. BH72 ref|YP_932962.1| 77 101 4.0E-20 GMRHGY403F9NHM 252 fusion protein of flavin-containing oxidoreductase and iron-sulfur-containing oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158925.1| 84 85.9 2.0E-15 GMRHGY403F8F3J 388 O-methyltransferase family protein Salinispora tropica CNB-440 ref|YP_001157067.1| 53 144 3.0E-33 GMRHGY403FYNIG 182 TonB TonB-like Nitrosospira multiformis ATCC 25196 ref|YP_411744.1| 78 63.9 7.0E-09 GMRHGY403GK8HO 200 cheA3 chemotaxis protein cheA Azoarcus sp. BH72 ref|YP_932966.1| 75 57.8 4.0E-18 GMRHGY403GSZWM 245 type II secretion system protein, probably involved in Flp pilus synthesis Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159109.1| 67 90.9 5.0E-17 GMRHGY403GJP0U 90 putative methylase Azoarcus sp. BH72 ref|YP_932267.1| 96 57 8.0E-07 GMRHGY403GJAAZ 278 hipO4 hippurate hydrolase Achromobacter piechaudii ATCC 43553 ref|ZP_06685858.1| 68 90.1 7.0E-24 GMRHGY403GRXIX 201 diaminohydroxyphosphoribosylaminopyrimidine deaminase Alkalilimnicola ehrlichii MLHE-1 ref|YP_741220.1| 71 79 2.0E-13 GMRHGY403G5W4E 142 recC exodeoxyribonuclease V, gamma subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158556.1| 82 65.1 3.0E-09 GMRHGY403F76IV 298 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160614.1| 95 145 2.0E-33 GMRHGY403F5UGO 182 tRNA 2-selenouridine synthase Dinoroseobacter shibae DFL 12 ref|YP_001534683.1| 57 54.7 4.0E-06 GMRHGY403HBKMH 369 prfA peptide chain release factor 1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157645.1| 92 221 3.0E-56 GMRHGY403HCOMD 239 CoxG cytochrome C oxidase assembly protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159415.1| 80 123 9.0E-27 GMRHGY403F00LJ 402 LysR family transcriptional regulator Dechloromonas aromatica RCB ref|YP_283521.1| 52 79.7 1.0E-13 GMRHGY403GPKGY 222 MoxR-like ATPase in aerotolerance operon Flavobacteriaceae bacterium 3519-10 ref|YP_003096355.1| 77 63.9 7.0E-09 GMRHGY403GM0LW 393 ispG 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157699.1| 95 234 4.0E-60 GMRHGY403FWOE3 247 site-specific DNA-methyltransferase Bordetella petrii DSM 12804 ref|YP_001633049.1| 77 135 2.0E-30 GMRHGY403F9312 133 nuoN NADH dehydrogenase subunit N Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159778.1| 83 73.2 1.0E-11 GMRHGY403GD54V 353 cbbL ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit uncultured bacterium gb|AAX21118.1| 83 101 1.0E-38 GMRHGY403F9B1X 466 sulfatase Variovorax paradoxus S110 ref|YP_002945460.1| 79 137 6.0E-31 GMRHGY403G6F3X 167 thiF adenylyltransferase, putative Azoarcus sp. BH72 ref|YP_934307.1| 80 53.5 9.0E-06 GMRHGY403G2G1I 174 nosL putative lipoprotein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160608.1| 84 88.6 2.0E-16 GMRHGY403GD4YL 504 GCN5-related N-acetyltransferase Rhodopseudomonas palustris BisA53 ref|YP_780418.1| 57 139 1.0E-31 GMRHGY403FR12V 247 recB ATP-dependent exoDNAse beta subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158555.1| 50 61.6 3.0E-08 GMRHGY403G2JWI 318 NlpBDapX lipoprotein Dechloromonas aromatica RCB ref|YP_284073.1| 65 140 7.0E-32 GMRHGY403GKHH0 280 ATP-dependent protease domain protein Burkholderia thailandensis MSMB43 ref|ZP_02461956.1| 61 99.4 1.0E-19 GMRHGY403FMEPA 356 GTP-binding protein TypA Conexibacter woesei DSM 14684 ref|YP_003393807.1| 50 104 4.0E-21 GMRHGY403FOXYZ 110 HflK HflK Dechloromonas aromatica RCB ref|YP_286178.1| 75 55.5 2.0E-06 GMRHGY403HEL69 176 adenylyl cyclase class-3/4/guanylyl cyclase Dechloromonas aromatica RCB ref|YP_286613.1| 73 82.8 1.0E-41 GMRHGY403F3VWC 273 Peptidase M23 Methylovorus sp. SIP3-4 ref|YP_003049842.1| 61 102 2.0E-20 GMRHGY403FY2FH 173 rpmG 50S ribosomal protein L33 Azoarcus sp. BH72 ref|YP_932639.1| 90 55.8 2.0E-06 GMRHGY403GFO6J 307 2-oxoisovalerate dehydrogenase subunit beta Ochrobactrum intermedium LMG 3301 ref|ZP_04682180.1| 85 112 1.0E-36 GMRHGY403F75N9 397 ISRSO18-transposase protein Ralstonia solanacearum GMI1000 ref|NP_522697.1| 71 101 3.0E-43 GMRHGY403HEASO 170 phaP granule-associated protein (phasin) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160836.1| 80 50.8 6.0E-05 GMRHGY403G9FX3 329 CobN cobaltochelatase subunit Pseudomonas stutzeri A1501 ref|YP_001172312.1| 45 55.5 9.0E-16 GMRHGY403F61GV 210 copper-translocating P-type ATPase Pseudomonas putida GB-1 ref|YP_001666283.1| 90 60.8 8.0E-14 GMRHGY403FZAQA 195 two component LuxR family transcriptional regulator Burkholderia phymatum STM815 ref|YP_001862021.1| 48 38.9 7.0E-05 GMRHGY403GCGYI 306 amino acid-binding protein Azoarcus sp. BH72 ref|YP_934437.1| 50 90.9 5.0E-17 GMRHGY403FWVHJ 354 octaprenyl-diphosphate synthase Bordetella bronchiseptica RB50 ref|NP_886865.1| 64 60.1 9.0E-08 GMRHGY403HE1KX 201 toxin secretion ABC transporter permease and ATP-binding protein Azoarcus sp. BH72 ref|YP_932150.1| 68 80.9 5.0E-14 GMRHGY403FXD9R 209 peptidase Pseudomonas stutzeri A1501 ref|YP_001171545.1| 57 49.3 4.0E-06 GMRHGY403F5QSU 208 pilT twitching motility protein Azoarcus sp. BH72 ref|YP_934970.1| 90 117 4.0E-25 GMRHGY403G5O18 330 hemC porphobilinogen deaminase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157636.1| 95 192 9.0E-48 GMRHGY403HARAX 204 fructose-bisphosphate aldolase Marinomonas sp. MED121 ref|ZP_01077077.1| 88 65.9 2.0E-09 GMRHGY403GHHRT 257 gapA glyceraldehyde 3-phosphate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157603.1| 88 149 2.0E-34 GMRHGY403GWP0O 206 cysP periplasmic thiosulfate-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160570.1| 92 72 7.0E-20 GMRHGY403GY0QI 331 Pyruvate carboxylase Oceanicola granulosus HTCC2516 ref|ZP_01157011.1| 49 64.3 5.0E-09 GMRHGY403HAKFY 264 tauC nitrate/sulfonate/bicarbonate ABC transporter permease Herbaspirillum seropedicae SmR1 ref|YP_003777060.1| 88 149 9.0E-35 GMRHGY403F4SZN 367 rpmB 50S ribosomal protein L28 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159568.1| 98 143 6.0E-33 GMRHGY403G1CYE 372 homocysteine S-methyltransferase domain-containing protein Geobacter sulfurreducens PCA ref|NP_952380.1| 50 58.9 2.0E-07 GMRHGY403GMBL0 482 major facilitator transporter Syntrophobacter fumaroxidans MPOB ref|YP_847476.1| 72 148 3.0E-34 GMRHGY403FX5OL 448 cysA ATP-binding protein of thiosulfate ABC transporter Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160573.1| 84 183 3.0E-59 GMRHGY403G9MGX 342 50S ribosomal protein L1 Burkholderia oklahomensis EO147 ref|ZP_02357343.1| 74 82 2.0E-14 GMRHGY403HD2CR 110 HflK HflK Dechloromonas aromatica RCB ref|YP_286178.1| 75 52.8 2.0E-05 GMRHGY403GYXY4 114 DNA-directed RNA polymerase, beta subunit/140 kd subunit uncultured Pseudomonadales bacterium HF0010_05E14gb|ADI18882.1| 91 65.5 2.0E-09 GMRHGY403F34WG 488 hydroxylamine reductase Actinobacillus minor 202 ref|ZP_05628925.1| 52 102 1.0E-20 GMRHGY403GC3GJ 314 aspartate kinase Acidovorax delafieldii 2AN ref|ZP_04762184.1| 68 108 2.0E-25 GMRHGY403GB7FE 433 GTP-binding protein LepA Burkholderia pseudomallei 305 ref|ZP_01764368.1| 45 88.2 3.0E-16 GMRHGY403GN7VC 407 high-affinity nickel-transporter Rhodopseudomonas palustris BisA53 ref|YP_783705.1| 72 130 7.0E-29
! ! 204!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GIDD5 355 octaprenyl-diphosphate synthase Bordetella bronchiseptica RB50 ref|NP_886865.1| 64 60.1 9.0E-08 GMRHGY403GIPXO 181 ptsI PEP-utilizing enzyme, component EI of PTS system Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158597.1| 88 68.2 5.0E-15 GMRHGY403FTVW0 251 Electron transfer flavoprotein alpha/beta-subunit Hyphomicrobium ref|YP_003754342.1| 76 72.8 1.0E-11 GMRHGY403GNLSL 270 cell wall/surface repeat protein Thermotogales bacterium mesG1.Ag.4.2 ref|ZP_07577646.1| 36 50.4 7.0E-05 GMRHGY403GK9BX 465 pnp polynucleotide phosphorylase/polyadenylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160348.1| 98 289 7.0E-77 GMRHGY403G1Y1A 365 transport system ATP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159809.1| 83 136 8.0E-31 GMRHGY403GVV7X 421 pyrG CTP synthase Burkholderia phymatum STM815 ref|YP_001857669.1| 73 200 4.0E-50 GMRHGY403GNFFC 255 membrane protein involved in cell envelope biogenesis Azoarcus sp.BH72 ref|YP_935084.1| 42 54.3 5.0E-06 GMRHGY403HAAN4 468 gdhA glutamate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160107.1| 89 139 9.0E-32 GMRHGY403GJ6HX 450 dehydrogenase subunit, putative Methylococcus capsulatus str.Bath ref|YP_114984.1| 57 167 4.0E-40 GMRHGY403FKEBZ 268 uncharacterized lipoprotein Thiobacillus denitrificans ATCC 25259 ref|YP_314857.1| 64 51.6 5.0E-15 GMRHGY403GDLYE 125 rpoH RNA polymerase factor sigma-32 Azoarcus sp. BH72 ref|YP_934794.1| 87 59.3 2.0E-07 GMRHGY403G7G06 470 SecA preprotein translocase subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157807.1| 95 277 3.0E-73 GMRHGY403GL0BH 242 gltA citrate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160848.1| 96 124 3.0E-27 GMRHGY403F32F5 313 rpe putative ribulose-phosphate 3-epimerase Azoarcus sp. BH72 ref|YP_934829.1| 59 92.8 1.0E-17 GMRHGY403GONSJ 389 serine/threonine-protein kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159742.1| 87 176 3.0E-54 GMRHGY403FI6DA 424 icd isocitrate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157458.1| 93 258 2.0E-67 GMRHGY403F1SXH 421 fixC Electron-transferring-flavoprotein Magnetospirillum gryphiswaldense MSR-1 emb|CAM76746.1| 76 199 1.0E-49 GMRHGY403GABGU 269 dTDP-glucose 4,6-dehydratase Methanosarcina barkeri str. Fusaro ref|YP_304692.1| 50 56.6 1.0E-06 GMRHGY403F5I8I 402 Hydroxymethylbilane synthase Truepera radiovictrix DSM 17093 ref|YP_003704017.1| 42 91.7 3.0E-17 GMRHGY403HC9WK 372 diguanylate cyclase/phosphodiesterase with PAS/PAC sensor(s) Geobacter lovleyi SZ ref|YP_001950859.1| 45 115 2.0E-24 GMRHGY403G1A00 335 rpoN1 RNA polymerase factor sigma-54 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158944.1| 97 68.2 3.0E-10 GMRHGY403FSWRB 379 ABC transporter related Chloroflexus aurantiacus J-10-fl ref|YP_001634116.1| 55 137 4.0E-31 GMRHGY403G72HX 357 aceF dihydrolipoamide acetyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157095.1| 61 77.4 2.0E-29 GMRHGY403F9Q8Z 204 ascD CDP-6-deoxy-delta-3,4-glucoseen reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157989.1| 97 85.5 2.0E-15 GMRHGY403GKGKW 319 ggt putative sensory box/GGDEF family protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158656.1| 75 107 5.0E-22 GMRHGY403GJ0JI 485 aldehyde dehydrogenase 16 family, member A1 Xenopus (Silurana)tropicalis gb|AAI21296.1| 65 204 3.0E-51 GMRHGY403HBRGD 383 cyanophycin synthetase Ralstonia eutropha JMP134 ref|YP_296791.1| 78 192 2.0E-47 GMRHGY403GN7CV 470 FlhA flagellar biosynthesis protein Thauera sp. MZ1T ref|YP_002889913.1| 75 119 2.0E-51 GMRHGY403GNVE7 424 GTPase ObgE Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157468.1| 65 121 1.0E-26 GMRHGY403FZBX5 323 putative sulphurtransferase protein Azoarcus sp. BH72 ref|YP_932244.1| 69 137 5.0E-31 GMRHGY403G100I 98 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157499.1| 100 61.2 4.0E-08 GMRHGY403F9BYX 341 ATP-dependent helicase HrpA Cupriavidus metallidurans CH34 ref|YP_584217.1| 44 82 2.0E-14 GMRHGY403FURR4 146 cofE F420-0:gamma-glutamyl ligase Thermomicrobium roseum DSM 5159 ref|YP_002523830.1| 80 53.5 9.0E-06 GMRHGY403GBY2H 91 ABC transporter related protein Geobacter bemidjiensis Bem ref|YP_002138541.1| 88 50.1 1.0E-04 GMRHGY403G9EAP 407 ABC transporter, ATP-binding protein Thiobacillus denitrificans ATCC 25259 ref|YP_314006.1| 51 117 6.0E-25 GMRHGY403F64FV 94 NAD-dependent epimerase/dehydratase Dehalococcoides sp. BAV1 ref|YP_001214784.1| 81 52 3.0E-05 GMRHGY403GEXWW 440 NADH dehydrogenase I, D subunit Ktedonobacter racemifer DSM 44963 ref|ZP_06970159.1| 59 164 3.0E-39 GMRHGY403GVMFN 462 clpA ATP-dependent protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159563.1| 97 280 6.0E-74 GMRHGY403FQV5U 248 two component transcriptional regulator, winged helix family Thauera sp. MZ1T ref|YP_002355696.1| 73 103 6.0E-21 GMRHGY403F2EJT 446 sdhB succinate dehydrogenase iron-sulfur subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160850.1| 95 296 5.0E-79 GMRHGY403GZ3JG 398 MutS2 family protein Thermosediminibacter oceani DSM 16646 ref|YP_003825770.1| 52 81.6 3.0E-14 GMRHGY403HIFFU 397 Flp/Fap pilin component Ralstonia eutropha JMP134 ref|YP_296844.1| 65 66.6 1.0E-09 GMRHGY403GU31V 485 hupI rubredoxin protein Azoarcus sp. BH72 ref|YP_935298.1| 70 109 1.0E-22 GMRHGY403F022L 492 crp catabolite gene activator, (cAMP receptor protein) (cAMP-regulatory protein) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161022.1| 68 184 2.0E-45 GMRHGY403FO3NK 407 pstS1b protein sphX [precursor], phosphate transporter, signal peptide Herminiimonas arsenicoxydans ref|YP_001098835.1| 66 95.1 3.0E-18 GMRHGY403GT4G9 446 putative N-acetylneuraminate synthase Streptomyces scabiei 87.22 ref|YP_003489144.1| 62 102 2.0E-20 GMRHGY403FUURY 416 Peptidase S24/S26A/S26B, conserved region Halothiobacillus neapolitanus c2 ref|YP_003262775.1| 55 116 8.0E-25 GMRHGY403F96A9 152 glnA glutamine synthetase I Azoarcus sp. BH72 ref|YP_932242.1| 92 97.8 4.0E-19 GMRHGY403FZHQW 443 groeS chaperonins cpn10 (10 kDa subunit) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157651.1| 100 188 2.0E-46 GMRHGY403F2AZJ 421 CTP synthase Burkholderia phymatum STM815 ref|YP_001857669.1| 73 200 4.0E-50 GMRHGY403F0NH4 209 glyS glycyl-tRNA synthetase beta chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157746.1| 61 71.6 3.0E-11 GMRHGY403F64SW 451 putative S-adenosyl-L-methionine-dependent methyltransferase Ralstonia solanacearum CFBP2957 ref|YP_003745406.1| 47 125 1.0E-27 GMRHGY403FNECT 247 methanol/ethanol family PQQ-dependent dehydrogenase Leptothrix cholodnii SP-6 ref|YP_001790843.1| 82 90.5 2.0E-28 GMRHGY403HAYOM 362 ilvH acetolactate synthase 3 regulatory subunit Janthinobacterium sp. Marseille ref|YP_001353839.1| 82 154 4.0E-36 GMRHGY403GEHPE 423 lactoylglutathione lyase Escherichia coli MS 187-1 ref|ZP_07146251.1| 61 110 8.0E-23 GMRHGY403HEXG4 467 cytochrome B561 Sideroxydans lithotrophicus ES-1 ref|YP_003522801.1| 75 197 3.0E-49 GMRHGY403FI31H 194 ilvI thiamine pyrophosphate dependent acetolactate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161069.1| 87 108 3.0E-22 GMRHGY403GM5WR 449 Twin-arginine translocation pathway signal Limnobacter sp. MED105 ref|ZP_01916612.1| 80 237 3.0E-61 GMRHGY403FO7NH 378 tldD Zn-dependent peptidase, potential modulator of DNA gyrase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157538.1| 96 100 5.0E-34 GMRHGY403GI1LL 483 threonyl-tRNA synthetase Herpetosiphon aurantiacus ATCC 23779 ref|YP_001545616.1| 57 175 2.0E-42 GMRHGY403F3FCZ 496 Rho transcription termination factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159706.1| 94 311 2.0E-83 GMRHGY403FY30B 119 norB Nitric-oxide reductase Thauera sp. MZ1T ref|YP_002889577.1| 94 40 2.0E-07 GMRHGY403GAT7B 342 50S ribosomal protein L1 Burkholderia oklahomensis EO147 ref|ZP_02357343.1| 74 82 2.0E-14 GMRHGY403GYBQ6 361 fusion protein of flavin-containing oxidoreductase and iron-sulfur-containing oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158925.1| 92 201 3.0E-50 GMRHGY403G5MJ6 377 phasin; polyhydroxyalkanoate synthesis and granule formationregulator/factor Cupriavidus taiwanensis ref|YP_002005324.1| 40 80.1 9.0E-14 GMRHGY403FXHXT 519 heavy metal translocating P-type ATPase Nocardioides sp. JS614 ref|YP_922862.1| 48 148 2.0E-34 GMRHGY403FVJIT 450 putative ABC-type transport system, periplasmic component Ralstonia solanacearum PSI07 ref|YP_003752843.1| 48 131 3.0E-29 GMRHGY403G64RU 180 glycosyl transferase group 1 Chthoniobacter flavus Ellin428 ref|ZP_03131719.1| 55 56.6 1.0E-06 GMRHGY403GREUR 360 peptidase M48, Ste24p Solibacter usitatus Ellin6076 ref|YP_824973.1| 62 91.3 3.0E-28 GMRHGY403G5GR5 140 cbf2 PpiC-type peptidyl-prolyl cis-trans isomerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160742.1| 81 74.7 4.0E-12 GMRHGY403FUS9G 142 hflX GTP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157693.1| 92 51.6 3.0E-05 GMRHGY403FVPJO 398 fhs formyl tetrahydrofolate synthetase uncultured microorganism gb|ADE42995.1| 88 223 8.0E-57 GMRHGY403G6LW0 145 PpiC-type peptidyl-prolyl cis-trans isomerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160742.1| 83 59.8 2.0E-08 GMRHGY403HB80Y 425 putative short-chain alcohol dehydrogenase oxidoreductase protein Ralstonia solanacearum emb|CBJ38190.1| 49 97.1 7.0E-19 GMRHGY403FP555 309 formaldehyde-activating enzyme Burkholderia sp. CCGE1001 ref|ZP_06292380.1| 91 190 6.0E-47
! ! 205!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403FLGTD 324 putative sulphurtransferase protein Azoarcus sp. BH72 ref|YP_932244.1| 69 137 5.0E-31 GMRHGY403GFBOF 335 aroG phospho-2-dehydro-3-deoxyheptonate aldolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157562.1| 87 184 3.0E-45 GMRHGY403G4VQV 435 acyl-CoA dehydrogenase Janthinobacterium sp. Marseille ref|YP_001352277.1| 94 78.2 4.0E-24 GMRHGY403HETX9 298 helicase, putative, RecD/TraA family Ruminococcus sp. SR1/5 emb|CBL20603.1| 68 56.2 5.0E-09 GMRHGY403F6RHJ 439 Nitrous-oxide reductase Ferroglobus placidus DSM 10642 ref|YP_003434591.1| 59 172 2.0E-41 GMRHGY403GO9YK 481 FtsW cell division protein Oxalobacter formigenes HOxBLS ref|ZP_04577497.1| 62 201 3.0E-50 GMRHGY403FV0IV 260 Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP(+)), Phosphate acetyltransferasePedobacter heparinus DSM 2366 ref|YP_003094086.1| 70 43.5 5.0E-08 GMRHGY403GIM8R 302 nuoD NADH dehydrogenase subunit D Azoarcus sp. BH72 ref|YP_932903.1| 58 79 2.0E-15 GMRHGY403FN9A2 400 cardiolipin synthase 2 Polaromonas sp. JS666 ref|YP_546890.1| 67 182 2.0E-44 GMRHGY403GDM6Q 506 aspartokinase/homoserine dehydrogenase Gemmatimonas aurantiaca T-27 ref|YP_002760879.1| 79 60.8 1.0E-12 GMRHGY403GY97S 455 3-oxoacyl-(acyl-carrier-protein) reductase Rhodoferax ferrireducens T118 ref|YP_525276.1| 64 101 7.0E-39 GMRHGY403F06BE 459 decaheme c-type cytochrome, DmsE family Gallionella capsiferriformans ES-2 ref|YP_003847774.1| 55 93.2 1.0E-17 GMRHGY403FL21C 351 tig trigger factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159853.1| 87 176 1.0E-42 GMRHGY403G8L2S 425 diguanylate cyclase/phosphodiesterase with PAS/PAC sensor(s) Methylobacter tundripaludum SV96 ref|ZP_07655318.1| 70 190 6.0E-47 GMRHGY403GEAWM 504 molybdenum cofactor biosynthesis protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158179.1| 73 237 4.0E-61 GMRHGY403F0DAZ 482 methyl-accepting chemotaxis sensory transducer Geobacter bemidjiensis Bem ref|YP_002140664.1| 33 61.6 3.0E-08 GMRHGY403G4IH6 502 rubrerythrin Rhodopseudomonas palustris BisA53 ref|YP_779538.1| 44 76.3 1.0E-12 GMRHGY403F4GUW 447 Glutathione synthase/Ribosomal protein S6 modification enzyme Dyadobacter fermentans DSM 18053 ref|YP_003084871.1| 72 72 7.0E-20 GMRHGY403G35NY 452 rpsE 30S ribosomal protein S5 Azoarcus sp. BH72 ref|YP_934902.1| 75 142 1.0E-32 GMRHGY403GGOV0 138 glyoxalase/bleomycin resistance protein/dioxygenase Pseudomonas mendocina ymp ref|YP_001187506.1| 68 58.9 2.0E-07 GMRHGY403GP6JT 509 ugd UDP-glucose dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157507.1| 84 289 8.0E-77 GMRHGY403FUW0Y 326 NHL repeat containing protein Candidatus Methanosphaerula palustris E1-9c ref|YP_002465925.1| 54 120 4.0E-26 GMRHGY403G261N 194 phaZ AF474374_1 PHB depolymerase Azospirillum brasilense gb|AAQ05770.1| 64 67.8 4.0E-10 GMRHGY403F91FJ 522 rplE 50S ribosomal protein L5 Azoarcus sp. BH72 ref|YP_934907.1| 78 221 2.0E-56 GMRHGY403FRGHY 365 Methylmalonyl-CoA mutase Rhodopseudomonas palustris DX-1 ref|ZP_06360827.1| 66 89 1.0E-17 GMRHGY403GOUP4 532 suhB inositol monophosphatase (extragenic suppressor protein) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160693.1| 90 213 1.0E-53 GMRHGY403F4H95 466 phosphoribosylamine--glycine ligase Carboxydothermus hydrogenoformans Z-2901 ref|YP_359925.1| 52 92 2.0E-36 GMRHGY403GBXH4 495 purN phosphoribosylglycinamide formyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157363.1| 85 246 5.0E-64 GMRHGY403FVMEO 382 moaA molybdenum cofactor biosynthesis protein A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160111.1| 86 197 5.0E-49 GMRHGY403FXJPJ 325 rne ribonuclease E Azoarcus sp. BH72 ref|YP_933117.1| 92 50.4 2.0E-12 GMRHGY403GN1VK 441 soxC sulfite oxidase molybdopterin subunit Bradyrhizobium japonicum USDA 110 ref|NP_770156.1| 77 186 9.0E-46 GMRHGY403GPN19 410 cell divisionFtsK/SpoIIIE Sphingomonas wittichii RW1 ref|YP_001260209.1| 65 171 4.0E-41 GMRHGY403GMKSI 493 putative exported solute binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157567.1| 71 205 2.0E-56 GMRHGY403GUDM2 472 acetolactate synthase large subunit Nitrosococcus oceani ATCC 19707 ref|YP_343297.1| 67 211 2.0E-53 GMRHGY403HHMOB 442 lipoprotein, putative Dictyoglomus turgidum DSM 6724 ref|YP_002353178.1| 44 109 1.0E-22 GMRHGY403HHDMF 204 ascD CDP-6-deoxy-delta-3,4-glucoseen reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157989.1| 97 85.5 2.0E-15 GMRHGY403F3FRU 498 cysI putative sulfite reductase Azoarcus sp. BH72 ref|YP_931936.1| 75 216 8.0E-55 GMRHGY403FXSYW 295 exonuclease V subunit alpha Lawsonia intracellularis PHE/MN1-00 ref|YP_594664.1| 76 47.8 6.0E-07 GMRHGY403FRU2Y 519 putative oxidoreductase Streptomyces ambofaciens ATCC 23877 emb|CAJ90087.1| 44 71.6 3.0E-11 GMRHGY403HD0AQ 284 permease Thauera sp. MZ1T ref|YP_002355503.1| 92 164 5.0E-39 GMRHGY403F490H 475 panB 3-methyl-2-oxobutanoate hydroxymethyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161062.1| 75 222 1.0E-56 GMRHGY403FVGMA 414 argF ornithine carbamoyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159917.1| 94 257 4.0E-67 GMRHGY403G7FIB 261 L-serine ammonia-lyase Polaromonas sp. JS666 ref|YP_551156.1| 76 82.8 1.0E-14 GMRHGY403GJAVH 457 NUDIX family hydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159779.1| 87 217 4.0E-55 GMRHGY403G6I2I 347 ABC transporter, ATPase subunit Nitrobacter winogradskyi Nb-255 ref|YP_316794.1| 86 190 6.0E-47 GMRHGY403GIFY1 437 thms1 succinate semialdehyde dehydrogenase, [NAD(P)+] Azoarcus sp. BH72 ref|YP_933754.1| 74 210 4.0E-53 GMRHGY403G9214 360 IS3 family transposase Rhizobium etli CIAT 894 ref|ZP_03528191.1| 85 79.3 2.0E-29 GMRHGY403F63S7 254 4Fe-4S ferredoxin iron-sulfur binding domain protein Alicycliphilus denitrificans BC ref|ZP_07023175.1| 89 81.6 2.0E-23 GMRHGY403F1U7P 494 Two component transcriptional regulator, winged helix Bacillus mycoides Rock1-4 ref|ZP_04166437.1| 46 50.4 7.0E-05 GMRHGY403HGMXY 188 nosZ putative nitrous oxide reductase uncultured bacterium gb|ACJ02319.1| 88 114 3.0E-24 GMRHGY403G94X8 356 pyruvate phosphate dikinase Candidatus Koribacter versatilis Ellin345 ref|YP_590273.1| 67 80.1 9.0E-14 GMRHGY403GC7KL 484 trpS tryptophanyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160528.1| 95 164 3.0E-63 GMRHGY403GS1YU 493 pstS phosphate ABC transporter, periplasmic phosphate-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159759.1| 71 70.1 9.0E-11 GMRHGY403GXKWD 356 oligopeptidase A Burkholderia multivorans ATCC 17616 ref|YP_001579316.1| 68 164 5.0E-39 GMRHGY403F5MPP 519 glnS glutaminyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159333.1| 82 188 2.0E-46 GMRHGY403GEA8Y 518 leuC isopropylmalate isomerase large subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159717.1| 81 230 6.0E-59 GMRHGY403GST95 459 clpX ATP-dependent protease ATP-binding subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159855.1| 93 171 9.0E-52 GMRHGY403GOUNJ 113 murG N-acetylglucosaminyl transferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157816.1| 96 61.2 4.0E-08 GMRHGY403FM04P 481 pyrG CTP synthase Bordetella avium 197N ref|YP_785687.1| 77 212 1.0E-53 GMRHGY403HB65S 210 Wzz Pseudomonas aeruginosa C3719 ref|ZP_04929445.1| 50 62.8 1.0E-08 GMRHGY403GPUJ0 505 lpxD UDP-3-O-[3-hydroxymyristol] glucosamine N-acyltransferase Azoarcus sp. BH72 ref|YP_933403.1| 87 257 5.0E-72 GMRHGY403FPKDV 511 extracellular solute-binding protein family 5 Halothermothrix orenii H 168 ref|YP_002508243.1| 41 58.5 3.0E-16 GMRHGY403GO6KA 334 Cytochrome c oxidase, subunit II:Cytochrome c, class I:Cytochrome C oxidase subunit II Limnobacter sp. MED105 ref|ZP_01913893.1| 69 147 6.0E-34 GMRHGY403GQCY4 337 metZ O-succinylhomoserine sulfhydrylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159732.1| 77 37 2.0E-08 GMRHGY403FTYP2 82 rpsJ 30S ribosomal protein S10 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159182.1| 92 52.8 1.0E-05 GMRHGY403F1EIF 299 helicase, putative, RecD/TraA family Ruminococcus sp. SR1/5 emb|CBL20603.1| 68 56.2 6.0E-11 GMRHGY403HCEEE 454 istA transposase, [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158631.1| 70 124 5.0E-27 GMRHGY403GRZ58 482 putative transport ATPase Azoarcus sp. BH72 ref|YP_933793.1| 56 136 8.0E-31 GMRHGY403FMPIJ 444 argG Argininosuccinate synthase Candidatus Accumulibacter phosphatis clade IIA str.ref|YP_003168285.1| UW-1 90 139 9.0E-32 GMRHGY403F1SUW 509 lepB signal peptidase I (SPase I) transmembrane protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160177.1| 87 254 2.0E-72 GMRHGY403GEFX0 299 7TM receptor with intracellular metal dependent phosphohydrolase Rhodothermus marinus DSM 4252 ref|YP_003290506.1| 52 54.3 5.0E-06 GMRHGY403GG5E9 494 carboxyl-terminal protease Halothermothrix orenii H 168 ref|YP_002509379.1| 51 144 5.0E-33 GMRHGY403GQFSE 265 cell division protein FtsI/penicillin-binding protein 2 Kytococcus sedentarius DSM 20547 ref|YP_003149442.1| 66 60.8 5.0E-08 GMRHGY403GNCM5 464 thiC thiamine biosynthesis protein ThiC Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157949.1| 91 271 2.0E-71 GMRHGY403GOK48 434 metZ O-succinylhomoserine sulfhydrylase Azoarcus sp. BH72 ref|YP_932558.1| 73 202 1.0E-50 GMRHGY403G0KA3 399 Flp pilin component Cupriavidus taiwanensis ref|YP_002004748.1| 73 62.4 2.0E-08
! ! 206!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403G0UGN 491 cation transport ATPase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159373.1| 89 275 1.0E-72 GMRHGY403FZ5FV 447 ABC-2 type transporter Lutiella nitroferrum 2002 ref|ZP_03698777.1| 90 135 1.0E-30 GMRHGY403FYLOC 516 hisF imidazole glycerol phosphate synthase subunit HisF Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157717.1| 86 244 3.0E-63 GMRHGY403GOC0N 514 response regulator receiver protein Geobacter sp. M21 ref|YP_003021645.1| 33 66.6 1.0E-09 GMRHGY403G6JT6 406 threonine dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158940.1| 93 238 2.0E-61 GMRHGY403GM3SN 468 multi-sensor hybrid histidine kinase Methylobacter tundripaludum SV96 ref|ZP_07652558.1| 33 82.4 2.0E-14 GMRHGY403GNR9T 522 rpsO 30S ribosomal protein S15 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160347.1| 100 176 8.0E-43 GMRHGY403FJAKG 414 UDP-glucose 6-dehydrogenase Leptospirillum ferrodiazotrophum gb|EES52303.1| 46 57 3.0E-10 GMRHGY403GNV2M 448 glycogen phosphorylase Leptotrichia hofstadii F0254 ref|ZP_05901605.1| 67 134 3.0E-30 GMRHGY403GPQC2 518 dctQ C4-dicarboxylate transport system, permease small subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159381.1| 71 128 2.0E-49 GMRHGY403FT7AL 487 pip proline iminopeptidase Azoarcus sp. BH72 ref|YP_932689.1| 59 162 2.0E-38 GMRHGY403GH4K9 469 Ap4A phosphorylase II Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160008.1| 60 139 1.0E-31 GMRHGY403FQL1F 463 thiD putative phosphomethylpyrimidine kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157948.1| 56 149 9.0E-35 GMRHGY403HGL3G 520 recC exodeoxyribonuclease V, gamma subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158556.1| 65 169 2.0E-40 GMRHGY403F3TCD 215 cysK cysteine synthase A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159672.1| 70 60.5 7.0E-08 GMRHGY403FMCIH 504 phosphoribosylanthranilate isomerase Thiobacillus denitrificans ATCC 25259 ref|YP_315673.1| 45 120 6.0E-26 GMRHGY403GIK5Y 118 deoxyguanosinetriphosphate triphosphohydrolase-like protein Bordetella pertussis Tohama I ref|NP_882166.1| 86 66.6 1.0E-09 GMRHGY403GF0B6 464 sspA stringent starvation protein a Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157657.1| 88 271 2.0E-71 GMRHGY403GAELC 498 response regulator receiver protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157436.1| 60 185 2.0E-45 GMRHGY403GGZBY 430 TRAP dicarboxylate transporter, DctM subunit Prosthecochloris aestuarii DSM 271 ref|YP_002015097.1| 76 137 4.0E-34 GMRHGY403HEI9L 133 radical SAM family protein Dechloromonas aromatica RCB ref|YP_285185.1| 82 48.9 4.0E-06 GMRHGY403HD9GN 226 acetyl-CoA carboxylase, biotin carboxylase Thauera sp. MZ1T ref|YP_002890454.1| 95 131 3.0E-29 GMRHGY403HAKIZ 493 glutaredoxin-related protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157643.1| 90 165 2.0E-39 GMRHGY403F9LWO 115 trmA tRNA (uracil-5-)-methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157330.1| 82 60.5 7.0E-08 GMRHGY403G5G2S 476 lolE3 permease component of an ABC transporter system Azoarcus sp. BH72 ref|YP_934680.1| 63 182 1.0E-45 GMRHGY403GVZ6Y 487 glyA serine hydroxymethyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157571.1| 97 315 2.0E-84 GMRHGY403F8GLR 434 nitric oxide reductase activation protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157132.1| 77 109 1.0E-22 GMRHGY403FVYB9 548 TonB-dependent receptor Candidatus Koribacter versatilis Ellin345 ref|YP_589862.1| 25 60.1 1.0E-07 GMRHGY403GKXRV 120 putative tungsten-containing aldehyde ferredoxin oxidoreductase (AOR-1) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160067.1| 66 53.9 7.0E-06 GMRHGY403FOSWS 459 RpoS RNA polymerase sigma factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157427.1| 86 152 1.0E-35 GMRHGY403GB9HT 485 cysM cysteine synthase B Azoarcus sp. BH72 ref|YP_932581.1| 90 263 3.0E-74 GMRHGY403FYR6L 497 sucB dihydrolipoamide acetyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160846.1| 94 310 3.0E-83 GMRHGY403FRX6R 470 nuoN NADH dehydrogenase I chain N Herminiimonas arsenicoxydans ref|YP_001100093.1| 46 93.6 8.0E-18 GMRHGY403FK2Q9 384 PrkA PrkA protein Azoarcus sp. BH72 ref|YP_933577.1| 84 216 6.0E-55 GMRHGY403GJ5DR 432 pykA pyruvate kinase Thauera sp. MZ1T ref|YP_002355144.1| 69 177 5.0E-43 GMRHGY403GDTRX 503 msbA2 lipid A export ATP-binding/permease protein Azoarcus sp. BH72 ref|YP_935074.1| 72 150 9.0E-58 GMRHGY403FMONK 480 ccoN cytochrome-cbb3 oxidase, subunit I Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159939.1| 97 289 1.0E-82 GMRHGY403GRQUL 357 Extracellular ligand-binding receptor Alicycliphilus denitrificans BC ref|ZP_07023282.1| 71 152 1.0E-35 GMRHGY403GVG5P 429 LysR family transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159878.1| 78 205 2.0E-51 GMRHGY403FWF5E 512 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157499.1| 95 337 3.0E-91 GMRHGY403G5N93 441 aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit C Dechloromonas aromatica RCB ref|YP_283346.1| 62 107 5.0E-22 GMRHGY403FNFTM 202 penicillin-binding protein 7 Achromobacter piechaudii ATCC 43553 ref|ZP_06689381.1| 67 81.3 4.0E-14 GMRHGY403G1ICY 333 Pyruvate carboxylase Oceanicola granulosus HTCC2516 ref|ZP_01157011.1| 47 57.8 5.0E-07 GMRHGY403FTOGH 505 rlpB putative lipoprotein B precursor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159512.1| 74 103 8.0E-21 GMRHGY403GN8I1 477 rseA anti sigma-E protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160137.1| 54 120 6.0E-26 GMRHGY403FOO7J 481 arsB putative arsenical-resistance protein Azoarcus sp. BH72 ref|YP_933858.1| 83 248 2.0E-64 GMRHGY403F1UAO 515 ABC transporter ATP-binding protein Bordetella petrii DSM 12804 ref|YP_001631848.1| 85 110 8.0E-23 GMRHGY403GVCOW 496 PlsX putative glycerol-3-phosphate acyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160128.1| 68 172 9.0E-46 GMRHGY403GERS4 289 zinc-containing alcohol dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158774.1| 93 150 2.0E-38 GMRHGY403GL0GU 355 lasT RNA methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160692.1| 88 75.9 1.0E-16 GMRHGY403GONQY 501 PlsY putative glycerol-3-phosphate acyltransferase Rhodopseudomonas palustris BisB5 ref|YP_570157.1| 58 174 9.0E-43 GMRHGY403GPJJW 450 ABC transporter related protein Thermincola sp. JR ref|YP_003641208.1| 45 63.5 8.0E-09 GMRHGY403F96MV 488 TonB-dependent receptor plug Chloroherpeton thalassium ATCC 35110 ref|YP_001996949.1| 44 108 2.0E-22 GMRHGY403FM0QY 363 cardiolipin synthase 2 Azotobacter vinelandii DJ ref|YP_002799641.1| 68 122 2.0E-31 GMRHGY403FVGWW 174 asnB amidotransferase, asparagine synthase, (glutamine-hydrolyzing) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159440.1| 92 90.1 8.0E-17 GMRHGY403GQEGN 251 putative esterase Acidobacterium sp. SP1PR4 ref|ZP_07650024.1| 31 47.8 1.0E-05 GMRHGY403GV2TV 424 organic solvent resistance ABC transporter permease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157733.1| 94 162 1.0E-38 GMRHGY403G3ABK 330 cheA1 putative chemotaxis protein histidine kinase Azoarcus sp. BH72 ref|YP_931912.1| 78 144 1.0E-33 GMRHGY403GQMKF 496 ilvI thiamine pyrophosphate dependent acetolactate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161069.1| 87 108 3.0E-22 GMRHGY403G7ML8 477 rfaG glycosyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160967.1| 80 246 9.0E-64 GMRHGY403GRTSZ 497 hydroxyacylglutathione hydrolase Polaromonas sp. JS666 ref|YP_548883.1| 84 206 8.0E-52 GMRHGY403FN7FW 499 AMP-dependent synthetase and ligase Thauera sp. MZ1T ref|YP_002890336.1| 83 223 6.0E-57 GMRHGY403G8G8E 272 SecA preprotein translocase subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157807.1| 88 163 8.0E-39 GMRHGY403GO57U 507 putative phospholipid biosynthesis acyltransferase Azoarcus sp. BH72 ref|YP_935420.1| 64 183 8.0E-45 GMRHGY403HATBD 270 fcbC thioesterase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160542.1| 75 82.4 4.0E-25 GMRHGY403F2D73 465 putative indolepyruvate oxidoreductase subunit B Ralstonia solanacearum GMI1000 ref|NP_519949.1| 36 75.1 3.0E-12 GMRHGY403F9B1C 497 secretion protein HlyD family protein Thauera sp. MZ1T ref|YP_002354679.1| 85 67.8 4.0E-10 GMRHGY403GLEL7 281 FtsY cell division protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157778.1| 69 74.3 5.0E-12 GMRHGY403F9Z2S 498 glycosyl transferase family 39 Methylovorus sp. SIP3-4 ref|YP_003052464.1| 56 159 8.0E-38 GMRHGY403FWIZY 476 frr ribosome recycling factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160443.1| 89 154 2.0E-65 GMRHGY403GWNRD 468 infB translation initiation factor IF-2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160344.1| 64 107 5.0E-22 GMRHGY403F4OHH 98 argH argininosuccinate lyase Thauera sp. MZ1T ref|YP_002355088.1| 96 65.1 3.0E-09 GMRHGY403G5PS4 497 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 83 274 3.0E-72 GMRHGY403GNR35 386 substrate-binding protein Bradyrhizobium japonicum USDA 110 ref|NP_774479.1| 80 218 2.0E-55 GMRHGY403GXQVM 492 dhaL aldehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158262.1| 94 305 1.0E-81 GMRHGY403G7NR2 165 TonB-dependent receptor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159258.1| 82 91.3 4.0E-17
! ! 207!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GZ1DI 464 putative acetolactate synthase (acetohydroxy-acid synthase) (ALS)TPP-requiring enzyme Bradyrhizobium sp. BTAi1 ref|YP_001238124.1| 80 84 3.0E-30 GMRHGY403F691I 485 periplasmic nitrate (or nitrite) reductase c-type cytochrome,NapC/NirT family Burkholderia sp. H160 ref|ZP_03269642.1| 86 50.8 6.0E-05 GMRHGY403G6RR2 504 phbC2 poly-beta-hydroxybutyrate synthase Azoarcus sp. BH72 ref|YP_932854.1| 73 257 4.0E-67 GMRHGY403FT0UA 465 Na+/H+ antiporter Kribbella flavida DSM 17836 ref|YP_003381602.1| 63 77.4 6.0E-13 GMRHGY403GL87V 505 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157499.1| 75 221 3.0E-63 GMRHGY403GW9JC 421 serine/threonine protein kinase Janthinobacterium sp. Marseille ref|YP_001352544.1| 67 205 2.0E-51 GMRHGY403FQIO1 479 major facilitator transporter Syntrophobacter fumaroxidans MPOB ref|YP_847476.1| 72 148 3.0E-34 GMRHGY403G8RKW 437 rplS 50S ribosomal protein L19 Methylobacillus flagellatus KT ref|YP_546439.1| 92 148 9.0E-39 GMRHGY403GZ4WH 372 rplT large subunit ribosomal protein L20 Azospirillum sp. B510 ref|YP_003449782.1| 72 140 1.0E-36 GMRHGY403G0F7Y 495 rumA RNA methyltransferase, TrmA family Thauera sp. MZ1T ref|YP_002889979.1| 63 202 9.0E-51 GMRHGY403FYHE9 447 ABC transporter related Opitutaceae bacterium TAV2 ref|ZP_03726640.1| 68 187 4.0E-46 GMRHGY403G6E0Y 164 twin-arginine translocation pathway signal Dechloromonas aromatica RCB ref|YP_285108.1| 79 76.3 1.0E-12 GMRHGY403GYPEE 516 two component transcriptional regulator, LuxR family Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003168625.1| 62 73.2 1.0E-11 GMRHGY403GWTSO 516 TRAP transporter solute receptor TAXI family protein Polaromonas sp. JS666 ref|YP_548832.1| 84 302 9.0E-81 GMRHGY403FWFBN 183 PglZ domain family Octadecabacter antarcticus 238 ref|ZP_05064540.1| 68 65.9 6.0E-10 GMRHGY403GKC43 281 pyruvate oxidoreductase uncultured Chloroflexi bacterium emb|CAI78535.1| 77 76.6 5.0E-16 GMRHGY403GB4YP 509 putative lauroyl/myristoyl acyltransferase involved in LPS biosynthesis Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161032.1| 56 81.3 4.0E-14 GMRHGY403FU2UC 272 lysA1 diaminopimelate decarboxylase Azoarcus sp. BH72 ref|YP_934750.1| 58 95.5 2.0E-18 GMRHGY403GHK2B 441 flagellar hook-associated protein FlgK Citrobacter koseri ATCC BAA-895 ref|YP_001453539.1| 44 57 8.0E-07 GMRHGY403FMPAR 341 COG4177: ABC-type branched-chain amino acid transport system, permease component Magnetospirillum magnetotacticum ref|ZP_00053654.1| 50 108 2.0E-22 GMRHGY403HBE0U 501 lysC aspartate kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157354.1| 96 285 2.0E-78 GMRHGY403GJPK9 474 dgt1 deoxyguanosinetriphosphate triphosphohydrolase-like protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158269.1| 79 243 5.0E-63 GMRHGY403GOR44 332 General secretion pathway protein K Ralstonia pickettii 12J ref|YP_001900948.1| 87 73.9 3.0E-15 GMRHGY403GXG30 461 cytochrome C oxidase assembly transmembrane protein Herbaspirillum seropedicae SmR1 ref|YP_003777537.1| 68 67.8 4.0E-10 GMRHGY403F0TPC 481 cation transport P-type ATPase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159937.1| 86 274 3.0E-72 GMRHGY403GUT0V 485 mrdB rod shape-determining protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158740.1| 78 221 2.0E-56 GMRHGY403GPV1B 319 alpha/beta family hydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159785.1| 65 133 7.0E-30 GMRHGY403HFTL4 526 phbC poly-beta-hydroxybutyrate polymerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159697.1| 89 286 7.0E-76 GMRHGY403GAW49 511 glycosyl transferase group 1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159437.1| 71 249 1.0E-64 GMRHGY403HFPBV 464 CTP synthase Burkholderia sp. CCGE1002 ref|YP_003605448.1| 80 100 6.0E-41 GMRHGY403HCPW9 481 elmS putative two-component sensor histidine kinase Azoarcus sp. BH72 ref|YP_934482.1| 54 152 1.0E-35 GMRHGY403F7EZ5 483 methylmalonyl-CoA mutase Methylibium petroleiphilum PM1 ref|YP_001020100.1| 96 311 2.0E-83 GMRHGY403FQU6Q 493 fbaA fructose-1,6-bisphosphate aldolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157606.1| 93 309 9.0E-83 GMRHGY403G6BD8 326 thrC threonine synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159845.1| 90 194 3.0E-48 GMRHGY403GYWPP 269 kup2 potassium uptake protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159072.1| 89 137 5.0E-31 GMRHGY403FR5J3 174 leucine rich protein Escherichia sp. 3_2_53FAA ref|ZP_04532939.1| 59 55.1 3.0E-06 GMRHGY403GIF7V 516 glycosyl transferase, group 1 Acidothermus cellulolyticus 11B ref|YP_873687.1| 65 121 4.0E-37 GMRHGY403GMMLB 171 MORN repeat variant Fusobacterium periodonticum ATCC 33693 ref|ZP_06027764.1| 51 53.1 1.0E-05 GMRHGY403F5Q8C 512 ChaB family protein Micromonospora sp. L5 ref|ZP_06397383.1| 70 69.3 4.0E-12 GMRHGY403GP7IX 483 phosphotransferase domain-containing protein Thermofilum pendens Hrk 5 ref|YP_920831.1| 39 95.9 2.0E-18 GMRHGY403FVIY2 486 conserved hypothetical protein bacterium Ellin514 ref|ZP_03630806.1| 89 170 5.0E-41 GMRHGY403G4R0U 468 transcriptional regulator, TetR family Gallionellacapsiferriformans ES-2 ref|YP_003848250.1| 53 85.9 6.0E-28 GMRHGY403F1LVG 342 COG4177: ABC-type branched-chain amino acid transport system, permease component Magnetospirillum magnetotacticum MS-1 ref|ZP_00053654.1| 49 108 2.0E-22 GMRHGY403GKUH5 483 serine/threonine protein kinase Haliangium ochraceum DSM 14365 ref|YP_003268182.1| 44 92.4 2.0E-17 GMRHGY403GVJ4N 387 lpxH UDP-2,3-diacylglucosamine hydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159733.1| 65 101 3.0E-20 GMRHGY403FRGCY 460 fumA fumarate hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157121.1| 90 267 3.0E-70 GMRHGY403GJ4XJ 550 type III restriction protein res subunit Geobacter lovleyi SZ ref|YP_001953735.1| 73 74.7 5.0E-12 GMRHGY403FT64R 520 purF amidophosphoribosyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159731.1| 90 307 4.0E-82 GMRHGY403FRVS4 215 cysK cysteine synthase A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159672.1| 70 60.5 7.0E-08 GMRHGY403GTXFE 274 inner-membrane translocator Rhodoferax ferrireducens T118 ref|YP_521500.1| 55 60.1 9.0E-08 GMRHGY403F1YNI 446 putative cytochrome c-type protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160605.1| 92 156 1.0E-36 GMRHGY403FPBTE 303 nuoG NADH dehydrogenase subunit G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159771.1| 58 61.6 1.0E-16 GMRHGY403FK90M 421 phosphoserine aminotransferase Alicycliphilus denitrificans BC ref|ZP_07024126.1| 70 81.6 3.0E-11 GMRHGY403F71DW 468 phenylalanyl-tRNA synthetase, alpha subunit Thermoanaerobacter mathranii subsp.mathranii str.A3 ref|YP_003677244.1| 49 134 1.0E-33 GMRHGY403HHTRU 503 nolU carbamoyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159283.1| 94 332 8.0E-90 GMRHGY403F9CXM 500 rplD 50S ribosomal protein L4 Achromobacter piechaudii ATCC 43553 ref|ZP_06685077.1| 78 181 4.0E-44 GMRHGY403G9KIT 534 ribosomal-protein-alanine acetyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160734.1| 77 77.8 5.0E-13 GMRHGY403GRKT4 539 ATP-dependent DNA ligase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160827.1| 77 180 6.0E-44 GMRHGY403F5NON 403 L-asparaginase I Oceanospirillum sp. MED92 ref|ZP_01165373.1| 53 102 4.0E-24 GMRHGY403GB8NO 495 ilvE Branched-chain-amino-acid transaminase Ralstonia solanacearum emb|CBJ39080.1| 78 273 4.0E-72 GMRHGY403GIKME 561 40-residue YVTN family beta-propeller repeat protein Acidobacterium sp. MP5ACTX8 ref|ZP_07030988.1| 47 75.9 2.0E-12 GMRHGY403GAPDK 489 sbmB methylmalonyl-CoA mutase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159130.1| 98 319 8.0E-86 GMRHGY403G8X5D 468 excinuclease ABC subunit B Ralstonia eutropha JMP134 ref|YP_295274.1| 82 260 5.0E-68 GMRHGY403FIZJJ 464 acriflavin resistance protein Methylobacter tundripaludum SV96 ref|ZP_07655104.1| 46 139 2.0E-31 GMRHGY403GVP48 448 ABC-2 type transporter Lutiella nitroferrum 2002 ref|ZP_03698777.1| 90 135 1.0E-30 GMRHGY403HEMH5 502 trxB FAD-dependent pyridine nucleotide-disulphide oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161023.1| 92 231 2.0E-59 GMRHGY403HBLV7 488 bcp alkyl hydroperoxide reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160725.1| 66 218 2.0E-55 GMRHGY403HE70V 368 putative circadian clock protein, KaiC Desulfonatronospirathiodismutans ASO3-1 ref|ZP_07017763.1| 70 90.1 8.0E-17 GMRHGY403FU6GE 491 cation transport ATPase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159373.1| 89 275 1.0E-72 GMRHGY403GN4UW 475 RND family efflux transporter MFP subunit Acidovorax sp. JS42 ref|YP_984656.1| 63 184 3.0E-45 GMRHGY403F1L1C 366 nuoD NADH dehydrogenase subunit D Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159768.1| 94 248 2.0E-64 GMRHGY403GM5JQ 475 int integrase Pseudomonas putida gb|AAQ17120.1| 84 92.4 2.0E-17 GMRHGY403GEIAE 558 cutA2 thiol-disulfide interchange protein, potentially involved in divalent cation resistance Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159045.1| 72 237 8.0E-63 GMRHGY403G7F68 482 czcA2 putative cation efflux system protein Azoarcus sp. BH72 ref|YP_931772.1| 67 204 2.0E-51 GMRHGY403GVMWB 352 NosR transcription regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160613.1| 91 129 3.0E-50 GMRHGY403FYGX0 527 PilQ fimbrial type-IV assembly protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158272.1| 85 299 1.0E-79
! ! 208!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GVUXO 444 Argininosuccinate synthase Candidatus Accumulibacter phosphatisclade IIA str. UW-1 ref|YP_003168285.1| 90 139 9.0E-32 GMRHGY403HCDK0 476 cheV1 chemotaxis protein CheV-like Azoarcus sp. BH72 ref|YP_932962.1| 71 220 5.0E-56 GMRHGY403HEFFF 479 AraC family transcriptional regulator Chromobacterium violaceum ATCC 12472 ref|NP_902162.1| 40 107 7.0E-22 GMRHGY403GWSUO 508 Alcohol dehydrogenase GroES domain protein Arthrobacter chlorophenolicus A6 ref|YP_002488148.1| 73 120 6.0E-26 GMRHGY403GDVOF 528 radical SAM domain-containing protein Syntrophobacter fumaroxidans MPOB ref|YP_847165.1| 68 241 2.0E-62 GMRHGY403GKNJ2 499 lpxA UDP-N-acetylglucosamine acyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160452.1| 87 236 7.0E-61 GMRHGY403GI8HW 483 succinate dehydrogenase flavoprotein subunit Burkholderia ambifaria AMMD ref|YP_775202.1| 66 157 8.0E-38 GMRHGY403GIO09 489 glcB malate synthase G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157451.1| 88 291 1.0E-77 GMRHGY403F8721 533 fusion Na-dependent permease/histidine kinase domain-containing protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157117.1| 69 213 8.0E-54 GMRHGY403HAKVI 496 spooJ ParB-like partition protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158662.1| 58 129 2.0E-32 GMRHGY403HAXVQ 294 sensory box histidine kinase/response regulator,C-terminal part Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159144.1| 78 70.9 5.0E-11 GMRHGY403GU3FC 332 clpX ATP-dependent protease ATP-binding subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159855.1| 97 132 4.0E-44 GMRHGY403GSMJ6 518 ApaH bis(5'nucleosyl)-tetraphosphatase Fibrobacter succinogenes subsp. succinogenes S85 ref|YP_003248119.1| 38 113 1.0E-23 GMRHGY403G72H0 415 trmU tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase Azoarcus sp. BH72 ref|YP_934275.1| 96 213 5.0E-54 GMRHGY403G5JS7 511 glycosyl transferase group 1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159437.1| 71 249 1.0E-64 GMRHGY403G51L6 510 topB DNA topoisomerase III Bordetella avium 197N ref|YP_787875.1| 77 98.6 2.0E-19 GMRHGY403G1BEK 497 purL phosphoribosylformylglycinamidine synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160535.1| 91 305 2.0E-81 GMRHGY403F58SM 503 UDP-glucose/GDP-mannose dehydrogenase family protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157038.1| 82 272 9.0E-72 GMRHGY403FQGYO 439 rpsP 30S ribosomal protein S16 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161088.1| 96 129 1.0E-28 GMRHGY403FR0PK 352 clpS ATP-dependent Clp protease adaptor protein ClpS Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159564.1| 94 181 3.0E-44 GMRHGY403GRKOF 504 ZZ-type zinc finger-containing protein Dictyostelium discoideum AX4 ef|XP_646538.1| 35 53.1 1.0E-05 GMRHGY403GN5AK 349 phnF transcription regulator protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160854.1| 52 65.5 2.0E-09 GMRHGY403HANH5 504 dapE succinyl-diaminopimelate desuccinylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160678.1| 89 233 2.0E-67 GMRHGY403GNL94 485 fusA elongation factor G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159180.1| 96 317 4.0E-85 GMRHGY403G67D9 512 aldo/keto reductase Burkholderia multivorans CGD2M ref|ZP_03571565.1| 50 87.8 4.0E-16 GMRHGY403FYPD8 497 6-phosphogluconate dehydrogenase-like protein Anaeromyxobacter sp. Fw109-5 ref|YP_001377794.1| 68 134 5.0E-30 GMRHGY403GX9RL 391 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160614.1| 54 97.4 5.0E-19 GMRHGY403F165Y 433 ribosomal-protein-alanine acetyltransferase Thauera sp. MZ1T ref|YP_002355891.1| 69 68.9 2.0E-10 GMRHGY403GNCO5 479 clpP ATP-dependent Clp protease proteolytic subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159854.1| 93 145 4.0E-63 GMRHGY403GF433 185 putative molybdopterin-containing oxidoreductase Burkholderiacenocepacia J2315 ref|YP_002232577.1| 51 52.4 2.0E-05 GMRHGY403FYGUH 477 high-affinity branched-chain amino acid transport, ATP-binding protein Magnetospirillum gryphiswaldense MSR-1 emb|CAM75967.1| 74 80.9 6.0E-18 GMRHGY403FZKMS 296 putative deoxyribonuclease Brevibacillus brevis NBRC 100599 ref|YP_002769568.1| 37 66.2 1.0E-09 GMRHGY403F269T 477 nuoN NADH dehydrogenase subunit N Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159778.1| 76 225 2.0E-57 GMRHGY403FL6V6 489 zinc-containing alcohol dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158774.1| 94 252 1.0E-65 GMRHGY403GKC2S 483 UbiB putative ubiquinone biosynthesis protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159067.1| 80 248 2.0E-64 GMRHGY403F73QL 497 putative E1-E2 type ATPase Sinorhizobium meliloti ref|YP_001965581.1| 70 216 8.0E-55 GMRHGY403GA4AE 467 exoenzymes regulatory protein AepA precursor Streptomyces sp. AA4 ref|ZP_05483384.1| 48 82.4 2.0E-14 GMRHGY403FRYTR 455 sensory box protein Colwellia psychrerythraea 34H ref|YP_268068.1| 50 161 3.0E-38 GMRHGY403GQXCB 485 pal outer membrane protein, porin-associated lipoprotein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158518.1| 83 209 2.0E-59 GMRHGY403F97SK 407 norD nitric oxide reductase activation protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157132.1| 90 101 6.0E-31 GMRHGY403FRL1U 529 hisB imidazoleglycerol-phosphate dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157720.1| 91 331 2.0E-89 GMRHGY403GXMXR 510 rplA 50S ribosomal protein L1 Bordetella avium 197N ref|YP_784548.1| 77 123 9.0E-27 GMRHGY403F3V5O 429 tyrosyl-tRNA synthetase Ralstonia eutropha JMP134 ref|YP_294702.1| 65 56.6 9.0E-16 GMRHGY403G1AAD 438 peptidase C15, pyroglutamyl peptidase I Rhodopseudomonas palustris HaA2 ref|YP_487149.1| 50 130 4.0E-29 GMRHGY403HAG5K 302 gltD glutamate synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158267.1| 80 147 3.0E-34 GMRHGY403G7D81 468 lig DNA ligase Chromobacterium violaceum ATCC 12472 ref|NP_903573.1| 52 61.2 2.0E-14 GMRHGY403FXVWY 357 amine oxidase Xanthobacter autotrophicus Py2 ref|YP_001415018.1| 59 131 3.0E-29 GMRHGY403GGRVS 477 CRP/FNR family transcriptional regulator Burkholderia vietnamiensis G4 ref|YP_001115351.1| 56 75.9 2.0E-12 GMRHGY403GGPB4 508 xylose isomerase domain-containing protein Solibacter usitatus Ellin6076 ref|YP_827184.1| 63 176 9.0E-43 GMRHGY403G2KHM 397 lpxB lipid-A-disaccharide synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160453.1| 62 106 1.0E-21 GMRHGY403G3SQM 462 rnfA NADH:ferredoxin oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159805.1| 85 245 2.0E-63 GMRHGY403FXICT 353 cyt2 cytochrome c-552 precursor Azoarcus sp. BH72 ref|YP_935002.1| 70 93.6 8.0E-18 GMRHGY403GCA5G 480 complex regulator protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157435.1| 59 176 9.0E-43 GMRHGY403FRJ7K 454 dnaE DNA polymerase III alpha subunit Gemmatimonas aurantiaca T-27 ref|YP_002760249.1| 57 71.2 4.0E-11 GMRHGY403F2GRL 239 D-isomer specific 2-hydroxyacid dehydrogenase NAD-binding Rhodobacter sp. SW2 ref|ZP_05843400.1| 56 82.8 1.0E-14 GMRHGY403GG607 477 glcB malate synthase G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157451.1| 81 259 8.0E-68 GMRHGY403F8184 540 methionine sulfoxide reductase A Roseiflexus sp. RS-1 ref|YP_001277674.1| 56 187 5.0E-46 GMRHGY403FPWUJ 475 putative nucleotidyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159838.1| 69 214 3.0E-54 GMRHGY403G0LOZ 496 sucC succinyl-CoA synthetase subunit beta Azoarcus sp. BH72 ref|YP_934835.1| 85 221 2.0E-56 GMRHGY403F7NHY 490 oxidoreductase FAD/NAD(P)-binding domain protein Thauera sp. MZ1T ref|YP_002890694.1| 44 117 6.0E-25 GMRHGY403HFZRL 485 nadE NAD synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161026.1| 92 263 2.0E-69 GMRHGY403F6YCL 395 translation initiation factor eIF-2B alpha subunit Synechococcus elongatus PCC 6301 ref|YP_172814.1| 65 175 2.0E-42 GMRHGY403G75IU 485 purL phosphoribosylformylglycinamidine synthase Bordetella avium 197N ref|YP_786072.1| 46 90.9 1.0E-26 GMRHGY403FT65H 482 dnaB replicative DNA helicase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159344.1| 96 303 5.0E-81 GMRHGY403GUZZD 393 phenylacetate-CoA ligase Rhodospirillum rubrum ATCC 11170 ref|YP_427063.1| 68 187 5.0E-46 GMRHGY403FTYFH 500 fadH 2,4-dienoyl-CoA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160341.1| 79 263 4.0E-69 GMRHGY403F4CQ0 519 adiA Orn/Lys/Arg decarboxylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159530.1| 95 343 6.0E-93 GMRHGY403GF7BK 514 ffH signal recognition particle protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159492.1| 86 281 3.0E-74 GMRHGY403GQ5M8 477 lipopolysaccharide heptosyltransferase II Dechloromonas aromatica RCB ref|YP_286496.1| 38 110 8.0E-23 GMRHGY403FTTTX 501 rpoB DNA-directed RNA polymerase subunit beta Azoarcus sp. BH72 ref|YP_934926.1| 93 308 2.0E-82 GMRHGY403F6DFY 426 AAA family ATPase Prevotella ruminicola 23 ref|YP_003573975.1| 33 42.4 1.0E-08 GMRHGY403GMV1Q 502 xthA2 exodeoxyribonuclease III Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158898.1| 80 165 2.0E-39 GMRHGY403GHR3K 444 GroEL chaperonin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157650.1| 100 127 1.0E-57 GMRHGY403GYZN3 523 sspB ClpXP, protease specificity-enhancing factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157656.1| 89 158 3.0E-37 GMRHGY403FNKWA 205 ABC transporter related protein Burkholderia sp. CCGE1001 ref|ZP_06292042.1| 79 97.8 4.0E-19 GMRHGY403GV46Q 497 FtsZ cell division protein Oceanobacillus iheyensis HTE831 ref|NP_692394.1| 67 108 1.0E-40
! ! 209!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403HAVDL 497 putative membrane protein (TRAP dicarboxylate transporter DctMsubunit) Bordetella petrii DSM 12804 ref|YP_001630846.1| 72 99 2.0E-19 GMRHGY403FXUL5 330 guaB IMP dehydrogenase Azoarcus sp. BH72 ref|YP_933084.1| 90 166 9.0E-40 GMRHGY403G38LN 430 histidine kinase Pectobacterium carotovorum subsp. Brasiliensis PBR1692 ref|ZP_03825881.1| 41 51.2 2.0E-09 GMRHGY403F5JVX 531 Activator of Hsp90 ATPase 1 family protein bacterium Ellin514 ref|ZP_03629245.1| 73 200 7.0E-50 GMRHGY403FLMN7 511 TonB-dependent receptor plug Pseudomonas putida GB-1 ref|YP_001666818.1| 43 130 8.0E-29 GMRHGY403F5NSW 513 ftsH cell division protein ftsH-like protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159974.1| 62 144 1.0E-33 GMRHGY403GI5KE 379 ABC transporter related Chloroflexus aurantiacus J-10-fl ref|YP_001634116.1| 55 137 4.0E-31 GMRHGY403F2YA4 487 hyuB hydantoin utilization protein B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158162.1| 40 135 2.0E-30 GMRHGY403G06VK 454 rpoB DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159176.1| 97 189 1.0E-46 GMRHGY403GWZ0K 504 serC phosphoserine aminotransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157515.1| 76 253 6.0E-66 GMRHGY403FYRJW 500 infB translation initiation factor IF-2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160344.1| 87 271 2.0E-71 GMRHGY403G4CRJ 478 cbbL ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit form IA uncultured bacterium gb|ABY77404.1| 96 307 3.0E-82 GMRHGY403HCWFY 497 decaheme c-type cytochrome, DmsE family Gallionella capsiferriformans ES-2 ref|YP_003847774.1| 65 194 2.0E-48 GMRHGY403FNBL7 515 slyB outer membrane lipoprotein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157841.1| 50 95.1 9.0E-27 GMRHGY403HGCSU 429 aat leucyl/phenylalanyl-tRNA--protein transferase Azoarcus sp. BH72 ref|YP_933712.1| 68 78.2 3.0E-22 GMRHGY403FO2AO 494 TPR repeat-containing protein Herpetosiphon aurantiacus ATCC 23779 ref|YP_001544385.1| 41 65.1 3.0E-09 GMRHGY403FI817 486 cysM cysteine synthase B Azoarcus sp. BH72 ref|YP_932581.1| 90 296 6.0E-79 GMRHGY403F190G 507 clpA ATP-dependent protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159563.1| 91 237 4.0E-75 GMRHGY403FIJ5A 355 methanol/ethanol family PQQ-dependent dehydrogenase Leptothrix cholodnii SP-6 ref|YP_001790843.1| 78 196 1.0E-48 GMRHGY403HEBI1 409 ABC-2 type transporter Thauera sp. MZ1T ref|YP_002889821.1| 73 63.2 1.0E-08 GMRHGY403HG1AA 453 infB translation initiation factor IF-2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160344.1| 97 277 4.0E-73 GMRHGY403GSBXM 487 PilO fimbrial type-IV assembly protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158274.1| 87 211 2.0E-53 GMRHGY403GS3UU 486 D-alanine aminotransferase Paenibacillus larvae subsp. Larvae BRL-230010 ref|ZP_02328380.1| 43 96.7 9.0E-19 GMRHGY403FPW61 203 3-isopropylmalate dehydratase, large subunit Burkholderia sp. Ch1-1 ref|ZP_06842528.1| 79 104 3.0E-21 GMRHGY403FZ6YP 507 acxB acetone carboxylase alpha subunit Azoarcus communis gb|ABE73732.1| 91 323 5.0E-87 GMRHGY403F6A0K 481 extracellular solute-binding protein family 3 Thermotogales bacterium mesG1.Ag.4.2 ref|ZP_07579278.1| 31 46.2 8.0E-07 GMRHGY403G26MK 499 transcriptional regulatory protein Azoarcus sp. BH72 ref|YP_933316.1| 93 308 1.0E-82 GMRHGY403G0ZIP 497 wza putative polysaccharide export protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159428.1| 61 135 1.0E-30 GMRHGY403GM4B2 448 kinesin-like protein Leishmania major ref|XP_001687553.1| 34 53.5 9.0E-06 GMRHGY403F5OFJ 506 afg ATPase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160844.1| 92 76.3 1.0E-28 GMRHGY403G4FAO 399 peptidase S15 Solibacter usitatus Ellin6076 ref|YP_822114.1| 67 132 1.0E-29 GMRHGY403GG5Q0 477 fabF beta-ketoacyl-(acyl-carrier-protein) synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160133.1| 86 285 2.0E-75 GMRHGY403GW8J3 538 3-deoxy-D-manno-octulosonic-acid transferase Zunongwangia profunda SM-A87 ref|YP_003586191.1| 39 44.3 2.0E-09 GMRHGY403FV3JT 490 rne ribonuclease E, (RNase E) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160120.1| 76 87.8 4.0E-16 GMRHGY403F8DK9 488 metG methionyl-tRNA synthetase Azoarcus sp. BH72 ref|YP_934705.1| 78 254 3.0E-66 GMRHGY403GXXNS 481 ATPase, P-type (transporting), HAD superfamily, subfamily IC Polaromonas naphthalenivorans CJ2 ref|YP_973494.1| 76 234 4.0E-60 GMRHGY403F5EX3 495 PREDICTED: 30S ribosomal protein S3-like Xenopus (Silurana) tropicalis ref|XP_002944239.1| 89 173 6.0E-42 GMRHGY403HDHIQ 463 riboflavin biosynthesis protein RibD Burkholderia sp. CCGE1002 ref|YP_003605878.1| 61 164 3.0E-39 GMRHGY403G0JW9 234 RND family efflux transporter MFP subunit Anaeromyxobacter sp. Fw109-5 ref|YP_001381257.1| 53 45.8 7.0E-07 GMRHGY403F09SW 472 livG branched-chain amino acid transport system ATP-binding protein Azospirillum sp. B510 ref|YP_003449640.1| 62 189 1.0E-64 GMRHGY403GMGQF 508 Rieske (2Fe-2S) domain-containing protein Novosphingobium aromaticivorans DSM 12444 ref|YP_001165920.1| 52 177 4.0E-43 GMRHGY403FNFU8 499 mcrA penicillin-binding 1 (peptidoglycan synthetase) transmembrane protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158277.1| 78 264 2.0E-69 GMRHGY403GC51X 523 pcm1 protein-L-isoaspartate(D-aspartate) O-methyltransferase Dechloromonas aromatica RCB ref|YP_285726.1| 66 73.9 5.0E-36 GMRHGY403G94D1 500 putative glycine cleavage T-protein (aminomethyl transferase) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160100.1| 76 256 9.0E-67 GMRHGY403HD12H 504 NosR transcription regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160613.1| 84 100 5.0E-20 GMRHGY403G1T1I 475 livH ABC transporter permease protein Azoarcus sp. BH72 ref|YP_935233.1| 43 54.3 3.0E-15 GMRHGY403FQDOP 500 fructose-bisphosphate aldolase Polaromonas sp. JS666 ref|YP_551518.1| 66 78.6 2.0E-13 GMRHGY403HEMIE 475 complex regulator protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157435.1| 91 209 9.0E-53 GMRHGY403FT7JZ 483 carB carbamoyl phosphate synthase large subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159751.1| 98 312 8.0E-84 GMRHGY403GSMWE 466 FdhC formate transporter Methanothermobacter thermautotrophicus gb|AAC44819.1| 41 113 7.0E-24 GMRHGY403GVH7N 531 acsB acetyl-CoA synthetase Azoarcus sp. BH72 ref|YP_933918.1| 94 352 1.0E-95 GMRHGY403FM8MG 415 pstA phosphate ABC transporter, permease protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159761.1| 96 202 3.0E-57 GMRHGY403GI6L9 491 tryptophan synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159353.1| 85 274 3.0E-72 GMRHGY403FU97M 354 phospholipase D/transphosphatidylase Anaeromyxobacter dehalogenans 2CP-C ref|YP_464202.1| 79 53.5 5.0E-13 GMRHGY403FSPO6 469 excinuclease ABC subunit B Herpetosiphon aurantiacus ATCC 23779 ref|YP_001547725.1| 67 103 6.0E-21 GMRHGY403G0S8J 190 rplR 50S ribosomal protein L18 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159199.1| 73 50.1 1.0E-04 GMRHGY403GPDY0 502 response regulator receiver protein Herpetosiphon aurantiacus ATCC 23779 ref|YP_001543994.1| 66 233 5.0E-60 GMRHGY403HCUXD 470 gapD succinate semialdehyde dehydrogenase Candidatus Nitrospira defluvii ref|YP_003799569.1| 64 197 4.0E-49 GMRHGY403GFGGG 458 lon ATP-dependent protease La Bordetella petrii DSM 12804 ref|YP_001631399.1| 62 185 2.0E-45 GMRHGY403GF9M0 484 alanine dehydrogenase/PNT-like Frankia sp. CcI3 ref|YP_482033.1| 68 71.6 3.0E-11 GMRHGY403FRSSP 503 extracellular solute-binding protein family 1 Halothermothrix orenii H 168 ref|YP_002509199.1| 54 183 7.0E-45 GMRHGY403G0576 462 lysR transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160731.1| 90 208 2.0E-52 GMRHGY403G9URQ 338 salicylyl-CoA 5-hydroxylase Streptomyces sp. AA4 ref|ZP_07282785.1| 42 56.2 1.0E-06 GMRHGY403FKP0S 485 organic acid-CoA ligase (ADP forming) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160094.1| 70 179 1.0E-44 GMRHGY403GARHT 528 hisC histidinol-phosphate aminotransferase [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157721.1| 75 214 5.0E-54 GMRHGY403FP30I 503 argH argininosuccinate lyase Ralstonia solanacearum PSI07 ref|YP_003751766.1| 95 149 2.0E-43 GMRHGY403HCB3N 365 two component, sigma54 specific, transcriptional regulator, Fis family Thauera sp. MZ1T ref|YP_002355251.1| 78 145 2.0E-42 GMRHGY403GELYU 339 gatC aspartyl/glutamyl-tRNA amidotransferase subunit C Azoarcus sp. BH72 ref|YP_931678.1| 70 119 2.0E-25 GMRHGY403FLPRH 482 iron-sulfur cluster-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157131.1| 92 307 3.0E-82 GMRHGY403GZZ3R 485 oppB oligopeptide transport system permease protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160830.1| 97 94.7 3.0E-18 GMRHGY403GRIJ4 485 serine protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157036.1| 82 82.8 1.0E-14 GMRHGY403HC5IQ 483 integral membrane sensor signal transduction histidine kinase Afipia sp. 1NLS2 ref|ZP_07027450.1| 76 245 2.0E-63 GMRHGY403FL3J9 480 ilvD dihydroxy-acid dehydratase Aromatoleum aromaticum EbN1 ref|YP_158011.1| 96 300 3.0E-80 GMRHGY403G1VAU 516 HisF imidazoleglycerol phosphate synthase, cyclase subunit Thauera sp. MZ1T ref|YP_002354572.1| 81 167 5.0E-40 GMRHGY403GA36H 472 livG branched-chain amino acid transport system ATP-binding protein Azospirillum sp. B510 ref|YP_003449640.1| 62 189 1.0E-46 GMRHGY403G8PIV 486 penicillin-binding protein 6 Dechloromonas aromatica RCB ref|YP_283520.1| 65 110 2.0E-33
! ! 210!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403G10V3 496 greA transcription elongation factor Aromatoleum aromaticum EbN1 ref|YP_159752.1| 92 243 8.0E-63 GMRHGY403GV5VO 270 segregation and condensation protein B Chloroherpeton thalassium ATCC 35110 ref|YP_001995984.1| 50 40 1.0E-07 GMRHGY403GNFT1 503 rpoE2 RNA polymerase sigma factor RpoE Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160136.1| 98 185 1.0E-78 GMRHGY403FMTYZ 514 ugd UDP-glucose dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157507.1| 79 174 4.0E-42 GMRHGY403FVSPS 462 PilQ fimbrial type-IV assembly protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158272.1| 92 245 2.0E-64 GMRHGY403GVFQO 491 glycosyl transferase family 51 Sphaerobacter thermophilus DSM 20745 ref|YP_003321480.1| 44 139 9.0E-32 GMRHGY403FQIZ6 394 rdgC recombination associated protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158704.1| 77 189 1.0E-46 GMRHGY403GQ9E0 361 ribosomal large subunit pseudouridine synthase D Oxalobacter formigenes OXCC13 ref|ZP_04579442.1| 57 124 5.0E-27 GMRHGY403F7D6E 487 cbiD cobalamin biosynthesis protein CbiD Thauera sp. MZ1T ref|YP_002890709.1| 62 159 1.0E-37 GMRHGY403FW5E7 526 ABC transporter related Solibacter usitatus Ellin6076 ref|YP_825659.1| 37 105 3.0E-21 GMRHGY403F2F47 511 aminotransferase class-IV Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158743.1| 82 142 1.0E-32 GMRHGY403GQMUK 488 helicase-related protein Pseudomonas aeruginosa gb|ACD39011.1| 52 154 4.0E-36 GMRHGY403F2A1B 508 acnA2 aconitate hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160900.1| 76 265 2.0E-69 GMRHGY403F4658 512 Cobyrinic acid ac-diamide synthase Rhodothermus marinus DSM 4252 ref|YP_003289742.1| 60 187 3.0E-46 GMRHGY403HAC02 511 alpha-amylase family protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161007.1| 71 181 4.0E-44 GMRHGY403FKW1X 492 hisS histidyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157698.1| 84 265 1.0E-70 GMRHGY403GNT7J 473 adenosylhomocysteinase Thauera sp. MZ1T ref|YP_002890808.1| 96 308 2.0E-82 GMRHGY403F1PHO 498 ctpa carboxy-terminal processing protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157576.1| 82 236 5.0E-61 GMRHGY403GL45L 498 pyrG CTP synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160544.1| 96 318 1.0E-85 GMRHGY403GZHU2 499 hslU ATP-dependent protease ATP-binding subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158510.1| 95 209 7.0E-53 GMRHGY403GL1E2 495 rplY 50S ribosomal protein L25/general stress protein Ctc Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157789.1| 78 244 3.0E-63 GMRHGY403GSZIX 523 gltA citrate synthase Deferribacter desulfuricans SSM1 ref|YP_003496048.1| 64 231 3.0E-59 GMRHGY403GNA4W 504 NUDIX hydrolase Roseiflexus sp. RS-1 ref|YP_001275649.1| 44 118 3.0E-25 GMRHGY403GYLWO 537 heat shock protein HslVU, ATPase subunit HslU Chloroherpeton thalassium ATCC 35110 ref|YP_001995100.1| 74 248 2.0E-64 GMRHGY403G8HCM 515 VACJ lipoprotein precursor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157731.1| 81 231 7.0E-74 GMRHGY403GSJRA 485 clpA ATP-dependent protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159563.1| 94 291 2.0E-77 GMRHGY403HDTWY 439 FeS assembly protein SufB Thermobaculum terrenum ATCC BAA-798 ref|YP_003323426.1| 66 141 2.0E-40 GMRHGY403FN6F8 432 glyS glycyl-tRNA synthetase beta chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157746.1| 70 167 3.0E-40 GMRHGY403F58PB 483 Smr protein/MutS2 Leptothrix cholodnii SP-6 ref|YP_001789736.1| 62 189 1.0E-46 GMRHGY403GSNET 491 glS1 ferredoxin-dependent glutamate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158268.1| 89 278 1.0E-73 GMRHGY403F2GXM 507 putative transferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159400.1| 72 212 6.0E-60 GMRHGY403GCCMY 330 SecA preprotein translocase subunit Azoarcus sp. BH72 ref|YP_932397.1| 53 79.3 1.0E-13 GMRHGY403F2MGG 503 GTPase EngB Burkholderia sp. 383 ref|YP_367724.1| 52 141 3.0E-32 GMRHGY403FZHE2 493 glS1 ferredoxin-dependent glutamate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158268.1| 98 283 4.0E-75 GMRHGY403GZM53 489 tolC type I secretion outer membrane efflux protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157920.1| 54 155 2.0E-36 GMRHGY403HC1A5 497 dhpS dihydropteroate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159757.1| 68 216 6.0E-55 GMRHGY403FTVF7 506 Prephenate dehydrogenase Sphaerobacter thermophilus DSM 20745 ref|YP_003319062.1| 38 82 2.0E-14 GMRHGY403FPQEZ 540 DNA helicase uncultured archaeon GZfos32E7 gb|AAU83667.1| 62 92.4 2.0E-21 GMRHGY403HD90T 533 TonB-dependent copper receptor Sideroxydans lithotrophicus ES-1 ref|YP_003523357.1| 72 247 8.0E-66 GMRHGY403HHJPK 592 PemK-like protein Beggiatoa sp. PS ref|ZP_02001028.1| 55 139 2.0E-31 GMRHGY403FR6YX 469 porin Gram-negative type Thauera sp. MZ1T ref|YP_002354740.1| 41 77 7.0E-13 GMRHGY403F1A66 468 periplasmic lipoprotein Spirochaeta smaragdinae DSM 11293 ref|YP_003801981.1| 33 81.6 3.0E-14 GMRHGY403GLRYD 494 LuxR family DNA-binding response regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160938.1| 85 171 4.0E-41 GMRHGY403HHSXS 469 multicomponent K+:H+ antiporter subunit E, pH adaptation Thauera sp. MZ1T ref|YP_002889422.1| 78 151 2.0E-35 GMRHGY403F4DO9 538 short-chain dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159936.1| 96 206 8.0E-52 GMRHGY403F68AO 513 etfA electron transfer flavoprotein alpha subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160745.1| 91 103 2.0E-41 GMRHGY403FJU31 492 gcvP1 glycine dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160522.1| 85 237 9.0E-65 GMRHGY403FOXAI 515 twin-arginine translocation pathway signal Citreicella sp. SE45 ref|ZP_05782186.1| 71 254 3.0E-66 GMRHGY403FOIVS 481 cysM cysteine synthase B Azoarcus sp. BH72 ref|YP_932581.1| 91 291 4.0E-78 GMRHGY403FPFDU 434 phoB phosphate regulon transcriptional regulatory protein Azoarcus sp. BH72 ref|YP_931669.1| 84 233 6.0E-60 GMRHGY403GOONV 507 ampG muropeptide transporter Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158605.1| 72 194 4.0E-48 GMRHGY403FLYYC 447 glmS glutamine amidotransferase class-II Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158263.1| 100 144 3.0E-33 GMRHGY403GHBL9 203 ppcK phosphoenolpyruvate carboxykinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159317.1| 88 103 6.0E-21 GMRHGY403FJO0I 172 trmU tRNA 5-methylaminomethyl-2-thiouridylate-methyltransferase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157545.1| 90 94.7 3.0E-18 GMRHGY403GHQ43 483 insecticidal toxin protein, C-terminal Rhodococcus jostii RHA1 ref|YP_708348.1| 61 180 4.0E-51 GMRHGY403FV3VB 514 ABC transporter ATP-binding protein Bordetella petrii DSM 12804 ref|YP_001631848.1| 110 9.0E-23 GMRHGY403FP57F 491 Integrase catalytic region Variovorax paradoxus S110 ref|YP_002944153.1| 40 48.1 4.0E-04 GMRHGY403G4NRT 489 rhlE2 ATP-dependent RNA helicase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159102.1| 73 226 8.0E-58 GMRHGY403G7AA8 500 glcB malate synthase G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157451.1| 93 208 2.0E-52 GMRHGY403GV08M 523 cysK cysteine synthase A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159672.1| 86 291 2.0E-77 GMRHGY403FQSTS 502 putative pyruvate dehydrogenase E1 component (alpha subunit)oxidoreductase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158234.1| 66 124 3.0E-27 GMRHGY403GVDWK 501 drug resistance transporter, EmrB/QacA subfamily protein alpha proteobacterium BAL199 ref|ZP_02189198.1| 45 162 1.0E-38 GMRHGY403G2RQL 460 gltK glutamate/aspartate ABC transporter transmembrane protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159610.1| 96 206 5.0E-63 GMRHGY403FU8VA 528 petA iron-sulfur subunit of cytochrome bc1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157660.1| 91 317 5.0E-85 GMRHGY403FREPT 491 dihydropyrimidinase Moorella thermoacetica ATCC 39073 ref|YP_430836.1| ] 45 88.2 3.0E-16 GMRHGY403GWU34 496 prmA ribosomal protein L11 methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157837.1| 90 288 2.0E-76 GMRHGY403GJF1G 457 lon ATP-dependent protease La Bordetella petrii DSM 12804 ref|YP_001631399.1| 63 189 1.0E-46 GMRHGY403FVDB8 488 CbiD cobalamin biosynthesis protein CbiD Thauera sp. MZ1T ref|YP_002890709.1| 61 150 5.0E-35 GMRHGY403HAF09 514 cysE O-acetylserine synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160691.1| 77 248 3.0E-64 GMRHGY403G51MT 509 rpoB DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159176.1| 95 316 6.0E-85 GMRHGY403G3BKC 515 S9C family peptidase Myxococcus xanthus DK 1622 ref|YP_634720.1| 56 120 1.0E-41 GMRHGY403G3QJ2 518 putative acyl CoA dehydrogenase oxidoreductase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159083.1| 94 169 9.0E-41 GMRHGY403FT5FE 475 coxB2 cytochrome-c oxidase subunit II Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159093.1| 61 167 4.0E-40 GMRHGY403GL1US 512 ileS isoleucyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159544.1| 81 271 3.0E-71 GMRHGY403FKILT 508 relA GTP pyrophosphokinase (ATP:GTP 3'-pyrophosphotransferase) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160757.1| 75 214 4.0E-59 GMRHGY403FJ9DA 552 extracellular solute-binding protein family 1 Halothermothrix orenii H 168 ref|YP_002509199.1| 36 103 8.0E-21
! ! 211!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GW9LM 492 pyridoxal-dependent decarboxylase, exosortase system type 1 associated Thauera sp. MZ1T ref|YP_002355047.1| 83 57 8.0E-07 GMRHGY403GH76X 478 NusA transcription elongation factor NusA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160343.1| 92 281 1.0E-74 GMRHGY403GN7RG 502 ispG 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157699.1| 92 132 4.0E-58 GMRHGY403G9HTG 519 putative carbon monoxide dehydrogenase accessory protein (coxI) Ralstonia solanacearum PSI07 ref|YP_003749904.1| 79 135 3.0E-30 GMRHGY403FMH4H 490 elongation factor Tu Nitrosomonas eutropha C91 ref|YP_748001.1| 89 118 8.0E-50 GMRHGY403GHKZH 339 gltD glutamate synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158267.1| 47 86.3 1.0E-15 GMRHGY403GFIX5 474 cobyric acid synthase Azoarcus sp. BH72 ref|YP_935019.1| 83 118 6.0E-43 GMRHGY403GOG73 483 prfC peptide chain release factor 3 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158855.1| 91 296 8.0E-79 GMRHGY403HCPZW 285 cueO multicopper oxidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158047.1| 64 80.5 7.0E-14 GMRHGY403GBOFS 505 argG argininosuccinate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159918.1| 96 333 3.0E-90 GMRHGY403F1GGU 477 relA GTP pyrophosphokinase (ATP:GTP 3'-pyrophosphotransferase) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160757.1| 85 254 2.0E-66 GMRHGY403G8MOS 483 two component, sigma54 specific, Fis family transcriptional regulator Geobacter metallireducens GS-15 ref|YP_384356.1| 54 108 4.0E-33 GMRHGY403F1XGI 495 degQ serine protease DegQ/MucD Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160140.1| 70 129 2.0E-28 GMRHGY403G1NFY 511 phage integrase Methylobacterium extorquens DM4 ref|YP_003066791.1| 67 241 2.0E-62 GMRHGY403GWCKA 302 leuS leucyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159513.1| 93 91.3 5.0E-36 GMRHGY403G6NRP 487 DegT/DnrJ/EryC1/StrS aminotransferase Polaromonas naphthalenivorans CJ2 ref|YP_981820.1| 35 84 6.0E-15 GMRHGY403GUZEQ 501 type IV pilus assembly PilZ Thauera sp. MZ1T ref|YP_002889870.1| 49 122 2.0E-26 GMRHGY403HBBEB 493 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160614.1| 66 201 3.0E-50 GMRHGY403GLHVQ 491 complex regulator protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157435.1| 62 191 3.0E-47 GMRHGY403FL2I8 523 RecQ domain protein Oceanobacter sp. RED65 ref|ZP_01306596.1| 50 67.8 3.0E-37 GMRHGY403FV2MO 526 thyA thymidylate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159532.1| 87 303 4.0E-81 GMRHGY403HD1J3 477 transcriptional regulator, AraC family protein Alcanivorax sp. DG881 ref|ZP_05043610.1| 65 109 7.0E-35 GMRHGY403GQY8A 479 gacA LuxR family transcriptional regulator Azoarcus sp. BH72 ref|YP_934483.1| 73 185 2.0E-45 GMRHGY403G8W69 499 pheA chorismate mutase/prephenate dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157514.1| 81 263 5.0E-69 GMRHGY403G06VD 342 short chain dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160492.1| 58 109 1.0E-22 GMRHGY403G9U4K 484 PdxJ pyridoxal phosphate biosynthetic protein Azoarcus sp. BH72 ref|YP_933149.1| 85 275 1.0E-72 GMRHGY403GQL11 469 gdH NAD-glutamate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159071.1| 83 256 7.0E-67 GMRHGY403G8A3A 503 integral membrane protein Fusobacterium gonidiaformans ATCC 25563 ref|ZP_05631424.1| 42 83.6 3.0E-26 GMRHGY403G37TW 552 two component regulator propeller domain-containing protein Clostridium phytofermentans ISDg ref|YP_001558799.1| 26 67.8 6.0E-10 GMRHGY403FVZS3 495 fer21 putative ferredoxin 2Fe-2S protein Azoarcus sp. BH72 ref|YP_931867.1| 59 134 5.0E-30 GMRHGY403G03T6 501 FeS assembly SUF system protein SufT Thauera sp. MZ1T ref|YP_002355541.1| 59 192 1.0E-47 GMRHGY403G2IBR 478 aminoglycoside phosphotransferase Sphingopyxis alaskensis RB2256 ref|YP_615945.1| 42 102 1.0E-20 GMRHGY403GKMN2 508 17 kDa surface antigen Alicycliphilus denitrificans BC ref|ZP_07022749.1| 41 70.1 1.0E-10 GMRHGY403F50B7 507 yidC preprotein translocase subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158619.1| 66 162 3.0E-58 GMRHGY403G2KN7 500 ftsH cell division protein ftsH-like protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159974.1| 69 217 5.0E-55 GMRHGY403GB2IM 499 hemC porphobilinogen deaminase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157636.1| 89 150 5.0E-35 GMRHGY403GTIG5 498 methyl-accepting chemotaxis sensory transducer Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003168630.1| 58 150 5.0E-35 GMRHGY403GY8MK 523 TonB-dependent receptor Rhodothermus marinus DSM 4252 ref|YP_003290439.1| 34 65.9 2.0E-09 GMRHGY403FZBI2 505 ABC transporter related Acidovorax ebreus TPSY ref|YP_002553883.1| 68 140 6.0E-32 GMRHGY403FRKU6 517 peptidase family protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157521.1| 63 201 2.0E-50 GMRHGY403GYF9E 499 Anhydro-N-acetylmuramic acid kinase Laribacter hongkongensis HLHK9 ref|YP_002796393.1| 53 142 1.0E-32 GMRHGY403F8ZVX 499 glS1 ferredoxin-dependent glutamate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158268.1| 88 186 2.0E-67 GMRHGY403GJZHN 308 Cho nuclease subunit Cho of the excinuclease complex, (UvrC homolog protein) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158999.1| 66 61.6 2.0E-10 GMRHGY403HBZHT 543 aroC chorismate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159714.1| 85 320 4.0E-86 GMRHGY403G93XK 502 trxB FAD-dependent pyridine nucleotide-disulphide oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161023.1| 92 231 2.0E-59 GMRHGY403HA80F 549 oligopeptidase F. Metallo peptidase. MEROPS family M03B Roseburia intestinalis M50/1 emb|CBL09349.1| 63 105 3.0E-24 GMRHGY403G1LOJ 366 Threonine aldolase Conexibacter woesei DSM 14684 ref|YP_003397348.1| 50 97.1 7.0E-19 GMRHGY403HDNI2 455 fimV1 putative type 4 pilus biogenesis Azoarcus sp. BH72 ref|YP_932547.1| 51 84.3 5.0E-17 GMRHGY403F8UT7 506 Alpha/beta hydrolase fold-3 domain protein Thauera sp. MZ1T ref|YP_002889491.1| 64 133 9.0E-49 GMRHGY403FMQ03 524 fabF beta-ketoacyl-(acyl-carrier-protein) synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160133.1| 90 231 2.0E-59 GMRHGY403GV9G3 517 Cho nuclease subunit Cho of the excinuclease complex (UvrC homolog protein) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158999.1| 69 226 6.0E-58 GMRHGY403G6Y97 499 Alkyl hydroperoxide reductase/ Thiol specific antioxidant/ Mal allergen Nitrosospira multiformis ATCC 25196 ref|YP_413068.1| 74 221 3.0E-56 GMRHGY403GJC1S 499 Acyl-CoA dehydrogenase, type 2-like Mesorhizobium sp. BNC1 ref|YP_674648.1| 72 221 3.0E-56 GMRHGY403GXVGN 501 ribosomal protein L13a Cyanophora paradoxa emb|CAA71090.1| 67 231 2.0E-59 GMRHGY403F70QE 491 hemA glutamyl-tRNA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157646.1| 88 281 2.0E-74 GMRHGY403FPBVP 500 nitric oxide reductase activation protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157132.1| 95 208 1.0E-69 GMRHGY403GZBHK 503 moaA molybdenum cofactor biosynthesis protein A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160111.1| 89 206 2.0E-73 GMRHGY403FNPN3 341 serine/threonine-protein kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159742.1| 34 54.3 5.0E-06 GMRHGY403GBK8Y 463 thiC thiamine biosynthesis protein ThiC [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157949.1| 99 315 1.0E-84 GMRHGY403GVMJH 504 wbpI UDP-N-acetylglucosamine 2-epimerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159433.1| 73 187 4.0E-46 GMRHGY403GDZLV 515 cytochrome b5 Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003167890.1| 59 109 1.0E-22 GMRHGY403FT0R0 511 glycosyl transferase group 1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159437.1| 71 249 1.0E-64 GMRHGY403GKXRX 510 alpha/beta fold family hydrolase Myxococcus xanthus DK 1622 ref|YP_628502.1| 44 73.6 9.0E-12 GMRHGY403HEJZA 451 phasin; polyhydroxyalkanoate synthesis and granule formationregulator/factor Cupriavidus taiwanensis ref|YP_002005324.1| 40 119 1.0E-25 GMRHGY403FXTFS 557 N-acetylmuramoyl-L-alanine amidase family 2 bacterium Ellin514 ref|ZP_03627606.1| 35 60.1 1.0E-07 GMRHGY403FOPM2 513 ChaB family protein Micromonospora sp. L5 ref|ZP_06397383.1| 76 112 2.0E-23 GMRHGY403GYY4L 499 UDP-glucose/GDP-mannose dehydrogenase family protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157038.1| 90 300 3.0E-80 GMRHGY403HAEB5 524 argD acetylornithine aminotransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159916.1| 83 294 3.0E-78 GMRHGY403FKD90 435 Flp/Fap pilin component Lutiella nitroferrum 2002 ref|ZP_03697317.1| 58 48.1 4.0E-04 GMRHGY403G9FSW 502 imidazole glycerol phosphate synthase subunit HisH Geobacter uraniireducens Rf4 ref|YP_001230459.1| 58 79.7 1.0E-17 GMRHGY403FY7VU 332 livK branched-chain amino acid transport system substrate-binding protein Azospirillum sp. B510 ref|YP_003450458.1| 57 129 1.0E-28 GMRHGY403G9OMI 506 dctP C4-dicarboxylate-binding periplasmic protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159380.1| 91 303 7.0E-81 GMRHGY403FLUS1 462 putative penicillin-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158742.1| 82 201 2.0E-55 GMRHGY403GC1RP 479 gyrB DNA gyrase subunit B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158625.1| 81 248 2.0E-64 GMRHGY403FLGFT 462 polynucleotide phosphorylase/polyadenylase Limnobacter sp. MED105 ref|ZP_01915791.1| 97 96.3 4.0E-50 GMRHGY403GAJF3 476 30S ribosomal protein S13 Cupriavidus metallidurans CH34 ref|YP_585435.1| 81 211 2.0E-53
! ! 212!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403FOUI0 501 pfkB carbohydrate kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157842.1| 95 128 3.0E-28 GMRHGY403FZPHM 460 rpoC DNA-directed RNA polymerase subunit beta Azoarcus sp. BH72 ref|YP_934925.1| 85 274 3.0E-72 GMRHGY403G486R 495 maeB1 malic Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159517.1| 95 309 9.0E-38 GMRHGY403FNA6P 480 PilO type IV pilus assembly protein Janthinobacterium sp. Marseille ref|YP_001355056.1| 64 86.3 1.0E-16 GMRHGY403G1VZ4 518 Superoxide dismutase Methylotenera mobilis JLW8 ref|YP_003049626.1| 76 251 3.0E-65 GMRHGY403HCT7D 508 protein translocase subunit yidC Methylibium petroleiphilum PM1 ref|YP_001023013.1| 61 212 2.0E-53 GMRHGY403GS42G 434 icd1 isocitrate dehydrogenase [NADP] Azoarcus sp. BH72 ref|YP_932651.1| 86 259 1.0E-67 GMRHGY403FYH00 492 nudH dinucleoside polyphosphate hydrolase Azoarcus sp. BH72 ref|YP_934267.1| 80 259 1.0E-67 GMRHGY403GKDF9 475 hflB cell division protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159756.1| 95 171 1.0E-58 GMRHGY403HGDRY 496 capD putative capsular polysaccharide biosynthesis protein Azoarcus sp. BH72 ref|YP_935083.1| 72 240 4.0E-62 GMRHGY403GXGUW 493 secretion protein HlyD Geobacter metallireducens GS-15 ref|YP_383776.1| 65 159 1.0E-37 GMRHGY403FXZ42 516 peptidase S9B dipeptidylpeptidase IV domain protein Rhodothermus marinus DSM 4252 ref|YP_003291671.1| 54 47.4 7.0E-04 GMRHGY403GQPC1 502 rnc ribonuclease III Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160179.1| 81 218 2.0E-55 GMRHGY403GMPJI 505 mandelate racemase/muconate lactonizing enzyme family protein Bacillus sp. B14905 ref|ZP_01721889.1| 32 80.5 7.0E-14 GMRHGY403HCIQL 508 iorB Isoquinoline 1-oxidoreductase subunit beta Ralstonia solanacearumPSI07 ref|YP_003749903.1| 76 187 3.0E-46 GMRHGY403FM8EN 500 ATP synthase F1, gamma subunit Dehalococcoides sp. GT ref|YP_003462323.1| 29 74.7 4.0E-12 GMRHGY403F02XL 522 M23 family peptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157577.1| 71 152 2.0E-35 GMRHGY403FQ2C9 496 5-oxoprolinase (ATP-hydrolyzing) Conexibacter woesei DSM 14684 ref|YP_003393931.1| 41 126 8.0E-28 GMRHGY403F0MDV 553 nudH dinucleoside polyphosphate hydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157530.1| 89 211 3.0E-53 GMRHGY403FJFCJ 358 PilO fimbrial type-IV assembly protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158274.1| 83 117 6.0E-25 GMRHGY403GXXTT 332 molybdopterin oxidoreductase, iron-sulfur binding subunit Truepera radiovictrix DSM 17093 ref|YP_003704696.1| 60 183 6.0E-20 GMRHGY403GZ0T5 468 mraW S-adenosyl-methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157823.1| 83 128 7.0E-33 GMRHGY403GBRFG 516 cycH putative cyctochrome C biogenesis protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159013.1| 71 63.9 2.0E-10 GMRHGY403GBUX9 515 narK nitrate/nitrite antiporter Thauera sp. MZ1T ref|YP_002889615.1| 70 235 2.0E-60 GMRHGY403F9G8P 531 tig trigger factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159853.1| 82 286 1.0E-75 GMRHGY403GRAL6 497 amidohydrolase Conexibacter woesei DSM 14684 ref|YP_003392865.1| 56 191 3.0E-47 GMRHGY403FLOVG 140 EngB GTPase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157590.1| 89 48.9 9.0E-05 GMRHGY403FKZKL 475 integrase family protein Thauera sp. MZ1T ref|YP_002354590.1| 81 241 2.0E-62 GMRHGY403HAWL8 503 cysT membrane component of thiosulfate ABC-transporter Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160571.1| 77 258 2.0E-67 GMRHGY403GVGCG 504 paaR paa operon transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159031.1| 83 127 6.0E-28 GMRHGY403F82GA 500 phasin; polyhydroxyalkanoate synthesis and granule formationregulator/factor Cupriavidus taiwanensis ref|YP_002005324.1| 43 141 2.0E-32 GMRHGY403GQLWJ 488 L-asparaginase I Oceanospirillum sp. MED92 ref|ZP_01165373.1| 49 156 1.0E-36 GMRHGY403FRG0E 489 phosphoribosyl transferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157788.1| 96 241 2.0E-62 GMRHGY403F5NKO 489 aspS aspartyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159485.1| 97 278 4.0E-78 GMRHGY403FUAUE 382 acetoacetyl-CoA synthase Parvibaculum lavamentivorans DS-1 ref|YP_001411834.1| 78 113 9.0E-24 GMRHGY403G1KO3 491 rep similar to ATP-dependent DNA helicase,putative replication protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158748.1| 92 303 6.0E-81 GMRHGY403GL8LM 504 boxZ aldehyde dehydrogenase Azoarcus evansii gb|AAN39373.1| 76 209 3.0E-55 GMRHGY403HDNBO 447 inner-membrane translocator:ABC transporter related Dechloromonas aromatica RCB ref|YP_284134.1| 44 112 2.0E-23 GMRHGY403GK01H 503 rpoC DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159177.1| 95 308 1.0E-82 GMRHGY403F9GWX 472 EF hand family protein Tetrahymena thermophila ref|XP_001031900.2| 30 63.5 8.0E-09 GMRHGY403FV890 500 speE1 putative spermidine synthase Azoarcus sp. BH72 ref|YP_933557.1| 64 140 8.0E-40 GMRHGY403GYMQY 495 DNA gyrase, A subunit Roseiflexus castenholzii DSM 13941 ref|YP_001430478.1| 65 222 1.0E-56 GMRHGY403FIQ40 203 3-isopropylmalate dehydratase, large subunit Burkholderia sp.Ch1-1 ref|ZP_06842528.1| 79 104 3.0E-21 GMRHGY403FLB03 418 moaC molybdenum cofactor biosynthesis protein C Burkholderia multivorans CGD1 ref|ZP_03584322.1| 72 196 8.0E-49 GMRHGY403FMU6E 534 rnpA ribonuclease P protein component Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158621.1| 73 162 2.0E-38 GMRHGY403FR4LY 496 ribonuclease activity regulator protein RraA Ralstonia eutropha JMP134 ref|YP_296171.1| 59 150 5.0E-35 GMRHGY403FUNSV 515 nlaB phospholipid and glycerol acyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157744.1| 90 313 5.0E-84 GMRHGY403FVEJ7 372 cfa1 cyclopropane fatty acyl phospholipid synthase Azoarcus sp. BH72 ref|YP_932460.1| 49 60.1 9.0E-08 GMRHGY403FS9R6 512 exodeoxyribonuclease V, gamma subunit Methylococcus capsulatus str. Bath ref|YP_113201.1| 67 87.4 2.0E-34 GMRHGY403GKUEZ 515 AlgW putative HTRA-like serine protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157710.1| 88 225 7.0E-62 GMRHGY403F8BX2 499 histidine kinase Acidobacterium sp. MP5ACTX8 ref|ZP_07030700.1| 41 114 4.0E-24 GMRHGY403GDXGV 489 gacA LuxR family transcriptional regulator Azoarcus sp. BH72 ref|YP_934483.1| 63 131 2.0E-40 GMRHGY403GYU3W 136 putative cytosolic aminopeptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161094.1| 80 80.1 9.0E-14 GMRHGY403FK3U0 533 putative TONB-dependent receptor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159304.1| 72 251 4.0E-65 GMRHGY403GMZL7 386 murD UDP-N-acetylmuramoylalanine-D-glutamate ligase Azoarcus sp. BH72 ref|YP_932386.1| 50 105 3.0E-21 GMRHGY403F9CZN 538 nuoG NADH dehydrogenase subunit G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159771.1| 83 251 2.0E-65 GMRHGY403G7XZL 469 nuoG NADH-quinone oxidoreductase, chain G Thauera sp. MZ1T ref|YP_002355396.1| 68 197 3.0E-49 GMRHGY403GG1IJ 494 Catalase Desulfomicrobium baculatum DSM 4028 ref|YP_003157700.1| 63 116 1.0E-24 GMRHGY403G0ITH 493 Amino acid permease-associated region NC10 bacterium 'Dutch sediment' emb|CBE67489.1| 60 176 9.0E-43 GMRHGY403GHXJD 311 trmD tRNA (guanine-N1)-methyltransferase Thauera sp. MZ1T ref|YP_002889760.1| 88 41.2 4.0E-08 GMRHGY403F0QOR 447 isocitrate lyase Burkholderia sp. H160 ref|ZP_03266852.1| 74 109 2.0E-50 GMRHGY403FS5ZA 331 ribAB bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTPcyclohydrolase II protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159043.1| 80 114 3.0E-24 GMRHGY403GJPAO 486 cbiD cobalamin biosynthesis protein CbiD Thauera sp. MZ1T ref|YP_002890709.1| 62 159 1.0E-37 GMRHGY403GFO6S 519 Outer membrane porin protein 32 precursor Ralstonia solanacearum UW551 ref|ZP_00946041.1| 46 107 8.0E-22 GMRHGY403GXFWN 497 aroC chorismate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159714.1| 85 196 8.0E-49 GMRHGY403GS7YY 126 cstA carbon starvation A transmembrane protein Herbaspirillum seropedicae SmR1 ref|YP_003775375.1| 81 58.2 4.0E-07 GMRHGY403FRHVX 487 trpS tryptophanyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160528.1| 92 308 2.0E-82 GMRHGY403G8RSR 519 suhB inositol monophosphatase (extragenic suppressor protein) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160693.1| 83 205 2.0E-51 GMRHGY403FJVET 505 fbp fructose-1,6-bisphosphatase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157654.1| 95 218 2.0E-55 GMRHGY403F72B3 492 typA GTP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160670.1| 91 297 3.0E-79 GMRHGY403F2M26 494 glycosyltransferase group I Vibrio cholerae gb|ADF81003.1| 57 181 4.0E-44 GMRHGY403FN19Y 514 leuS leucyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159513.1| 88 316 8.0E-85 GMRHGY403GRU6W 381 gapA glyceraldehyde 3-phosphate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157603.1| 86 150 2.0E-46 GMRHGY403GAH0V 512 surface lipoprotein Maritimibacter alkaliphilus HTCC2654 ref|ZP_01011779.1| 51 167 4.0E-40 GMRHGY403GE4XG 378 phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase Chlorobium limicola DSM 245 ref|YP_001944010.1| 61 46.6 8.0E-08 GMRHGY403GPMES 505 padB2 molybdenum enzyme, large subunit,related to phenylacetyl-CoA: acceptor oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160228.1| 45 105 3.0E-21 GMRHGY403GVXIE 499 ribose 5-phosphate isomerase Desulfatibacillum alkenivorans AK-01 ref|YP_002432131.1| 65 145 5.0E-36
! ! 213!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403FU1U3 368 thioesterase superfamily protein Afipia sp. 1NLS2 ref|ZP_07026176.1| 37 50.8 6.0E-05 GMRHGY403HEDX5 495 arylsulfatase Methanosarcina barkeri str. Fusaro ref|YP_306549.1| 62 204 3.0E-51 GMRHGY403GS0DG 492 tryptophan synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159353.1| 84 268 2.0E-70 GMRHGY403F8NZE 489 marR MarR family transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159793.1| 91 165 7.0E-56 GMRHGY403FZUWE 471 tripeptide aminopeptidase Sphingobacterium spiritivorum ATCC 33861 ref|ZP_07081353.1| 53 139 2.0E-31 GMRHGY403FSSMV 538 alpha,alpha-trehalose-phosphate synthase Herbaspirillum seropedicae SmR1 ref|YP_003773630.1| 51 135 2.0E-30 GMRHGY403F97CF 469 recG RecG-like helicases Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158295.1| 75 221 3.0E-56 GMRHGY403FOEOT 514 ilvB acetolactate synthase catalytic subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159712.1| 86 266 5.0E-73 GMRHGY403GE5I4 500 Anhydro-N-acetylmuramic acid kinase Laribacter hongkongensis HLHK9 ref|YP_002796393.1| 60 111 6.0E-41 GMRHGY403GB17Q 482 ilvI thiamine pyrophosphate dependent acetolactate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161069.1| 70 244 2.0E-63 GMRHGY403GWMBV 534 putative homocysteine S-methyltransferase Bacteroides fragilis 3_1_12 ref|ZP_05284073.1| 41 85.5 3.0E-15 GMRHGY403HCV90 472 transcriptional regulator, SARP family Thioalkalivibrio sp. HL-EbGR7 ref|YP_002512370.1| 36 78.2 3.0E-13 GMRHGY403FSL6I 484 aggA putative outer membrane efflux protein Azoarcus sp. BH72 ref|YP_932158.1| 64 151 3.0E-53 GMRHGY403FSXTE 492 glS1 ferredoxin-dependent glutamate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158268.1| 89 282 1.0E-74 GMRHGY403FO4YM 498 leucyl-tRNA synthetase Thermotoga naphthophila RKU-10 ref|YP_003346306.1| 58 113 7.0E-24 GMRHGY403FJH9V 488 lysyl-tRNA synthetase Bordetella avium 197N ref|YP_786043.1| 84 244 2.0E-63 GMRHGY403G6M32 519 ribosomal protein S13 Actinomyces urogenitalis DSM 15434 ref|ZP_03927454.1| 68 124 8.0E-36 GMRHGY403HHTNU 434 RnfC electron transport complex protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159803.1| 84 165 6.0E-51 GMRHGY403G300B 362 transcriptional regulator, TetR family Thauera sp. MZ1T ref|YP_002354681.1| 71 118 3.0E-32 GMRHGY403GW7MS 480 gyrA DNA gyrase subunit A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157516.1| 93 299 7.0E-80 GMRHGY403G9DJ6 491 rpsU 30S ribosomal protein S21 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159503.1| 100 112 2.0E-23 GMRHGY403G3ZBX 470 lpxD UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (FirA protein) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160450.1| 75 162 2.0E-38 GMRHGY403HAD6I 603 ribosomal protein L24 Alicyclobacillus acidocaldarius LAA1 ref|ZP_03494172.1| 61 104 7.0E-21 GMRHGY403GAQOG 475 putative nucleotidyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159838.1| 69 214 3.0E-54 GMRHGY403HEYZR 428 ISRm25b transposase Sinorhizobium meliloti 1021 ref|NP_435784.1| 75 208 2.0E-52 GMRHGY403GBCMT 476 integral membrane sensor hybrid histidine kinase Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003166371.1| 50 115 1.0E-24 GMRHGY403GYEUN 484 oppB oligopeptide transport system permease protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160830.1| 97 93.2 1.0E-17 GMRHGY403GW2BJ 502 Glycosyltransferases involved in cell wall biogenesis Magnetospirillum gryphiswaldense MSR-1 emb|CAM77197.1| 32 58.9 2.0E-07 GMRHGY403FM0M7 484 transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158429.1| 68 178 9.0E-46 GMRHGY403GQBLD 501 putative metal cation transporter Sphaerobacter thermophilus DSM 20745 ref|YP_003321370.1| 38 74.7 7.0E-24 GMRHGY403HFSHP 510 etfA electron transfer flavoprotein alpha subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160745.1| 84 265 1.0E-69 GMRHGY403F4FN3 513 hydrolase or acytransferase Azoarcus sp. BH72 ref|YP_933620.1| 63 110 7.0E-23 GMRHGY403FPFHW 430 serine/threonine protein kinase Solibacter usitatus Ellin6076 ref|YP_827125.1| 55 62 2.0E-19 GMRHGY403FZNUF 492 ilvI thiamine pyrophosphate dependent acetolactate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161069.1| 91 311 2.0E-83 GMRHGY403F8DGM 488 gspD2 general (Type II) secretion pathway (GSP) D protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157682.1| 78 253 4.0E-66 GMRHGY403FZ63G 586 two component transcriptional regulator, LuxR family Chthoniobacter flavus Ellin428 ref|ZP_03132564.1| 54 157 5.0E-37 GMRHGY403FVVJY 252 nucleoid DNA-binding protein Lactobacillus gasseri ATCC 33323 ref|YP_814721.1| 59 72.8 1.0E-11 GMRHGY403G1LTJ 494 TPR repeat-containing protein Roseiflexus sp. RS-1 ref|YP_001277071.1| 43 61.2 4.0E-08 GMRHGY403FIXNS 499 helix-turn-helix, Fis-type Dechloromonas aromatica RCB ref|YP_284001.1| 53 101 2.0E-29 GMRHGY403FMF99 484 insecticidal toxin protein, C-terminal Rhodococcus jostii RHA1 ref|YP_708348.1| 64 220 4.0E-56 GMRHGY403FSA61 491 HsdR family type I site-specific deoxyribonuclease Rhodoferax ferrireducens T118 ref|YP_521321.1| 91 168 9.0E-71 GMRHGY403G4OON 477 ATP-dependent DNA helicase, RecQ family Allochromatium vinosum DSM 180 ref|YP_003445108.1| 56 154 5.0E-36 GMRHGY403F6RLI 474 putative transport ATPase Azoarcus sp. BH72 ref|YP_933793.1| 52 169 1.0E-40 GMRHGY403FL2FG 443 aspartokinase/homoserine dehydrogenase Gemmatimonas aurantiaca T-27 ref|YP_002760879.1| 79 60.8 1.0E-12 GMRHGY403FRPJR 513 TRAP transporter solute receptor TAXI family protein Polaromonas sp. JS666 ref|YP_548832.1| 84 302 1.0E-80 GMRHGY403GWPYC 484 sucD succinyl-CoA synthetase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157704.1| 92 236 1.0E-72 GMRHGY403G5CYQ 484 queA S-adenosylmethionine:tRNA ribosyltransferase-isomerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157791.1| 91 270 6.0E-71 GMRHGY403F1VI4 464 lipopolysaccharide biosynthesis protein Pseudomonas putida KT2440 ref|NP_745271.1| 28 61.6 3.0E-08 GMRHGY403FKGR5 509 methanol/ethanol family PQQ-dependent dehydrogenase Leptothrix cholodnii SP-6 ref|YP_001790843.1| 85 226 6.0E-58 GMRHGY403GI653 157 treS putative alpha amylase/trehalose synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161006.1| 81 94.4 4.0E-18 GMRHGY403GIC2U 466 narI nitrate reductase, gamma subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160618.1| 83 270 3.0E-71 GMRHGY403HCYMU 491 maeB1 malic Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159517.1| 85 271 2.0E-71 GMRHGY403GMD5J 508 ilvC ketol-acid reductoisomerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161071.1| 96 321 2.0E-86 GMRHGY403GY31J 492 fusion protein of flavin-containing oxidoreductase and iron-sulfur-containing oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158925.1| 90 301 1.0E-80 GMRHGY403FIG8T 498 RND efflux transporter, permease protein Azoarcus sp. BH72 ref|YP_931749.1| 73 200 6.0E-50 GMRHGY403G5D8M 501 cbf2 putative cell binding factor Azoarcus sp. BH72 ref|YP_933207.1| 66 152 1.0E-35 GMRHGY403GL9TH 278 MucD putative serine protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158697.1| 68 60.8 5.0E-08 GMRHGY403GRKG9 474 ispG 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157699.1| 66 185 2.0E-45 GMRHGY403G67IK 521 tRNA (guanine-N(7))-methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158321.1| 86 146 9.0E-34 GMRHGY403FS1C5 504 UvrD/REP helicase Geobacter uraniireducens Rf4 ref|YP_001230608.1| 56 169 9.0E-41 GMRHGY403GMWJS 585 nucleic acid-binding protein Actinobacillus pleuropneumoniae serovar 3 str. JL03 ref|YP_001652761.1| 43 44.3 2.0E-07 GMRHGY403GYFRP 499 ATP synthase F1, gamma subunit Dehalococcoides sp. GT ref|YP_003462323.1| 30 77 7.0E-13 GMRHGY403FM69T 492 pheA chorismate mutase/prephenate dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157514.1| 81 203 5.0E-51 GMRHGY403GWGF8 534 threonine dehydratase, biosynthetic Sideroxydans lithotrophicus ES-1 ref|YP_003525355.1| 75 162 2.0E-38 GMRHGY403FK1DC 476 Outer membrane receptor for ferrienterochelin and colicins Marinobacter algicola DG893 ref|ZP_01892594.1| 41 114 5.0E-24 GMRHGY403GG9F0 466 PREDICTED: similar to AER446Wp Hydra magnipapillata ref|XP_002155081.1| 80 103 2.0E-34 GMRHGY403FRISQ 303 fdhA1 formate dehydrogenase, alpha subunit Azoarcus sp. BH72 ref|YP_934541.1| 64 118 3.0E-25 GMRHGY403GC6U3 473 putative signaling protein Azoarcus sp. BH72 ref|YP_935001.1| 52 98.6 1.0E-40 GMRHGY403GS9H9 481 undecaprenyl-phosphate galactose phosphotransferase Clostridium sp. 7_2_43FAA ref|ZP_05129919.1| 64 51.6 4.0E-10 GMRHGY403FTPFK 414 denitrification system component cytochrome c-552 Thauera sp. MZ1T ref|YP_002889595.1| 82 229 7.0E-59 GMRHGY403HAEAK 485 MerR family transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159078.1| 80 224 4.0E-57 GMRHGY403HFPRH 492 hydroxylamine reductase Psychromonas sp. CNPT3 ref|ZP_01216027.1| 35 112 2.0E-23 GMRHGY403GW58B 494 padC phenylacetyl-CoA:acceptor oxidoreductase Azoarcus sp. BH72 ref|YP_933451.1| 52 134 3.0E-30 GMRHGY403GPF1H 506 4-diphosphocytidyl-2C-methyl-D-erythritol synthase Azoarcus sp. BH72 ref|YP_933186.1| 61 185 2.0E-45 GMRHGY403GW9SJ 502 recA recombinase A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158947.1| 93 258 2.0E-67
! ! 214!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403F3OP7 490 tetratricopeptide TPR_2 Trichodesmium erythraeum IMS101 ref|YP_722460.1| 33 95.5 2.0E-18 GMRHGY403GIVT5 499 dnaA chromosomal replication iniciator protein DnaA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158623.1| 99 261 2.0E-68 GMRHGY403F1C5Z 521 exbD2 biopolymer transport protein ExbD/TolR ref|YP_159906.1| 80 110 5.0E-23 GMRHGY403GR51Z 523 murF UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diamin opimelate--D-alanyl-D-alanyl ligase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157820.1| 69 141 4.0E-32 GMRHGY403GS9KR 512 rimM 16S rRNA processing protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161089.1| 82 147 4.0E-34 GMRHGY403FMQNE 527 serine protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157036.1| 81 71.2 5.0E-21 GMRHGY403G1QX3 496 peptidase U32 falily Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159056.1| 85 282 9.0E-75 GMRHGY403F0NMB 479 LysR family transcriptional regulator Nitrosospira multiformis ATCC 25196 ref|YP_411386.1| 35 68.9 4.0E-13 GMRHGY403GISVD 518 murB UDP-N-acetylmuramate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158953.1| 78 144 4.0E-33 GMRHGY403GSDML 493 nrdA ribonucleotide-diphosphate reductase subunit alpha Bordetella parapertussis 12822 ref|NP_886053.1| 75 132 1.0E-45 GMRHGY403G19HF 503 pheT phenylalanyl-tRNA synthetase beta chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157482.1| 71 179 8.0E-44 GMRHGY403G2QIE 501 Putative diguanylate cyclase/phosphodiesterase (GGDEF & EAL domains) with PAS/PAC sensorsLyngbya sp. PCC 8106 ref|ZP_01624062.1| 27 68.2 3.0E-10 GMRHGY403GH754 495 amidohydrolase Conexibacter woesei DSM 14684 ref|YP_003392865.1| 56 191 3.0E-47 GMRHGY403G40ZY 493 boxA benzoyl-CoA oxygenase component A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158581.1| 70 248 2.0E-64 GMRHGY403FYTLX 489 FliN flagellar motor switch Azoarcus sp. BH72 ref|YP_934227.1| 80 129 1.0E-28 GMRHGY403HGH8I 235 pnp polynucleotide phosphorylase/polyadenylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160348.1| 93 89.4 5.0E-22 GMRHGY403FT4KB 497 gapA putative glyceraldehyde 3-phosphate dehydrogenase Azoarcus sp.BH72 ref|YP_934340.1| 95 314 2.0E-84 GMRHGY403GPP6J 494 dhaL aldehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158262.1| 93 307 3.0E-82 GMRHGY403HH9QQ 505 transketolase Burkholderia ubonensis Bu ref|ZP_02378491.1| 83 256 9.0E-67 GMRHGY403F2G1K 502 degQ serine protease DegQ/MucD Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160140.1| 61 144 3.0E-33 GMRHGY403G2483 546 50S ribosomal protein L34P Rhodospirillum rubrum ATCC 11170 ref|YP_428417.1| 90 84 8.0E-15 GMRHGY403GCZFB 507 inner-membrane translocator Dictyoglomus turgidum DSM 6724 ref|YP_002352828.1| 44 78.6 7.0E-30 GMRHGY403GEX9K 505 aceA isocitrate lyase Dechloromonas aromatica RCB ref|YP_286298.1| 95 325 2.0E-87 GMRHGY403FTOPS 454 polyribonucleotide nucleotidyltransferase Leptothrix cholodnii SP-6 ref|YP_001790530.1| 90 211 8.0E-63 GMRHGY403G5SZQ 556 purF amidophosphoribosyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159731.1| 95 169 3.0E-47 GMRHGY403G22EP 482 rne ribonuclease E, (RNase E) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160120.1| 96 204 1.0E-73 GMRHGY403GVE5Y 510 putative oxidoreductase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157988.1| 61 194 3.0E-48 GMRHGY403FZMHE 383 putative PAS/PAC sensor protein Desulfotomaculum acetoxidans DSM 771 ref|YP_003192202.1| 47 105 2.0E-21 GMRHGY403F577P 497 putative E1-E2 type ATPase Sinorhizobium meliloti ref|YP_001965581.1| 70 216 8.0E-55 GMRHGY403HEYMW 500 phaR polyhydroxyalkanoate synthesis repressor PhaR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159701.1| 97 282 9.0E-75 GMRHGY403GF1GJ 485 putative ABC transporter permease protein Bordetella pertussis Tohama I ref|NP_881724.1| 50 122 2.0E-26 GMRHGY403F8YJ7 505 glmS glutamine amidotransferase class-II Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158263.1| 93 142 1.0E-64 GMRHGY403FWRHK 488 histidine kinase Acidobacterium sp. MP5ACTX8 ref|ZP_07030700.1| 57 131 3.0E-29 GMRHGY403GFUOG 529 putative serine/threonine kinase Azoarcus sp. BH72 ref|YP_935390.1| 58 152 2.0E-35 GMRHGY403GBYKH 504 accA acetyl-CoA carboxylase carboxyltransferase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159375.2| 87 288 2.0E-76 GMRHGY403F0AGA 502 OmpW outer membrane protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160324.1| 91 216 6.0E-55 GMRHGY403HA31X 484 type II secretion system protein Roseiflexus castenholzii DSM 13941 ref|YP_001431790.1| 59 181 3.0E-44 GMRHGY403G31XS 547 tRNA/rRNA methyltransferase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157686.1| 95 162 2.0E-38 GMRHGY403GAITX 530 Sun Sun protein Azoarcus sp. BH72 ref|YP_935487.1| 60 53.1 1.0E-05 GMRHGY403GDJZS 359 mpl putative UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diamin opimelate ligase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157831.1| 79 165 2.0E-39 GMRHGY403FM3AK 491 Holliday junction DNA helicase RuvA Herpetosiphon aurantiacus ATCC 23779 ref|YP_001545282.1| 45 103 4.0E-28 GMRHGY403FKD1Y 505 sucC succinyl-CoA synthetase subunit beta Azoarcus sp. BH72 ref|YP_934835.1| 89 289 8.0E-77 GMRHGY403GY0H0 314 magnesium Mg(2+)/cobalt Co(2+) transport protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159575.1| 63 52 3.0E-05 GMRHGY403GHQ4P 485 sensory transduction histidine kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160729.1| 63 193 7.0E-48 GMRHGY403F0GEY 490 acyl-CoA dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160743.1| 85 129 1.0E-28 GMRHGY403F3O1M 257 cadmium-translocating P-type ATPase Gallionella capsiferriformans ES-2 ref|YP_003847945.1| 62 99.8 1.0E-19 GMRHGY403GVGAO 494 ribD riboflavin biosynthesis bifunctional RIBD Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157573.1| 78 268 1.0E-70 GMRHGY403GY4GF 474 gluconate 2-dehydrogenase cytochrome c subunit Burkholderia multivorans CGD2M ref|ZP_03569461.1| 51 60.1 9.0E-08 GMRHGY403GU7O8 521 transcriptional regulator, IclR family Burkholderia ambifaria MEX-5 ref|ZP_02909195.1| 49 157 4.0E-37 GMRHGY403G9ES5 536 endoribonuclease L-PSP Azoarcus sp. BH72 ref|YP_931980.1| 87 207 3.0E-52 GMRHGY403HBEIE 495 complex regulator protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157435.1| 57 164 3.0E-39 GMRHGY403FM0LR 505 dsbC putative thiol:disulphide interchange protein (periplasmic) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159346.1| 83 271 2.0E-71 GMRHGY403F64TN 525 nosC putative cytochrome c-type protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160605.1| 91 157 4.0E-37 GMRHGY403G5GPO 538 yidC preprotein translocase subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158619.1| 73 270 6.0E-71 GMRHGY403HBFEO 167 TonB-dependent receptor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159258.1| 82 91.3 4.0E-17 GMRHGY403GUXLC 493 aceA isocitrate lyase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159559.1| 96 317 4.0E-85 GMRHGY403HIO46 194 gspA secretion ATPase, PEP-CTERM locus subfamily Thauera sp. MZ1T ref|YP_002890250.1| 77 50.8 6.0E-05 GMRHGY403GOCTK 500 int phage integrase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157407.1| 46 99.4 1.0E-19 GMRHGY403FQKCX 497 Poly(R)-hydroxyalkanoic acid synthase, class I Dechloromonas aromatica RCB ref|YP_284827.1| 61 215 2.0E-54 GMRHGY403G2Q2X 420 Formyl-CoA transferase Starkeya novella DSM 506 ref|YP_003696169.1| 58 143 3.0E-36 GMRHGY403G984F 506 leuS leucyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159513.1| 93 328 1.0E-88 GMRHGY403GHD48 485 acetyltransferase Azoarcus sp. BH72 ref|YP_933529.1| 81 228 2.0E-58 GMRHGY403GUT0K 538 putative DNA binding protein Parabacteroides distasonis ATCC 8503 ref|YP_001304963.1| 42 70.1 1.0E-10 GMRHGY403GC73K 526 aroK shikimate kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158271.1| 79 76.3 9.0E-20 GMRHGY403FRU37 524 nosL putative lipoprotein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160608.1| 83 189 9.0E-47 GMRHGY403GRH88 507 pabA anthranilate synthase (component II) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159402.1| 92 238 3.0E-61 GMRHGY403GV464 531 ribosomal protein L1 bacterium Ellin514 ref|ZP_03628350.1| 73 251 3.0E-65 GMRHGY403FXTKI 336 pilJ witching motility transmembrane protein PilJ Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157973.1| 85 97.8 4.0E-19 GMRHGY403GLGLK 467 pnp polynucleotide phosphorylase/polyadenylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160348.1| 97 262 1.0E-68 GMRHGY403HHQ8K 496 prpE propionyl-CoA synthetase Azoarcus sp. BH72 ref|YP_934682.1| 93 309 7.0E-83 GMRHGY403FMJAG 485 lipoate-protein ligase B Pseudomonas mendocina ymp ref|YP_001189272.1| 53 100 8.0E-20 GMRHGY403F6OU7 507 dcd deoxycytidine triphosphate deaminase Azoarcus sp. BH72 ref|YP_934702.1| 96 298 1.0E-79 GMRHGY403GZ9XO 493 probable glutathione S-transferase Pseudomonas aeruginosa PAO1 ref|NP_251503.1| 52 119 2.0E-35 GMRHGY403GH12E 487 amidase Roseomonas cervicalis ATCC 49957 ref|ZP_06898729.1| 57 164 3.0E-39 GMRHGY403GO4YD 506 ribulose-bisphosphate carboxylase Rhodobacter sphaeroides ATCC 17025 ref|YP_001168903.1| 80 147 5.0E-34 GMRHGY403HCSRE 484 rnhB ribonuclease HII Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160454.1| 84 160 4.0E-38
! ! 215!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GU53G 478 leuB 3-isopropylmalate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159719.1| 93 275 3.0E-75 GMRHGY403HC6LV 531 transcriptional regulator LysR Dechloromonas aromatica RCB ref|YP_284570.1| 77 155 2.0E-57 GMRHGY403HEU86 411 purine or other phosphorylase family 1 Thauera sp. MZ1T ref|YP_002355818.1| 73 99.8 1.0E-19 GMRHGY403G6OJW 494 putative transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157975.1| 90 301 2.0E-80 GMRHGY403G0QFQ 353 murF UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diamin opimelate--D-alanyl-D-alanyl ligase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157820.1| 80 125 4.0E-34 GMRHGY403G412S 537 6-hydroxy-D-nicotine oxidase Mycobacterium kansasii ATCC 12478 ref|ZP_04751313.1| 80 76.6 1.0E-24 GMRHGY403FXJ1S 527 hydrogenase 2 protein HybA Dechloromonas aromatica RCB ref|YP_287170.1| 75 298 2.0E-79 GMRHGY403GU78X 570 glycosyltransferase, group 2 family protein delta proteobacterium NaphS2 ref|ZP_07200181.1| 31 59.7 2.0E-07 GMRHGY403G2HZH 516 FlhC transcriptional activator Azoarcus sp. BH72 ref|YP_932951.1| 78 157 4.0E-37 GMRHGY403FIWGU 515 prfB peptide chain release factor 2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160323.1| 92 318 2.0E-85 GMRHGY403FUCI2 441 FtsN putative FtsN cell division protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159069.1| 71 94.7 4.0E-33 GMRHGY403GPUQ1 495 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157126.1| 96 295 1.0E-78 GMRHGY403G1C4A 505 response regulator receiver modulated CheB methylesterase Geobacter sp. M18 ref|ZP_05312622.1| 43 140 7.0E-32 GMRHGY403FO2OH 505 dsbC putative thiol:disulphide interchange protein (periplasmic) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159346.1| 83 271 2.0E-71 GMRHGY403GEUNW 482 cysM cysteine synthase B Azoarcus sp. BH72 ref|YP_932581.1| 90 296 4.0E-79 GMRHGY403F43NI 481 nitric-oxide reductase Nitrosococcus watsoni C-113 ref|YP_003760514.1| 78 79.7 8.0E-39 GMRHGY403GAJ72 492 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 91 320 5.0E-86 GMRHGY403FMZUJ 487 frr ribosome recycling factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160443.1| 93 269 8.0E-71 GMRHGY403HHKZB 511 Peptidase M1 membrane alanine aminopeptidase Oscillochloris trichoides DG6 gb|EFO79106.1| 43 48.1 4.0E-04 GMRHGY403GLGIU 496 cheR methylase of chemotaxis methyl-accepting protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157434.1| 85 159 1.0E-37 GMRHGY403HDT5B 528 Pyrrolo-quinoline quinone Verminephrobacter eiseniae EF01-2 ref|YP_999677.1| 75 199 3.0E-71 GMRHGY403FPR1V 362 clpA ATP-dependent protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159563.1| 98 150 2.0E-55 GMRHGY403GPRL2 432 putative heme/copper-type cytochrome/quinol oxidase subunit 2 uncultured bacterium 888 gb|ACF98041.1| 75 166 9.0E-42 GMRHGY403GDVKN 500 50S ribosomal protein L10 Bordetella petrii DSM 12804 ref|YP_001633586.1| 78 205 2.0E-51 GMRHGY403FOB6U 473 pntAA pyridine nucleotide transhydrogenase alpha subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159576.1| 89 197 3.0E-67 GMRHGY403FJGAL 499 thrS threonyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157487.1| 94 266 7.0E-83 GMRHGY403GH32H 505 ileS isoleucyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159544.1| 80 265 2.0E-69 GMRHGY403G2AUP 495 suhB myo-inositol-1(or 4)-monophosphatase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157600.1| 72 112 1.0E-23 GMRHGY403G0P4M 517 glycosyltransferase Azoarcus sp. BH72 ref|YP_934761.1| 55 107 2.0E-24 GMRHGY403HEFDL 502 tpX thiol peroxidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158670.1| 82 234 4.0E-60 GMRHGY403F295Q 499 GAF modulated sigma54 specific transcriptional regulator, Fis family Thauera sp. MZ1T ref|YP_002889789.1| 73 192 2.0E-47 GMRHGY403G82LJ 514 radical SAM-linked protein Propionibacterium acnes SK187 ref|ZP_06427920.1| 76 227 5.0E-58 GMRHGY403GRXR7 492 30S ribosomal protein S4 Ralstonia solanacearum GMI1000 ref|NP_521115.1| 76 257 3.0E-67 GMRHGY403G08GX 504 DNA polymerase-related protein,bacteriophage-type Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160733.1| 72 134 3.0E-30 GMRHGY403FPB1R 493 AAA ATPase central domain protein Anaeromyxobacter sp. K ref|YP_002132673.1| 38 55.8 1.0E-08 GMRHGY403GCKAF 505 hflX GTP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157693.1| 87 147 5.0E-34 GMRHGY403F15UR 465 ubiquinone biosynthesis protein Azoarcus sp. BH72 ref|YP_934265.1| 87 107 1.0E-32 GMRHGY403FRBMM 498 hisA 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]imidazole-4-carboxamideAromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157718.1| 94 186 9.0E-46 GMRHGY403FOWDG 515 ilvB acetolactate synthase catalytic subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159712.1| 87 293 4.0E-78 GMRHGY403F4MDF 537 strongly similar to glycogen phosphorylase Candidatus Kuenenia stuttgartiensis emb|CAJ73447.1| 59 162 1.0E-38 GMRHGY403GMY6O 474 fdhB formate dehydrogenase, NAD(P) reducing, beta subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158570.1| 54 111 4.0E-23 GMRHGY403FTP7A 519 AhpC alkyl hydroperoxide reductase Cellvibrio japonicus Ueda107 ref|YP_001980899.1| 90 155 4.0E-66 GMRHGY403HDDLE 508 rplP 50S ribosomal protein L16 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159190.1| 98 195 2.0E-48 GMRHGY403HH6CP 539 cytochrome c family protein Persephonella marina EX-H1 ref|YP_002731213.1| 50 61.2 5.0E-08 GMRHGY403FUON9 555 Extracellular ligand-binding receptor Archaeoglobus profundus DSM 5631 ref|YP_003401560.1| 55 145 3.0E-42 GMRHGY403GX3XC 474 fadL long chain fatty acid transport protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158308.1| 71 187 4.0E-46 GMRHGY403GY9DS 488 putative GTP cyclohydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159536.1| 81 210 3.0E-61 GMRHGY403GOZVO 509 rnK putative regulator of nucleoside diphosphate kinase Azoarcus sp. BH72 ref|YP_932320.1| 45 90.5 7.0E-17 GMRHGY403GTARH 541 pyridoxamine 5'-phosphate oxidase-related FMN-binding Geobacter sp. M21 ref|YP_003021855.1| 41 103 9.0E-21 GMRHGY403F4G32 529 Pyrrolo-quinoline quinone Verminephrobacter eiseniae EF01-2 ref|YP_999677.1| 75 285 2.0E-75 GMRHGY403G0JNZ 331 molybdopterin oxidoreductase, iron-sulfur binding subunit Truepera radiovictrix DSM 17093 ref|YP_003704696.1| 66 80.5 1.0E-21 GMRHGY403FHYL2 421 aceC pyruvate dehydrogenase subunit E1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157094.1| 96 276 5.0E-73 GMRHGY403GL4NT 534 metallophosphoesterase Solibacter usitatus Ellin6076 ref|YP_822237.1| 61 179 2.0E-34 GMRHGY403GOKSF 161 rpoC DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159177.1| 100 83.2 6.0E-16 GMRHGY403HFOBS 483 oma outer membrane protein/surface antigen Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160448.1| 87 285 2.0E-75 GMRHGY403F26U4 306 YajC preprotein translocase subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157793.1| 90 63.5 2.0E-14 GMRHGY403G8QH5 506 pilD type IV fimbrial biogenesis protein, prepilin cysteine protease (C20) PilD Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159488.1| 67 208 2.0E-52 GMRHGY403FRF5D 511 4-hydroxybenzoate 3-monooxygenase Polaromonas naphthalenivorans CJ2 ref|YP_982132.1| 69 255 1.0E-66 GMRHGY403FLJ6X 484 Transcriptional regulator, LysR family Erwinia billingiae Eb661 ref|YP_003740546.1| 34 100 6.0E-20 GMRHGY403G76V9 509 aceE pyruvate dehydrogenase subunit E1 Bordetella pertussis Tohama I ref|NP_879787.1| 85 302 9.0E-81 GMRHGY403GQ743 414 ribosomal-protein-alanine acetyltransferase Thauera sp. MZ1T ref|YP_002355891.1| 59 63.9 6.0E-09 GMRHGY403FL37X 505 SUF system FeS assembly protein, NifU family Thauera sp. MZ1T ref|YP_002355542.1| 75 223 7.0E-57 GMRHGY403GR32Z 563 diguanylate cyclase/phosphodiesterase with PAS/PAC sensor(s) Thauera sp. MZ1T ref|YP_002355296.1| 72 107 7.0E-22 GMRHGY403GBKOW 527 fabF beta-ketoacyl-(acyl-carrier-protein) synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160133.1| 89 242 1.0E-62 GMRHGY403GDMQ8 247 putative transposase Acetivibrio cellulolyticus CD2 ref|ZP_07327145.1| 75 78.6 5.0E-14 GMRHGY403FPRYU 512 phaC polyhydroxyalkanoate synthase Azoarcus sp. BH72 ref|YP_932525.1| 89 306 6.0E-82 GMRHGY403G6N66 502 Neisseria PilC domain protein Sideroxydans lithotrophicus ES-1 ref|YP_003524702.1| 35 56.6 1.0E-06 GMRHGY403FPFU6 531 HybD peptidase Dechloromonas aromatica RCB ref|YP_287167.1| 76 145 9.0E-36 GMRHGY403GQP7W 469 phbC poly-beta-hydroxybutyrate polymerase Azorhizobium caulinodans ORS 571 ref|YP_001524761.1| 78 179 4.0E-59 GMRHGY403GLBW5 516 adenosylcobinamide kinase Methylobacillus flagellatus KT ref|YP_544225.1| 51 131 3.0E-29 GMRHGY403G92R0 484 guaB inosine-5'-monophosphate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160821.1| 94 157 4.0E-71 GMRHGY403FMD46 531 Pyrrolo-quinoline quinone Verminephrobacter eiseniae EF01-2 ref|YP_999677.1| 82 318 2.0E-85 GMRHGY403G6A40 486 ABC transporter, ATP-binding/permease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158002.1| 75 168 2.0E-40 GMRHGY403GRHFF 517 glycosyl transferase, group 1 Solibacter usitatus Ellin6076 ref|YP_828319.1| 38 94.4 5.0E-18 GMRHGY403GP2X2 494 putative ATP-binding ABC transporter protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158594.1| 89 266 7.0E-70 GMRHGY403GOVOY 524 menG ribonuclease activity regulator protein RraA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159562.1| 79 220 8.0E-62
! ! 216!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403G6A3K 369 tig trigger factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159853.1| 92 154 2.0E-45 GMRHGY403GWAY5 456 transcriptional regulator, ArsR family Chloroflexus aggregans DSM 9485 ref|YP_002464947.1| 46 45.1 5.0E-08 GMRHGY403HDVGD 434 Poly-beta-hydroxybutyrate polymerase Bradyrhizobium sp. BTAi1 ref|YP_001239967.1| 66 137 1.0E-36 GMRHGY403G4F06 463 phbC putative poly-beta-hydroxybutyrate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159076.1| 75 164 3.0E-58 GMRHGY403F0MXX 258 int site-specific recombinase, prophage insertion Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161124.1| 61 228 2.0E-17 GMRHGY403GL413 519 transcriptional regulator, GntR family Rubrobacter xylanophilus DSM 9941 ref|YP_645915.1| 36 70.9 1.0E-12 GMRHGY403GJ143 496 2-oxo-acid dehydrogenase E1 subunit, homodimeric type Nitrosococcus halophilus Nc4 ref|YP_003528350.1| 63 166 1.0E-39 GMRHGY403GLC45 477 putative sulfonate ABC transporter, periplasmic sulfonate-binding protein Thauera sp. MZ1T ref|YP_002889828.1| 71 229 7.0E-59 GMRHGY403FUX4G 484 pal outer membrane protein, porin-associated lipoprotein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158518.1| 82 243 5.0E-63 GMRHGY403HEVDD 386 DNA adenine methylase Halothiobacillus neapolitanus c2 ref|YP_003262112.1| 51 73.9 5.0E-22 GMRHGY403FWBO2 499 gabD Succinate-semialdehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158713.1| 48 288 2.0E-76 GMRHGY403FVIA3 473 glutathione synthase Parvibaculum lavamentivorans DS-1 ref|YP_001412095.1| 68 125 1.0E-43 GMRHGY403G7G8F 500 putative Histidine kinase Oscillatoria sp. PCC 6506 ref|ZP_07113263.1| 42 100 8.0E-20 GMRHGY403GKEOZ 366 poxD phenol hydroxylase, subunit P3 Azoarcus sp. BH72 ref|YP_933945.1| 77 171 2.0E-43 GMRHGY403G7D5Y 264 RuBisCO operon transcriptional regulator putative Azoarcus sp. BH72 ref|YP_933219.1| 94 99.4 1.0E-19 GMRHGY403FVV1E 494 norB Nitric-oxide reductase Thauera sp. MZ1T ref|YP_002889577.1| 85 280 4.0E-74 GMRHGY403HCSRW 432 RND superfamily exporter Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159684.1| 87 232 1.0E-59 GMRHGY403GXX8Y 490 GTP-binding protein LepA Burkholderia thailandensis MSMB43 ref|ZP_02464233.1| 79 124 4.0E-74 GMRHGY403GIHW0 499 acetyl-CoA synthetase Bordetella avium 197N ref|YP_785693.1| 73 144 7.0E-52 GMRHGY403F02TW 470 transposase IS3/IS911 Nitrosospira multiformis ATCC 25196 ref|YP_412698.1| 91 167 5.0E-40 GMRHGY403GUOQ8 479 putative putative abortive infection phage resistance protein Burkholderia pseudomallei S13 ref|ZP_04904727.1| 79 143 8.0E-33 GMRHGY403FL1RP 496 D-amino acid dehydrogenase small subunit Acidovorax avenae subsp. citrulli AAC00-1 ref|YP_972818.1| 75 167 4.0E-40 GMRHGY403F8KMV 513 D,D-heptose 1,7-bisphosphate phosphatase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157745.1| 88 301 2.0E-80 GMRHGY403GVIMY 502 tyrA prephenate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157513.1| 84 273 4.0E-72 GMRHGY403G08HY 495 probable serine protease Lyngbya sp. PCC 8106 ref|ZP_01622065.1| 29 47.8 5.0E-04 GMRHGY403FMYGW 509 hydroxyacylglutathione hydrolase Polaromonas sp. JS666 ref|YP_548883.1| 71 89.4 9.0E-22 GMRHGY403FSBGJ 499 phosphoglucomutase Burkholderia xenovorans LB400 ref|YP_555795.1| 66 232 1.0E-59 GMRHGY403GHAQE 515 hydrophobe/amphiphile efflux-1 family protein Brucella pinnipedialis B2/94 ref|ZP_05173381.1| 55 164 3.0E-39 GMRHGY403GGFGT 513 oma outer membrane protein/surface antigen Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160448.1| 83 306 8.0E-82 GMRHGY403FYYDO 400 pccB propionyl-CoA carboxylase beta subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159133.1| 96 256 7.0E-67 GMRHGY403HBFKG 454 ABC transporter permease protein Azoarcus sp. BH72 ref|YP_931816.1| 37 87 7.0E-16 GMRHGY403GV5MI 271 radical SAM enzyme, Cfr family Thermobaculum terrenum ATCC BAA-798 ref|YP_003323131.1| 40 60.8 5.0E-08 GMRHGY403GKP95 514 binding-protein-dependent transport systems inner membrane component Herpetosiphon aurantiacus ATCC 23779 ref|YP_001547323.1| 42 118 3.0E-25 GMRHGY403G87FR 508 dcd deoxycytidine triphosphate deaminase Azoarcus sp. BH72 ref|YP_934702.1| 96 300 4.0E-80 GMRHGY403GXJTV 500 topB2 DNA topoisomerase III Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158693.1| 91 310 4.0E-83 GMRHGY403GBDJU 194 int phage integrase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157407.1| 48 52 3.0E-05 GMRHGY403G4RY9 468 nlaB phospholipid and glycerol acyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157744.1| 84 258 2.0E-67 GMRHGY403F3UPM 152 aminotransferase class I and II Leptothrix cholodnii SP-6 ref|YP_001789122.1| 87 70.9 7.0E-12 GMRHGY403FO6TS 419 metal dependent phosphohydrolase Candidatus Koribacter versatilis Ellin345 ref|YP_590488.1| 64 86.7 3.0E-16 GMRHGY403FXT03 478 efP elongation factor P Azoarcus sp. BH72 ref|YP_931591.1| 85 81.6 3.0E-14 GMRHGY403FMKS4 478 fmt methionyl-tRNA formyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158688.1| 85 162 1.0E-53 GMRHGY403F4X8U 458 Alpha,alpha-trehalose-phosphate synthase (UDP-forming) Pelobacter propionicus DSM 2379 ref|YP_902921.1| 69 213 5.0E-54 GMRHGY403F72T7 507 narH nitrate reductase, beta subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160620.1| 87 286 5.0E-76 GMRHGY403HDY1L 472 pcnB RNA-poly(A) polymerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161059.1| 96 149 2.0E-62 GMRHGY403FPWUN 456 DNA repair protein RecO Burkholderia oklahomensis C6786 ref|ZP_02363559.1| 71 113 9.0E-24 GMRHGY403FT6A2 486 DNA-directed RNA polymerase beta chain Bordetella avium 197N ref|YP_784551.1| 91 140 5.0E-68 GMRHGY403G3C53 523 Glycosyl transferase, family 2 gamma proteobacterium HdN1 ref|YP_003809850.1| 33 55.1 3.0E-06 GMRHGY403FK8QU 510 ribAB bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTPcyclohydrolase II protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159043.1| 86 286 5.0E-76 GMRHGY403GOJCE 532 rne ribonuclease E, (RNase E) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160120.1| 88 172 1.0E-41 GMRHGY403GR878 281 nahO acetaldehyde dehydrogenase Azoarcus sp. BH72 ref|YP_933477.1| 73 84.3 5.0E-15 GMRHGY403GD8RD 501 Rhodanese domain protein Thauera sp. MZ1T ref|YP_002355197.1| 65 169 1.0E-40 GMRHGY403HE3MZ 490 ATP-dependent Clp protease ATP-binding subunit ClpA Thiobacillus denitrificans ATCC 25259 ref|YP_314995.1| 67 203 5.0E-51 GMRHGY403GENWK 501 phosphoglucomutase Herpetosiphon aurantiacus ATCC 23779 ref|YP_001547581.1| 49 141 2.0E-32 GMRHGY403GM7QW 479 tyrosyl-tRNA synthetase Ralstonia solanacearum PSI07 ref|YP_003753491.1| 71 224 3.0E-57 GMRHGY403FLEF6 474 ribonuclease II Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157961.1| 69 172 1.0E-41 GMRHGY403FM6R7 512 acsB acetyl-CoA synthetase Azoarcus sp. BH72 ref|YP_933918.1| 75 222 7.0E-62 GMRHGY403GH6D9 479 ATP-dependent DNA helicase, RecQ family Allochromatium vinosum DSM 180 ref|YP_003445108.1| 60 180 5.0E-44 GMRHGY403GTUTK 454 translation initiation factor Caldicelulosiruptor becscii DSM 6725 ref|YP_002573233.1| 47 74.7 4.0E-12 GMRHGY403GJH3C 553 Activator of Hsp90 ATPase 1 family protein Dyadobacter fermentans DSM 18053 gb|ACT95478.1| 50 154 5.0E-36 GMRHGY403F3F7Z 437 glycosylasparaginase Solibacter usitatus Ellin6076 ref|YP_821783.1| 58 89 2.0E-16 GMRHGY403G0P7L 505 putative iron-sulfur binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159248.1| 71 237 4.0E-61 GMRHGY403HIR2R 306 threonine dehydratase Acidovorax sp. JS42 ref|YP_984411.1| 54 104 3.0E-21 GMRHGY403G3AAW 486 nadA quinolinate synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159361.1| 91 316 7.0E-85 GMRHGY403GHDH2 493 Holliday junction DNA helicase RuvA Herpetosiphon aurantiacus ATCC 23779 ref|YP_001545282.1| 51 136 8.0E-31 GMRHGY403HECBU 478 M23 family peptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157577.1| 69 127 4.0E-29 GMRHGY403HAAR2 359 HtpX heat shock protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158687.1| 89 159 9.0E-38 GMRHGY403G6N17 275 adenylate/guanylate cyclase Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003169071.1| 50 64.7 4.0E-09 GMRHGY403GL7S8 459 trpA tryptophan synthase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159726.1| 91 102 5.0E-33 GMRHGY403HB1ZO 506 putative membrane associated hydrolase uncultured bacterium gb|ABQ08717.1| 45 67.4 2.0E-23 GMRHGY403HF33R 491 ATP-dependent metalloprotease FtsH Thermobaculum terrenum ATCC BAA-798 ref|YP_003322790.1| 49 143 6.0E-33 GMRHGY403GM9ZZ 483 nuoM NADH dehydrogenase subunit M Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159777.1| 89 280 6.0E-74 GMRHGY403GEKWG 524 hCG1814203 Homo sapiens gb|EAW81162.1| 76 84.7 4.0E-15 GMRHGY403G929G 481 Band 7 protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157692.1| 86 125 2.0E-27 GMRHGY403G6JJG 487 SecA preprotein translocase subunit SecA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157807.1| 82 261 2.0E-68 GMRHGY403G0VFZ 341 gapA glyceraldehyde 3-phosphate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157603.1| 89 193 5.0E-48 GMRHGY403F6191 477 FAD dependent oxidoreductase Desulfomicrobium baculatum DSM 4028 ref|YP_003157704.1| 57 166 1.0E-39
! ! 217!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GVG6V 471 phoB phosphate regulon transcriptional regulatory protein Azoarcus sp. BH72 ref|YP_931669.1| 84 233 6.0E-60 GMRHGY403GV25J 484 glycosyl transferase family protein Leptothrix cholodnii SP-6 ref|YP_001790421.1| 59 98.6 2.0E-19 GMRHGY403HHNWD 584 putative type IIs restriction endonuclease Geobacillus stearothermophilus ref|YP_001716012.1| 31 55.1 5.0E-06 GMRHGY403F31YN 533 short chain dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158453.1| 76 167 7.0E-40 GMRHGY403GH2QE 448 pyruvate, phosphate dikinase Ktedonobacter racemifer DSM 44963 ref|ZP_06967291.1| 47 128 2.0E-28 GMRHGY403GG1LM 507 hupL ferredoxin hydrogenase, large chain Azoarcus sp. BH72 ref|YP_935289.1| 73 261 3.0E-68 GMRHGY403G73PM 511 transcription factor IIA Naegleria gruberi ref|XP_002672395.1| 45 65.1 3.0E-09 GMRHGY403FZ1OK 502 tpX thiol peroxidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158670.1| 82 234 4.0E-60 GMRHGY403GSE22 488 ZipA-like protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160673.1| 76 113 9.0E-24 GMRHGY403G8M7B 513 bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase Nitrosospira multiformis ATCC 25196 ref|YP_410834.1| 70 82.8 3.0E-29 GMRHGY403GRW11 262 translation initiation factor IF-3 Roseiflexus castenholzii DSM 13941 ref|YP_001431547.1| 60 106 1.0E-21 GMRHGY403FMX5I 552 Pyrrolo-quinoline quinone Limnobacter sp. MED105 ref|ZP_01915892.1| 80 316 1.0E-84 GMRHGY403G3MXC 570 Leucine rich repeat protein Dyadobacter fermentans DSM 18053 ref|YP_003088975.1| 46 63.2 1.0E-12 GMRHGY403FYA00 525 OsmC-like protein Rubrobacter xylanophilus DSM 9941 ref|YP_642933.1| 55 157 4.0E-37 GMRHGY403GZ9SM 501 nuoD NADH dehydrogenase subunit D Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159768.1| 96 176 7.0E-43 GMRHGY403HD8G6 562 diguanylate cyclase/phosphodiesterase with PAS/PAC sensor(s) Thauera sp. MZ1T ref|YP_002355296.1| 66 65.9 1.0E-12 GMRHGY403FM4XV 498 fumA fumarate hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157121.1| 86 280 6.0E-74 GMRHGY403GZT59 486 response regulator receiver protein Chloroherpeton thalassium ATCC 35110 ref|YP_001997521.1| 55 110 8.0E-23 GMRHGY403HANFY 482 ptsN phosphotransferase system mannitol/fructose-specific IIA domain-containing protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158942.1| 90 284 2.0E-75 GMRHGY403G765E 483 rpoB DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159176.1| 91 286 8.0E-76 GMRHGY403FH1RB 113 translation elongation factor Tu Thiomonas intermedia K12 ref|YP_003644770.1| 100 74.3 5.0E-12 GMRHGY403FTVVA 190 ABC transporter, permease protein, putative Flavobacteria bacterium BAL38 ref|ZP_01732766.1| 52 45.1 7.0E-04 GMRHGY403FS00G 521 asparagine synthase (glutamine-hydrolyzing) Methylobacter tundripaludum SV96 ref|ZP_07652868.1| 75 145 1.0E-33 GMRHGY403F4KRS 489 nuoM NADH dehydrogenase subunit M Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159777.1| 94 306 7.0E-82 GMRHGY403G4YQE 494 glyQ glycyl-tRNA synthetase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157747.1| 100 341 2.0E-92 GMRHGY403GGGHR 511 cpaB pilus assembly transmembrane protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159118.1| 98 199 1.0E-69 GMRHGY403GH9AY 514 infB translation initiation factor IF-2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160344.1| 56 63.5 9.0E-09 GMRHGY403GMC16 495 ProQ activator of osmoprotectant transporter ProP Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158568.1| 81 178 2.0E-43 GMRHGY403FLSJT 489 spoT guanosine-3',5'-bis(diphosphate) 3'-pyrophosphohydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158997.1| 95 313 6.0E-84 GMRHGY403GB98X 506 hemA glutamyl-tRNA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157646.1| 88 291 2.0E-77 GMRHGY403HIOKZ 293 F0F1 ATP synthase subunit alpha Lawsonia intracellularis PHE/MN1-00 ref|YP_594778.1| 93 179 1.0E-43 GMRHGY403HG8HU 485 NADH/Ubiquinone/plastoquinone (complex I) Thauera sp. MZ1T ref|YP_002889423.1| 75 235 1.0E-60 GMRHGY403HB9AR 243 gcvP1 glycine dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160522.1| 74 87 1.0E-18 GMRHGY403FQ7QH 448 hemN coproporphyrinogen III oxidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159954.1| 97 286 5.0E-76 GMRHGY403G696N 514 trxA thioredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159707.1| 96 216 6.0E-55 GMRHGY403HERF5 512 peptidase S8 and S53 subtilisin kexin sedolisin Chloroherpeton thalassium ATCC 35110 ref|YP_001996952.1| 35 97.1 7.0E-19 GMRHGY403G2RBR 544 ppiA peptidyl-prolyl cis-trans isomerase precursor (PPIase) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159735.1| 77 281 3.0E-74 GMRHGY403FRNEV 400 ExsB Chlorobium ferrooxidans DSM 13031 ref|ZP_01386277.1| 64 83.6 2.0E-15 GMRHGY403FQPOE 508 oligopeptide ABC transporter substrate-binding protein Bacillus clausii KSM-K16 ref|YP_174739.1| 45 139 1.0E-31 GMRHGY403GCFH7 437 rpsQ 30S ribosomal protein S17 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159192.1| 98 97.4 2.0E-35 GMRHGY403GIPM5 553 Leucine rich repeat protein Dyadobacter fermentans DSM 18053 ref|YP_003088975.1| 47 98.6 3.0E-19 GMRHGY403G68SK 187 16S ribosomal RNA methyltransferase RsmE Pseudomonas stutzeri A1501 ref|YP_001174410.1| 88 82.4 2.0E-14 GMRHGY403G9F7L 538 extracellular solute-binding protein Chloroflexus aurantiacus J-10-fl ref|YP_001634648.1| 46 142 2.0E-32 GMRHGY403F8CQY 519 transcriptional regulator, IclR family Burkholderia sp. Ch1-1 ref|ZP_06839430.1| 57 115 2.0E-24 GMRHGY403GTF5C 443 boxR anaerobic benzoate catabolism transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158578.1| 83 99.4 2.0E-39 GMRHGY403G9N22 470 MerR family transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157480.1| 66 162 2.0E-38 GMRHGY403G52VB 488 narL nitrate/nitrite response regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160616.1| 95 159 9.0E-38 GMRHGY403GO8R0 469 NusA transcription elongation factor NusA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160343.1| 96 248 1.0E-64 GMRHGY403GIVG4 506 RNA-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159754.1| 87 130 6.0E-29 GMRHGY403FOBQQ 473 BLIP-II repeat-containing protein Herminiimonas arsenicoxydans ref|YP_001100469.1| 31 62.4 2.0E-08 GMRHGY403HGDF5 499 Poly-beta-hydroxybutyrate polymerase domain protein Thauera sp.MZ1T ref|YP_002354682.1| 100 270 5.0E-84 GMRHGY403F6MFM 478 ATP-dependent DNA helicase, RecQ family Allochromatium vinosum DSM 180 ref|YP_003445108.1| 73 128 8.0E-41 GMRHGY403FTXOR 516 recJ exodeoxyribonuclease VII, single-stranded DNA-specific exonuclease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160326.1| 81 109 4.0E-24 GMRHGY403GJIY7 517 Tripartite ATP-independent periplasmic transporter DctQ component Anaeromyxobacter dehalogenans 2CP-1 ref|YP_002493484.1| 71 109 1.0E-22 GMRHGY403FV9YQ 495 ccmC heme exporter, permease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159007.1| 89 267 3.0E-70 GMRHGY403F5PQ0 487 LuxR family DNA-binding response regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160938.1| 82 130 1.0E-37 GMRHGY403HGSYQ 492 RnfF protein related to RnfF or ApbE [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158461.1| 84 244 2.0E-63 GMRHGY403G3IDW 477 cheV2 chemotaxis protein CheV-like Azoarcus sp. BH72 ref|YP_932963.1| 78 148 2.0E-34 GMRHGY403GTI5A 486 putative transcriptional regulator Azoarcus sp. BH72 ref|YP_933972.1| 74 239 1.0E-61 GMRHGY403GTKVP 510 argH argininosuccinate lyase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157639.1| 94 264 3.0E-69 GMRHGY403FLCZH 373 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 81 89.4 4.0E-30 GMRHGY403FI243 440 hupL ferredoxin hydrogenase, large chain Azoarcus sp. BH72 ref|YP_935289.1| 92 250 4.0E-65 GMRHGY403GEHDT 179 citrate synthase Herbaspirillum seropedicae SmR1 ref|YP_003776366.1| 79 89.7 1.0E-16 GMRHGY403GS0BG 494 peptide chain release factor 3 Thiomonas intermedia K12 ref|YP_003643080.1| 64 208 2.0E-52 GMRHGY403G99ME 295 formyl-tetrahydrofolate synthetase uncultured bacterium gb|ADK11025.1| 71 130 7.0E-29 GMRHGY403FOQC3 500 aldo/keto reductase Truepera radiovictrix DSM 17093 ref|YP_003706180.1| 82 170 3.0E-57 GMRHGY403FSPL4 498 glcD glycolate oxidase (FAD-linked subunit) oxidoreductase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159571.1| 84 282 9.0E-75 GMRHGY403HFSJC 475 thrS threonyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157487.1| 78 224 2.0E-60 GMRHGY403G097O 401 sspB ClpXP, sspB protease specificity-enhancing factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157656.1| 88 88.2 5.0E-36 GMRHGY403HGBLJ 504 msbA2 lipid A export ATP-binding/permease protein Azoarcus sp. BH72 ref|YP_935074.1| 72 150 4.0E-59 GMRHGY403HBMYQ 469 transposase, mutator type alpha proteobacterium BAL199 ref|ZP_02192301.1| 71 227 4.0E-58 GMRHGY403FIQ7L 510 metH B12-dependent methionine synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158816.1| 88 281 2.0E-74 GMRHGY403FIEVZ 510 leuS leucyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159513.1| 97 251 3.0E-92 GMRHGY403GMXZ3 502 CoA-transferase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158169.1| 75 130 4.0E-29 GMRHGY403GEQSF 495 yjjK putative ABC transporter ATP-binding protein Azoarcus sp. BH72 ref|YP_932779.1| 91 306 6.0E-82 GMRHGY403G5CJP 580 beta-lactamase domain protein Akkermansia muciniphila ATCC BAA-835 ref|YP_001876862.1| 65 78.2 1.0E-13
! ! 218!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GE7FD 489 sugar-phosphate isomerase Azoarcus sp. BH72 ref|YP_932298.1| 66 203 5.0E-51 GMRHGY403GWI1A 495 MerR family transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157480.1| 61 112 2.0E-23 GMRHGY403F3AD3 497 bifunctional 3-deoxy-7-phosphoheptulonate synthase/chorismate mutase Deinococcus geothermalis DSM 11300 ref|YP_605036.1| 67 87 2.0E-22 GMRHGY403G49YU 488 pntB pyridine nucleotide transhydrogenase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159578.1| 97 315 9.0E-85 GMRHGY403GU6F9 484 aspartokinase/homoserine dehydrogenase Chryseobacterium gleum ATCC 35910 ref|ZP_07086061.1| 62 132 3.0E-47 GMRHGY403FKQV6 486 transferase [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158055.1| 90 265 1.0E-69 GMRHGY403HBHFO 514 HAD family hydrolase Maricaulis maris MCS10 ref|YP_756288.1| 36 85.1 3.0E-15 GMRHGY403G5IEH 510 adenosylcobinamide kinase/adenosylcobinamide-phosphateguanylyltransferase Xanthomonas oryzae pv. oryzae PXO99A ref|YP_001914977.1| 61 68.6 1.0E-19 GMRHGY403FWYDW 509 Chaperone protein dnaK (Heat shock protein 70) (Heat shock 70 kDa protein) (HSP70) Thiomonas sp. 3As emb|CAZ88862.1| 90 300 6.0E-80 GMRHGY403GV75B 508 nirS putative dissimilatory nitrite reductase uncultured bacterium gb|AAL89549.1| 93 241 3.0E-63 GMRHGY403HG7UJ 427 cytochrome c biogenesis protein transmembrane region Desulfonatronospira thiodismutans ASO3-1 ref|ZP_07015491.1| 31 63.2 1.0E-08 GMRHGY403G1NW4 487 ribAB bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTPcyclohydrolase II protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159043.1| 79 159 1.0E-37 GMRHGY403FYLHF 480 norD nitric oxide reductase activation protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157132.1| 83 255 1.0E-66 GMRHGY403FKH5G 468 putative aminotransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159216.1| 84 275 1.0E-72 GMRHGY403F93IG 114 putative TonB-dependent receptor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160538.1| 74 54.3 5.0E-06 GMRHGY403F1PG4 480 phbC putative poly-beta-hydroxybutyrate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159076.1| 81 261 3.0E-68 GMRHGY403FPRJO 550 TolB translocation protein TolB Nitrosomonas eutropha C91 ref|YP_746538.1| 55 81.6 1.0E-29 GMRHGY403GBZVS 436 pnp polynucleotide phosphorylase/polyadenylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160348.1| 94 270 3.0E-71 GMRHGY403GA54K 494 isocitrate dehydrogenase Acinetobacter baumannii SDF ref|YP_001706521.1| 86 235 2.0E-60 GMRHGY403F0XIW 499 RecO DNA repair protein RecO Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160181.1| 78 205 3.0E-52 GMRHGY403GBT7A 469 icd2 isocitrate dehydrogenase[NADP] Azoarcus sp. BH72 ref|YP_932650.1| 89 285 2.0E-75 GMRHGY403G8MYT 459 sugar ABC transporter ATPase Herbaspirillum seropedicae SmR1 ref|YP_003776478.1| 79 238 2.0E-61 GMRHGY403FWQXI 486 DNA-directed RNA polymerase, beta' subunit Burkholderiales bacterium 1_1_47 ref|ZP_07342456.1| 88 186 2.0E-48 GMRHGY403GWQ09 260 ndK nucleoside diphosphate kinase [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157703.1| 93 127 5.0E-29 GMRHGY403GST6R 444 putative lipoprotein Kordia algicida OT-1 ref|ZP_02160575.1| 35 73.2 1.0E-11 GMRHGY403G9QOA 272 lysA1 diaminopimelate decarboxylase Azoarcus sp. BH72 ref|YP_934750.1| 58 95.5 2.0E-18 GMRHGY403GKQCM 497 paaK aerobic phenylacetate-CoA ligase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159025.1| 88 195 1.0E-48 GMRHGY403GBJ2M 511 prfB peptide chain release factor 2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160323.1| 93 244 2.0E-69 GMRHGY403F9SVM 514 alcohol dehydrogenase Solibacter usitatus Ellin6076 ref|YP_822543.1| 56 151 5.0E-54 GMRHGY403GEIYX 514 acetamidase/formamidase Dinoroseobacter shibae DFL 12 ref|YP_001542368.1| 57 207 4.0E-52 GMRHGY403FQKRQ 507 isochorismatase hydrolase Thauera sp. MZ1T ref|YP_002355310.1| 83 177 4.0E-43 GMRHGY403F4NZ0 508 purC phosphoribosylaminoimidazole-succinocarboxamide synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157615.1| 81 220 4.0E-56 GMRHGY403G09LQ 507 putative phospholipid biosynthesis acyltransferase Azoarcus sp. BH72 ref|YP_935420.1| 59 102 2.0E-41 GMRHGY403G0SZK 203 dioxygenase Bordetella petrii DSM 12804 ref|YP_001629080.1| 64 62.4 2.0E-08 GMRHGY403GVLKR 464 ABC transporter related Thauera sp. MZ1T ref|YP_002354557.1| 85 94 6.0E-18 GMRHGY403HD5H9 398 ThrS ThrS protein Azoarcus sp. BH72 ref|YP_932583.1| 79 214 4.0E-54 GMRHGY403GF7H1 497 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 93 325 1.0E-87 GMRHGY403GZOSB 477 short-chain dehydrogenase/reductase SDR Mycobacterium kansasii ATCC 12478 ref|ZP_04747366.1| 38 76.3 1.0E-12 GMRHGY403FQ41I 481 cobalamin synthesis protein P47K Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003168899.1| 65 202 1.0E-50 GMRHGY403GGX0B 502 NIRH NIRH Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157492.1| 75 250 4.0E-65 GMRHGY403GD3LK 512 argF ornithine carbamoyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159917.1| 94 300 3.0E-80 GMRHGY403G597I 497 phosphoribosylaminoimidazole-succinocarboxamide synthase Cupriavidus metallidurans CH34 ref|YP_582659.1| 71 243 6.0E-63 GMRHGY403HDW0P 496 phaC putative poly-beta-hydroxyalkanoate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159900.1| 60 139 3.0E-40 GMRHGY403GASK1 141 rne ribonuclease E Azoarcus sp. BH72 ref|YP_933117.1| 90 79 2.0E-13 GMRHGY403G4CKN 444 FAD linked oxidase-like protein Cupriavidus metallidurans CH34 ref|YP_585066.1| 72 142 1.0E-32 GMRHGY403F6RNJ 522 long-chain N-acyl amino acid synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159427.1| 63 183 6.0E-45 GMRHGY403FKDAE 470 putative glycosyl transferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157912.1| 57 148 2.0E-43 GMRHGY403HHZFD 500 putative lipase precursor (triacylglycerol lipase) Frankia alni ACN14a ref|YP_713995.1| 43 74.7 4.0E-12 GMRHGY403G2AOO 489 dxs 1-deoxy-D-xylulose-5-phosphate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159537.1| 67 160 5.0E-38 GMRHGY403GQ045 496 hemN coproporphyrinogen III oxidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159954.1| 92 299 9.0E-80 GMRHGY403FJ7BY 490 pntB pyridine nucleotide transhydrogenase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159578.1| 90 277 4.0E-73 GMRHGY403GPJ02 510 Extracellular ligand-binding receptor Desulfatibacillum alkenivorans AK-01 ref|YP_002432825.1| 41 98.2 3.0E-19 GMRHGY403F14DV 223 hflX GTP-binding protein hflX Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157693.1| 97 77.4 1.0E-25 GMRHGY403HBWKK 459 putative sulfonate ABC transporter, periplasmic sulfonate-binding protein Thauera sp. MZ1T ref|YP_002889828.1| 72 236 9.0E-61 GMRHGY403HC17V 338 OsmC family protein Polaromonas naphthalenivorans CJ2 ref|YP_983886.1| 86 143 8.0E-33 GMRHGY403F6FIB 183 isocitrate dehydrogenase (NAD+) Rhodoferax ferrireducens T118 ref|YP_523628.1| 79 97.1 7.0E-19 GMRHGY403FVF3Z 457 hcrA 4-hydroxybenzoyl-CoA reductase, alpha subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159062.1| 51 144 3.0E-33 GMRHGY403FNX1B 351 NDPsugar dehydrogenase Azoarcus sp. BH72 ref|YP_934692.1| 52 62.8 7.0E-21 GMRHGY403F0C3A 463 pgsA CDP-diacylglycerol--glycerol-3-phosphatidyltrans ferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160186.1| 79 228 2.0E-58 GMRHGY403GT60R 520 ccmH cytochrome C biogenesis protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159012.1| 68 210 6.0E-53 GMRHGY403F0ZYO 373 uridylate kinase Thermomonospora curvata DSM 43183 ref|YP_003300984.1| 66 53.5 5.0E-10 GMRHGY403F9O4Y 500 Rieske (2Fe-2S) domain-containing protein Novosphingobium aromaticivorans DSM 12444 ref|YP_001165925.1| 50 179 1.0E-43 GMRHGY403HCN6C 496 LPS biosynthesis protein WbpG Polaromonas naphthalenivorans CJ2 ref|YP_983341.1| 75 218 6.0E-63 GMRHGY403GY6TN 197 DNA-directed RNA polymerase, beta subunit Truepera radiovictrix DSM 17093 ref|YP_003706127.1| 86 57.4 1.0E-11 GMRHGY403GJWEA 549 dfrA dihydrofolate reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159531.1| 53 89 3.0E-16 GMRHGY403G10PW 403 dadA D-amino acid dehydrogenase small subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157655.1| 62 156 7.0E-37 GMRHGY403F8FK1 488 RpoD RNA polymerase sigma factor RpoD Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159500.1| 99 239 9.0E-62 GMRHGY403HALWZ 474 Holliday junction DNA helicase motor protein Rhodoferax ferrireducens T118 ref|YP_522020.1| 80 127 1.0E-40 GMRHGY403HEZWR 509 tktA transketolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157602.1| 87 140 5.0E-33 GMRHGY403FZSSP 500 nuoF putative NADH-ubiquinone oxidoreductase chain F Azoarcus sp. BH72 ref|YP_932905.1| 80 263 7.0E-69 GMRHGY403GLR5N 196 ATP-dependent DNA helicase, RecQ family Allochromatium vinosum DSM 180 ref|YP_003445108.1| 76 91.7 3.0E-17 GMRHGY403GQYWZ 496 ABC transporter Nitrosomonas europaea ATCC 19718 ref|NP_842307.1| 83 287 4.0E-76 GMRHGY403GVSBD 484 helX putative thiol:disulphide oxidoreductase Azoarcus sp. BH72 ref|YP_931607.1| 64 117 5.0E-25 GMRHGY403GG389 464 PAS/PAC sensor signal transduction histidine kinase CandidatusAccumulibacter phosphatis clade IIA str. UW-1 ref|YP_003168624.1| 44 111 4.0E-23 GMRHGY403F3MXC 176 oppB oligopeptide transport system permease protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160830.1| 85 104 4.0E-21 GMRHGY403HBJEZ 508 putative peptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157428.1| 93 94 6.0E-34
! ! 219!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403G3S04 340 secreted serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member Sorangium cellulosum 'So ce 56' ref|YP_001614739.1| 59 97.4 5.0E-19 GMRHGY403FM0VV 485 fabF beta-ketoacyl-(acyl-carrier-protein) synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160133.1| 86 249 3.0E-71 GMRHGY403HHXPG 479 TRAP dicarboxylate transporter, DctP subunit Acidovorax avenae subsp. citrulli AAC00-1 ref|YP_971778.1| 50 77.8 4.0E-13 GMRHGY403GM1D4 458 ffH signal recognition particle protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159492.1| 98 124 6.0E-65 GMRHGY403G4UAW 531 MarR family transcriptional regulator Leptospira biflexa serovar Patoc strain 'Patoc 1 (Paris)' ref|YP_001837491.1| 63 60.5 1.0E-16 GMRHGY403GXSB6 494 LPS biosynthesis protein WbpG Polaromonas naphthalenivorans CJ2 ref|YP_983341.1| 73 262 9.0E-69 GMRHGY403GC10W 497 sucC succinyl-CoA synthetase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157705.1| 93 248 2.0E-64 GMRHGY403FZFK6 478 mltE transglycosylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159316.1| 72 48.5 1.0E-06 GMRHGY403GDPW9 488 50S ribosomal protein L1 Achromobacter piechaudii ATCC 43553 ref|ZP_06685093.1| 82 89.4 3.0E-20 GMRHGY403G39BM 485 DNA ligase Cupriavidus taiwanensis ref|YP_002005704.1| 61 182 1.0E-44 GMRHGY403FJ81S 471 fpr2 ferredoxin-NADP+ reductase Azoarcus sp. BH72 ref|YP_934161.1| 92 257 3.0E-67 GMRHGY403GBLLQ 493 boxA benzoyl-CoA oxygenase component A Azoarcus evansii gb|AAN39377.1| 71 253 4.0E-66 GMRHGY403FKP6K 531 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 78 290 4.0E-77 GMRHGY403GQK8K 495 lldA L-lactate dehydrogenase (cytochrome) Azoarcus sp. BH72 ref|YP_933974.1| 55 176 7.0E-43 GMRHGY403F83UL 230 rpoD RNA polymerase sigma factor rpoD (Sigma-70) Pantoea vagans C9-1 ref|YP_003932277.1| 98 139 1.0E-31 GMRHGY403F7PJ9 256 short chain dehydrogenase Nocardia farcinica IFM 10152 ref|YP_116721.1| 70 124 3.0E-27 GMRHGY403FJ2WV 527 parA3 ParA family protein Azoarcus sp. BH72 ref|YP_934714.1| 87 89.4 3.0E-18 GMRHGY403HGBZV 541 lhpP putative inorganic pyrophosphate phosphatase Azoarcus sp. BH72 ref|YP_932618.1| 57 52.8 2.0E-05 GMRHGY403FPX61 487 xanthine dehydrogenase, molybdenum binding subunit apoprotein Nocardioides sp. JS614 ref|YP_922834.1| 63 186 7.0E-46 GMRHGY403HD1ZE 523 prlC M3 family peptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157617.1| 88 201 2.0E-50 GMRHGY403G6K0S 514 cystine ABC transporter, periplasmic cystine-binding protein Pseudovibrio sp. JE062 ref|ZP_05084968.1| 51 60.5 2.0E-14 GMRHGY403FUKIE 476 2-oxoisovalerate dehydrogenase, E2 component (dihydrolipoamide acetyltransferase) Aurantimonas manganoxydans SI85-9A1 ref|ZP_01228375.1| 80 116 4.0E-47 GMRHGY403G4RU3 479 multi-sensor hybrid histidine kinase Rhodopseudomonas palustris TIE-1 ref|YP_001993376.1| 41 44.7 7.0E-16 GMRHGY403FWAU3 481 Uncharacterized ABC-type transport system, periplasmic component/surface lipoprotein Coprococcus catus GD/7 emb|CBK78992.1| 32 51.6 3.0E-05 GMRHGY403GECSP 525 peptidase, M48 family delta proteobacterium NaphS2 ref|ZP_07200056.1| 68 250 4.0E-65 GMRHGY403F7SUE 513 permease component of an ABC-transporter system Azoarcus sp. BH72 ref|YP_934679.1| 54 103 3.0E-35 GMRHGY403GEQ2I 521 respiratory-chain NADH dehydrogenase, subunit 1 Syntrophobacter fumaroxidans MPOB ref|YP_844339.1| 51 110 8.0E-29 GMRHGY403GI53M 513 sodB superoxide dismutase (Fe) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159902.1| 93 340 4.0E-92 GMRHGY403GVLSX 529 fimV1 putative type 4 pilus biogenesis Azoarcus sp. BH72 ref|YP_932547.1| 52 168 2.0E-40 GMRHGY403F8J6T 467 LuxR family DNA-binding response regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160938.1| 92 246 1.0E-64 GMRHGY403FPT9S 475 sucD succinyl-CoA synthetase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157704.1| 96 121 5.0E-56 GMRHGY403F2OK0 319 phosphoglucomutase Rhodoferax ferrireducens T118 ref|YP_522414.1| 74 157 3.0E-37 GMRHGY403FUW05 504 heterodisulfide reductase, B subunit Thiobacillus denitrificans ATCC 25259 ref|YP_315403.1| 96 117 2.0E-45 GMRHGY403FV89J 497 pterin-4-alpha-carbinolamine dehydratase Cupriavidus metallidurans CH34 ref|YP_585675.1| 61 121 3.0E-26 GMRHGY403G62G6 243 solute-binding family 7 protein Roseobacter denitrificans OCh 114 ref|YP_683924.1| 71 79.3 2.0E-19 GMRHGY403GMHSZ 173 3-oxoacyl-(acyl carrier protein) synthase III Bordetella bronchiseptica RB50 ref|NP_890293.1| 77 59.7 1.0E-08 GMRHGY403F8T8B 210 Mfd transcription-repair coupling protein Mfd Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160773.1| 76 80.5 7.0E-14 GMRHGY403G4DXY 516 UvrA UvrABC system protein A (excinuclease ABC subunit A) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159211.1| 95 329 9.0E-89 GMRHGY403HFKGC 521 AMP-dependent synthetase and ligase Acidovorax ebreus TPSY ref|YP_002552012.1| 69 118 9.0E-44 GMRHGY403FSTG1 544 protoporphyrinogen oxidase Opitutus terrae PB90-1 ref|YP_001819575.1| 37 78.2 8.0E-22 GMRHGY403GLNMY 405 putative regulator protein Azoarcus sp. BH72 ref|YP_934999.1| 79 153 4.0E-41 GMRHGY403GM80Q 238 ClpXP protease specificity-enhancing factor Dechloromonas aromatica RCB ref|YP_284039.1| 63 102 2.0E-20 GMRHGY403G8F2P 528 topB2 DNA topoisomerase III Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158693.1| 90 97.8 5.0E-19 GMRHGY403HFRGG 480 polysaccharide chain length determinant protein Azoarcus sp. BH72 ref|YP_934784.1| 77 157 3.0E-37 GMRHGY403GI6FP 306 HET-R Podospora anserina gb|ADA68815.1| 44 59.3 5.0E-09 GMRHGY403F7CJM 256 ABC-3 protein bacterium Ellin514 ref|ZP_03631413.1| 73 86.3 2.0E-19 GMRHGY403GK52O 494 hyfF hydrogenase 4 subunit F Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159396.1| 83 259 6.0E-68 GMRHGY403G2GBB 495 threonyl-tRNA synthetase Oxalobacter formigenes HOxBLS ref|ZP_04575950.1| 72 246 7.0E-64 GMRHGY403G2DW1 396 manX phosphotransferase, mannose/fructose-specific component IIA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158595.1| 79 189 8.0E-47 GMRHGY403F7ACP 532 glycosyl transferase family protein Rhodospirillum rubrum ATCC 11170 ref|YP_428172.1| 47 98.2 4.0E-19 GMRHGY403FNVY3 508 circadian clock protein KaiC Roseiflexus castenholzii DSM 13941 ref|YP_001434197.1| 66 113 2.0E-33 GMRHGY403FODSJ 490 transcriptional regulator, AsnC family Thauera sp. MZ1T ref|YP_002354692.1| 60 64.3 9.0E-27 GMRHGY403FPBLU 497 purL phosphoribosylformylglycinamidine synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160535.1| 88 268 2.0E-70 GMRHGY403FO1IS 400 adenylosuccinate lyase Rhodopseudomonas palustris BisB18 ref|YP_530955.1| 72 74.7 2.0E-27 GMRHGY403FLG3L 502 amine oxidase Solibacter usitatus Ellin6076 ref|YP_824821.1| 32 59.7 1.0E-07 GMRHGY403GJPJV 536 FAD dependent oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159704.1| 79 118 3.0E-25 GMRHGY403F6BSQ 492 sdhA succinate dehydrogenase, flavoprotein subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160851.1| 95 204 4.0E-51 GMRHGY403G5R2E 335 SecG preprotein translocase subunit SecG Azoarcus sp. BH72 ref|YP_932899.1| 70 36.6 4.0E-07 GMRHGY403FITFR 503 carbamoyl-phosphate synthase large subunit Thiobacillus denitrificans ATCC 25259 ref|YP_314886.1| 87 227 5.0E-71 GMRHGY403GJ08Z 486 dxs 1-deoxy-D-xylulose-5-phosphate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159537.1| 88 286 8.0E-76 GMRHGY403FVRFS 499 rpoB DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159176.1| 88 226 3.0E-68 GMRHGY403GHN9S 493 pgK phosphoglycerate kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157604.1| 98 196 6.0E-49 GMRHGY403G6E5S 499 retrotransposon protein Oryza sativa Indica Group gb|ABR26094.1| 45 74.3 5.0E-12 GMRHGY403FOF3P 181 parA3 ParA family protein Azoarcus sp. BH72 ref|YP_934714.1| 78 65.1 6.0E-11 GMRHGY403G7MDH 494 argB acetylglutamate kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159479.1| 95 255 2.0E-66 GMRHGY403GEVXG 182 isocitrate dehydrogenase (NAD+) Rhodoferax ferrireducens T118 ref|YP_523628.1| 79 97.1 7.0E-19 GMRHGY403F4Y3D 524 fimV1 putative type 4 pilus biogenesis Azoarcus sp. BH72 ref|YP_932547.1| 72 174 2.0E-42 GMRHGY403HIIHB 490 component of cytosolic 80S ribosome and 60S large subunit Volvox carteri f. nagariensis ref|XP_002947651.1| 65 175 1.0E-57 GMRHGY403GC1IL 497 conserved hypothetical protein Candidatus Kuenenia stuttgartiensis emb|CAJ72785.1| 65 169 1.0E-40 GMRHGY403FRE36 536 Uroporphyrin-III C/tetrapyrrole (Corrin/Porphyrin) methyltransferase Syntrophothermus lipocalidus DSM12680 ref|YP_003701424.1| 41 58.2 4.0E-07 GMRHGY403F98UQ 163 Glycosyltransferase Butyrivibrio fibrisolvens 16/4 emb|CBK75367.1| 54 62.4 2.0E-08 GMRHGY403G4OFQ 161 hrpA ATP-dependent RNA helicase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159339.1| 91 83.6 8.0E-15 GMRHGY403G5EBB 464 xthA2 exodeoxyribonuclease III Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158898.1| 84 287 4.0E-76 GMRHGY403FUGJH 516 acnB bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase Azoarcus sp. BH72 ref|YP_933038.1| 87 300 6.0E-80 GMRHGY403FUO2P 471 O-antigen polymerase Nitrosomonas eutropha C91 ref|YP_748039.1| 60 121 1.0E-32 GMRHGY403GS2Z6 515 yfbQ aminotransferase AlaT Azoarcus sp. BH72 ref|YP_933586.1| 71 108 3.0E-22
! ! 220!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GIIBX 486 NusA antitermination factor Sphaerobacter thermophilus DSM 20745 ref|YP_003319512.1| 48 150 7.0E-35 GMRHGY403GQ5EL 490 6-hydroxynicotinate reductase Burkholderia sp. Ch1-1 ref|ZP_06839801.1| 77 258 1.0E-67 GMRHGY403F7E0M 482 mraW S-adenosyl-methyltransferase mraW Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157823.1| 67 155 2.0E-36 GMRHGY403HEAUB 484 ATP-dependent Clp protease, proteolytic subunit ClpP Polaromonas naphthalenivorans CJ2 ref|YP_983177.1| 84 113 9.0E-24 GMRHGY403F0Q12 493 hisZ ATP phosphoribosyltransferase regulatory subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157689.1| 70 205 1.0E-51 GMRHGY403HAHYH 481 histidine kinase Acidobacterium sp. MP5ACTX8 ref|ZP_07030700.1| 37 98.6 2.0E-19 GMRHGY403GW449 180 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 94 77 3.0E-18 GMRHGY403GRA42 508 complex regulator protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157435.1| 53 167 6.0E-40 GMRHGY403HD2F4 350 acnB bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160014.1| 84 94 2.0E-28 GMRHGY403GJDG7 456 ATP-dependent Clp protease, proteolytic subunit ClpP Polaromonas naphthalenivorans CJ2 ref|YP_983177.1| 84 113 9.0E-24 GMRHGY403FWUSP 527 ubiquinone biosynthesis protein Azoarcus sp. BH72 ref|YP_934265.1| 68 104 5.0E-21 GMRHGY403GGR2G 484 complex two-component hybrid sensor protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159164.1| 61 105 1.0E-21 GMRHGY403HB0JK 344 putative ATP-binding ABC transporter protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158594.1| 97 144 4.0E-37 GMRHGY403GN0FQ 318 diguanylate cyclase/phosphodiesterase domain-containing protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158705.1| 69 99.4 1.0E-30 GMRHGY403HFK4U 474 coxB2 cytochrome-c oxidase subunit II Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159093.1| 71 63.9 1.0E-21 GMRHGY403F67BN 499 gshB glutathione synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158462.1| 89 204 2.0E-51 GMRHGY403F7778 532 mltE transglycosylase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159316.1| 56 75.1 3.0E-12 GMRHGY403F3T7Z 456 uidR TetR family transcriptional regulator Azoarcus sp. BH72 ref|YP_931747.1| 57 55.1 3.0E-11 GMRHGY403FM70Z 470 hemY putative protein porphyrin biosynthesis Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157633.1| 68 191 3.0E-47 GMRHGY403GEZDS 380 cysP periplasmic thiosulfate-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160570.1| 62 102 5.0E-23 GMRHGY403GIDK3 467 cysS cysteinyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159737.1| 90 204 2.0E-51 GMRHGY403GW7LZ 522 putative porin signal peptide protein Ralstonia metallidurans CH34 ref|YP_145671.1| 28 55.8 2.0E-06 GMRHGY403HI1PH 347 trkA potassium transporter peripheral membrane component Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158611.1| 83 117 5.0E-25 GMRHGY403G63IL 300 ATP phosphoribosyltransferase Chitinophaga pinensis DSM 2588 ref|YP_003121514.1| 78 80.5 7.0E-14 GMRHGY403FM1KT 487 dgkA diacylglycerol kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157839.1| 58 100 8.0E-20 GMRHGY403HETXU 457 narH nitrate reductase, beta subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160620.1| 94 125 8.0E-44 GMRHGY403G0UBT 446 Alpha,alpha-trehalose-phosphate synthase (UDP-forming) Pelobacter propionicus DSM 2379 ref|YP_902921.1| 69 209 1.0E-52 GMRHGY403F7AXV 171 hemB delta-aminolevulinic acid dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157589.1| 92 72.4 5.0E-15 GMRHGY403F9PC3 419 peptidase M15D vanX D-ala-D-ala dipeptidase Pedobacter heparinus DSM 2366 ref|YP_003094538.1| 58 80.9 5.0E-14 GMRHGY403HG05S 483 pabA anthranilate synthase (component II) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159402.1| 95 168 2.0E-40 GMRHGY403GHK16 516 carbohydrate kinase Chlorobium tepidum TLS ref|NP_661869.1| 55 161 9.0E-41 GMRHGY403GE9P7 406 glS1 ferredoxin-dependent glutamate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158268.1| 87 143 8.0E-46 GMRHGY403GHQYO 404 gatA glutamyl-tRNA(Gln) amidotransferase subunit A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158734.1| 61 90.1 2.0E-32 GMRHGY403FPBKE 484 MotA2 flagellar motor protein MotA Azoarcus sp. BH72 ref|YP_932952.1| 75 154 3.0E-36 GMRHGY403FQZI8 383 integron integrase Dechloromonas aromatica RCB ref|YP_286626.1| 64 89.7 1.0E-26 GMRHGY403FNSGP 457 dnaN DNA polymerase III (Beta chain) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158624.1| 83 174 3.0E-42 GMRHGY403FZUOY 496 tatB putative Sec-independent protein translocase protein Azoarcus sp. BH72 ref|YP_934841.1| 76 47.8 1.0E-15 GMRHGY403FRC8S 509 typA GTP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160670.1| 90 305 1.0E-81 GMRHGY403GINIB 478 nuoG NADH dehydrogenase subunit G Azoarcus sp. BH72 ref|YP_932906.1| 68 118 1.0E-39 GMRHGY403FKDGX 526 metZ O-succinylhomoserine sulfhydrylase Thauera sp. MZ1T ref|YP_002890030.1| 78 267 5.0E-70 GMRHGY403GZRNP 521 multicopper oxidase type 2 Geobacter sp. M21 ref|YP_003019916.1| 59 195 2.0E-48 GMRHGY403G264T 507 putative heme/copper-type cytochrome/quinol oxidase subunit 2 uncultured bacterium 888 gb|ACF98041.1| 70 203 6.0E-51 GMRHGY403HHGX6 487 pntB pyridine nucleotide transhydrogenase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159578.1| 98 140 1.0E-71 GMRHGY403G5EM1 483 sulfur relay protein, TusE/DsrC/DsvC family Sideroxydans lithotrophicus ES-1 ref|YP_003524301.1| 74 103 1.0E-20 GMRHGY403GJH30 499 Extracellular ligand-binding receptor Methylobacterium nodulans ORS 2060 ref|YP_002501542.1| 50 163 8.0E-39 GMRHGY403G9E1Y 449 chaperonin, 10 kDa Chlorobium tepidum TLS ref|NP_661469.1| 65 57 3.0E-09 GMRHGY403GRDWG 539 L-malate dehydrogenase (AA 339) Methanothermus fervidus emb|CAA36133.1| 38 85.5 2.0E-19 GMRHGY403FSFAC 356 RNA binding S1 Dechloromonas aromatica RCB ref|YP_285944.1| 64 56.2 1.0E-06 GMRHGY403GRTT8 570 predicted exonuclease of the beta-lactamase fold involved in RNA processing uncultured gamma proteobacterium emb|CAI78693.1| 45 69.3 2.0E-17 GMRHGY403GB5A5 515 trpB tryptophan synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159725.1| 94 324 3.0E-87 GMRHGY403GHMVG 499 phaR polyhydroxyalkanoate synthesis repressor PhaR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159701.1| 97 282 9.0E-75 GMRHGY403FVJHD 512 EngA GTP-binding protein EngA Azoarcus sp. BH72 ref|YP_932435.1| 85 78.2 4.0E-22 GMRHGY403GE0T4 490 dnaG DNA primase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159501.1| 34 57 8.0E-07 GMRHGY403G5OX4 487 iron(III) ABC transporter, ATP-binding protein Edwardsiella tarda ATCC 23685 ref|ZP_06715935.1| 54 130 6.0E-29 GMRHGY403GS5AR 507 glyQ glycyl-tRNA synthetase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157747.1| 92 223 9.0E-57 GMRHGY403HF41P 516 Fe-S cluster redox protein Azoarcus sp. BH72 ref|YP_931627.1| 57 60.8 1.0E-15 GMRHGY403GXARE 456 phospholipase D/Transphosphatidylase Anaeromyxobacter dehalogenans 2CP-1 ref|YP_002491468.1| 56 105 1.0E-21 GMRHGY403FKEVV 445 acnB bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160014.1| 97 279 1.0E-73 GMRHGY403G05M6 478 argK arginine/ornithine transport system ATPase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159132.1| 72 186 1.0E-45 GMRHGY403G7OCE 321 putative electron transfer flavoprotein-ubiquinone oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160541.1| 85 185 2.0E-45 GMRHGY403FVHK4 409 threonine tRNA synthetase Thiomonas sp. 3As emb|CAZ88373.1| 77 96.7 4.0E-22 GMRHGY403F72ZA 477 argK arginine/ornithine transport system ATPase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159132.1| 81 246 7.0E-64 GMRHGY403GOHSH 516 RNA-directed DNA polymerase (Reverse transcriptase) Dechloromonas aromatica RCB ref|YP_283916.1| 88 250 5.0E-65 GMRHGY403GYYEW 486 isocitrate dehydrogenase Acinetobacter baumannii SDF ref|YP_001706521.1| 76 231 2.0E-59 GMRHGY403GQEVG 518 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 93 256 5.0E-69 GMRHGY403GKW09 489 dhaL aldehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158262.1| 70 141 3.0E-56 GMRHGY403GM2EX 446 ribosomal protein S19 Thermobaculum terrenum ATCC BAA-798 ref|YP_003322458.1| 73 105 2.0E-25 GMRHGY403GTKJ0 468 hrpA ATP-dependent RNA helicase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159339.1| 92 262 9.0E-69 GMRHGY403G19KT 363 succinyl-CoA:3-ketoacid-coenzyme A transferase 1 Mycobacterium intracellulare ATCC 13950 ref|ZP_05227871.1| 68 79.7 1.0E-22 GMRHGY403F66L0 485 NosR transcription regulator NosR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160613.1| 89 286 5.0E-76 GMRHGY403GHZT8 521 polysaccharide deacetylase Alkalilimnicola ehrlichii MLHE-1 ref|YP_740988.1| 65 67.8 3.0E-15 GMRHGY403FQ8ES 406 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 72 147 9.0E-42 GMRHGY403FRVK7 237 nuoE NADH dehydrogenase subunit E Azoarcus sp. BH72 ref|YP_932904.1| 90 57 8.0E-07 GMRHGY403GO2AB 510 kdsB 3-deoxy-manno-octulosonate cytidylyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159908.1| 70 159 9.0E-38 GMRHGY403F9LZH 459 ribosomal protein S9 Gracilaria tenuistipitata var. liui ref|YP_063584.1| 54 104 3.0E-21 GMRHGY403HESK8 496 purM phosphoribosylaminoimidazole synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161055.1| 96 310 3.0E-83
! ! 221!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GQJ24 517 rpoB DNA-directed RNA polymerase, beta subunit Thauera sp. MZ1T ref|YP_002890317.1| 98 112 1.0E-58 GMRHGY403FJIDM 508 dnaG DNA primase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159501.1| 80 221 1.0E-59 GMRHGY403GQRLE 519 ugpQ cytoplasmic glycerophosphodiester phosphodiesterase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159583.1| 69 68.2 1.0E-28 GMRHGY403FWEM4 403 proton-translocating NADH-quinone oxidoreductase, chain N Chlorobaculum parvum NCIB 8327 ref|YP_001998901.1| 59 99 1.0E-19 GMRHGY403G4KUG 471 TRAP dicarboxylate transporter Bordetella petrii DSM 12804 ref|YP_001631951.1| 80 192 1.0E-47 GMRHGY403HARD5 460 gbd1 alcohol dehydrogenase, iron-containing family protein Halomonas elongata DSM 2581 ref|YP_003896110.1| 55 148 1.0E-35 GMRHGY403FUVIP 419 aldA aldehyde dehydrogenase (NAD+) Azoarcus sp. BH72 ref|YP_934442.1| 90 103 7.0E-21 GMRHGY403GGPZ2 277 sdhA succinate dehydrogenase, flavoprotein subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160851.1| 98 133 3.0E-33 GMRHGY403FIUK7 395 5'-Nucleotidase domain protein Variovorax paradoxus S110 ref|YP_002945365.1| 64 79 2.0E-13 GMRHGY403GIST5 226 phbB acetoacetyl-CoA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159698.1| 97 98.6 2.0E-19 GMRHGY403F8I8P 158 fadH 2,4-dienoyl-CoA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160341.1| 83 83.2 1.0E-14 GMRHGY403F88EK 161 paaH1 3-hydroxybutyryl-CoA dehydrogenase Azoarcus sp. BH72 ref|YP_931805.1| 78 82 2.0E-14 GMRHGY403GUHNE 161 paaH1 3-hydroxybutyryl-CoA dehydrogenase Azoarcus sp. BH72 ref|YP_931805.1| 78 82 2.0E-14 GMRHGY403GJ3MN 157 gcdH1 glutaryl-CoA dehydrogenase Azoarcus sp. BH72 ref|YP_933428.1| 75 73.9 6.0E-12 GMRHGY403F7Y3X 353 hbdA 3-hydroxybutyryl-CoA dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159705.1| 80 149 2.0E-40 GMRHGY403FUWWY 454 hbdA 3-hydroxybutyryl-CoA dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159705.1| 61 156 7.0E-37 GMRHGY403FKGLO 240 phbB acetoacetyl-CoA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159698.1| 96 108 2.0E-22 GMRHGY403HCXP3 218 gcdH1 glutaryl-CoA dehydrogenase Azoarcus sp. BH72 ref|YP_933428.1| 78 113 7.0E-24 GMRHGY403FNYWL 320 acyl-CoA dehydrogenase domain protein Dethiobacter alkaliphilus AHT 1 ref|ZP_03729887.1| 56 52.4 2.0E-05 GMRHGY403GZRNS 404 gcdH glutaryl-CoA dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158711.1| 84 211 2.0E-53 GMRHGY403GO46J 527 two-component sensor histidine kinase PhoR Pelobacter carbinolicus DSM 2380 ref|YP_356083.1| 41 50.4 6.0E-07 GMRHGY403HDH1R 473 fusion of 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158155.1| 71 216 6.0E-55 GMRHGY403GJSUH 509 acetyl-CoA acetyltransferase Acinetobacter haemolyticus ATCC 19194 ref|ZP_06728722.1| 90 126 1.0E-27 GMRHGY403F4BJT 497 2,4-dienoyl-CoA reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160341.1| 78 263 7.0E-69 GMRHGY403GP7Z1 514 3-hydroxy-acyl-CoA dehydrogenase Acinetobacter sp. DR1 ref|YP_003732649.1| 58 164 5.0E-39 GMRHGY403FI2QN 501 phbB1 acetoacetyl-CoA reductase Azoarcus sp. BH72 ref|YP_932526.1| 92 311 1.0E-83 GMRHGY403F1CLQ 487 atoB acetyl-CoA acetyltransferase Methanothermobacter marburgensis str. Marburg ref|YP_003850092.1| 60 199 8.0E-50 GMRHGY403FYOL4 496 3-hydroxybutyryl-CoA dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158051.1| 79 268 2.0E-70 GMRHGY403GK509 477 paaF2 enoyl-CoA hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160749.1| 90 224 2.0E-60 GMRHGY403FRPKQ 488 fatty oxidation complex, alpha subunit Burkholderia oklahomensis C6786 ref|ZP_02361114.1| 57 137 4.0E-31 GMRHGY403FMNEX 479 enoyl-CoA hydratase Bordetella petrii DSM 12804 ref|YP_001631174.1| 54 173 7.0E-42 GMRHGY403FJALI 480 enoyl-CoA hydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158052.1| 75 154 1.0E-59 GMRHGY403HHQR1 375 acetyl-CoA acetyltransferase Frankia sp. CcI3 ref|YP_483526.1| 89 72.8 1.0E-23 GMRHGY403G9IX3 476 transcriptional regulator, BadM/Rrf2 family Thauera sp. MZ1T ref|YP_002355547.1| 65 189 1.0E-46 GMRHGY403G48AN 326 2-hydroxyglutaryl-CoA dehydratase D-component Geobacter lovleyi SZ ref|YP_001952636.1| 40 71.6 3.0E-11 GMRHGY403GSTMD 491 transcriptional regulator, BadM/Rrf2 family Thauera sp. MZ1T ref|YP_002355547.1| 65 163 8.0E-39 GMRHGY403FRBH3 497 fdx1 ferredoxin Azoarcus sp. BH72 ref|YP_932265.1| 85 160 5.0E-38 GMRHGY403G2JLD 473 fdxA ferredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159709.1| 93 205 2.0E-51 GMRHGY403GFUI3 495 transcriptional regulatory protein Serratia odorifera 4Rx13 ref|ZP_06192897.1| 69 122 3.0E-54 GMRHGY403FUL7H 482 transcriptional regulator, BadM/Rrf2 family Thauera sp. MZ1T ref|YP_002355547.1| 55 112 2.0E-33 GMRHGY403FYGJI 482 CoA-substrate-specific enzyme activase Geobacter lovleyi SZ ref|YP_001952634.1| 45 142 1.0E-32 GMRHGY403G50X3 525 aorA aldehyde ferredoxin oxidoreductase Azoarcus sp. BH72 ref|YP_934433.1| 78 153 3.0E-51 GMRHGY403FJVT0 489 fusion protein of flavin-containing oxidoreductase and iron-sulfur-containing oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158925.1| 91 265 2.0E-78 GMRHGY403FK3FK 437 RNA binding S1 domain protein Thauera sp. MZ1T ref|YP_002355683.1| 89 220 2.0E-56 GMRHGY403G247H 148 UbiD-like carboxylase subunit encoded in anaerobic phenol operon Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158782.1| 87 65.9 2.0E-12 GMRHGY403FUK1F 200 UDP-glucuronic acid decarboxylase/UDP-4-amino-4-deoxy-L-arabinose formyltransferase Edwardsiella tarda EIB202 ref|YP_003295347.1| 79 99.4 1.0E-19 GMRHGY403GJB17 514 exopolysaccharide export protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159456.1| 58 106 1.0E-21 GMRHGY403F9O9L 416 FdhD formate dehydrogenase family accessory protein FdhD Laribacter hongkongensis HLHK9 gb|ACO73798.1| 64 67.4 6.0E-10 GMRHGY403GL2O6 503 fumarylacetoacetase family protein Sorangium cellulosum 'So ce56' ref|YP_001618893.1| 59 162 4.0E-43 GMRHGY403GO8Q7 487 conserved hypothetical protein, putative Glycosyl transferase,family Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159228.1| 76 231 3.0E-59 GMRHGY403GZDYC 502 leuS leucyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159513.1| 70 120 2.0E-35 GMRHGY403GN90R 226 retrotransposon protein Oryza sativa Indica Group gb|ABR26094.1| 63 67.4 6.0E-10 GMRHGY403FY2YK 444 rplF 50S ribosomal protein L6 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159198.1| 100 87.8 7.0E-33 GMRHGY403FVTPW 496 narK1 nitrate/proton symporter Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160623.1| 55 99 2.0E-19 GMRHGY403G5D23 501 rpoB DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159176.1| 90 252 3.0E-72 GMRHGY403FTP86 422 glcB malate synthase G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157451.1| 86 221 2.0E-56 GMRHGY403GHBCT 490 NosR transcription regulator NosR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160613.1| 94 326 7.0E-88 GMRHGY403F7LHL 538 tufB elongation factor Tu Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159169.1| 96 157 4.0E-37 GMRHGY403FPUGQ 507 dhaL aldehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158262.1| 79 266 7.0E-70 GMRHGY403GCKHJ 524 nirS putative dissimilatory nitrite reductase nirS uncultured bacterium gb|AAL89549.1| 85 307 3.0E-82 GMRHGY403G31L0 494 norB putative nitric-oxide reductase subunit B Azoarcus sp. BH72 ref|YP_934592.1| 83 295 1.0E-78 GMRHGY403FVG4G 513 nirS cytochrome cd1 nitrite reductase precursor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157499.1| 98 343 5.0E-93 GMRHGY403HEXKF 501 atpD F0F1 ATP synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158722.1| 98 317 4.0E-85 GMRHGY403GNV5R 481 rplF 50S ribosomal protein L6 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159198.1| 80 135 1.0E-30 GMRHGY403FVHQL 529 cell wall-associated hydrolase Brucella abortus NCTC 8038 ref|ZP_05822976.1| 91 291 3.0E-77 GMRHGY403HF9A0 485 rpoC DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159177.1| 95 244 2.0E-76 GMRHGY403GIWZW 522 carA carbamoyl phosphate synthase small subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159750.1| 90 200 7.0E-50 GMRHGY403GSJSZ 517 rpsJ 30S ribosomal protein S10 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159182.1| 98 165 3.0E-40 GMRHGY403FZITN 505 rpsN 30S ribosomal protein S14 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159196.1| 79 164 3.0E-39 GMRHGY403GNCUY 511 rpoC DNA-directed RNA polymerase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159177.1| 97 173 1.0E-49 GMRHGY403FJVXZ 463 pstS phosphate ABC transporter, periplasmic phosphate-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159759.1| 81 238 2.0E-61 GMRHGY403GIMON 499 dhaL aldehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158262.1| 98 171 3.0E-76 GMRHGY403GQ680 530 rpsK 30S ribosomal protein S11 Azoarcus sp. BH72 ref|YP_934895.1| 89 203 6.0E-51 GMRHGY403GZXZQ 503 thrS threonyl-tRNA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157487.1| 90 227 3.0E-67 GMRHGY403G9BCW 527 parA3 ParA family protein Azoarcus sp. BH72 ref|YP_934714.1| 71 177 6.0E-43 GMRHGY403GC4PG 486 argH argininosuccinate lyase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157639.1| 93 268 2.0E-70
! ! 222!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GU9EX 421 infC translation initiation factor IF-3 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157486.1| 82 175 2.0E-42 GMRHGY403G1O0H 501 oma outer membrane protein/surface antigen Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160448.1| 87 303 5.0E-81 GMRHGY403GSZDQ 483 nuoG NADH dehydrogenase subunit G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159771.1| 72 200 4.0E-50 GMRHGY403FI7DV 507 rpoA DNA-directed RNA polymerase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159208.1| 86 169 1.0E-43 GMRHGY403FYWJB 483 tufB elongation factor Tu Azoarcus sp. BH72 ref|YP_934921.1| 81 249 1.0E-64 GMRHGY403HABSX 532 rpsA 30S ribosomal protein S1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157511.2| 96 263 7.0E-69 GMRHGY403GAR7K 518 tig trigger factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159853.1| 86 189 1.0E-46 GMRHGY403G174W 469 hflX GTP-binding protein hflX Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157693.1| 91 227 3.0E-58 GMRHGY403GQXUY 538 rplC 50S ribosomal protein L3 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159183.1| 93 327 4.0E-88 GMRHGY403GT8G0 491 Peptidase S14, ClpP Beggiatoa sp. PS ref|ZP_02000426.1| 64 108 6.0E-51 GMRHGY403FIUH7 498 gapA putative glyceraldehyde 3-phosphate dehydrogenase Azoarcus sp.BH72 ref|YP_934340.1| 95 312 1.0E-83 GMRHGY403FZLBU 484 rpsH 30S ribosomal protein S8 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159197.1| 98 166 9.0E-40 GMRHGY403F7V6R 473 gltA citrate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160848.1| 96 308 2.0E-82 GMRHGY403GIYLC 468 argininosuccinate lyase Bordetella avium 197N ref|YP_786155.1| 69 139 2.0E-31 GMRHGY403GDCKF 516 rimM 16S rRNA processing protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161089.1| 82 147 4.0E-34 GMRHGY403G69ND 508 rpsD 30S ribosomal protein S4 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159207.1| 95 318 2.0E-85 GMRHGY403G8YBZ 508 diguanylate cyclase Marinobacter algicola DG893 ref|ZP_01892225.1| 53 93.6 2.0E-22 GMRHGY403G5JQC 510 PpiC-type peptidyl-prolyl cis-trans isomerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160742.1| 81 280 4.0E-74 GMRHGY403HAPNI 505 glyS glycyl-tRNA synthetase beta chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157746.1| 86 203 5.0E-51 GMRHGY403GOALQ 484 ATP synthase beta subunit Citrobacter rodentium ICC168 ref|YP_003367444.1| 85 204 4.0E-68 GMRHGY403F8MH3 273 hypothetical protein ebA5142 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159950.1| 76 144 4.0E-33 GMRHGY403F1Y5T 265 hypothetical protein Sdel_2021 Sulfurospirillum deleyianum DSM 6946 ref|YP_003305070.1| 63 55.5 2.0E-07 GMRHGY403FZ6ZC 295 pirin domain-containing protein Sphingomonas wittichii RW1 ref|YP_001265046.1| 78 132 2.0E-29 GMRHGY403HBLVK 245 conserved hypothetical protein Streptomyces sp. C ref|ZP_07286019.1| 51 48.1 5.0E-08 GMRHGY403GWPFS 252 conserved hypothetical protein Achromobacter piechaudii ATCC 43553 ref|ZP_06686492.1| 68 105 3.0E-21 GMRHGY403FYG66 202 conserved hypothetical protein Parachlamydia acanthamoebae str. Hall's coccus ref|ZP_06300078.1| 37 56.6 1.0E-06 GMRHGY403GFNMD 410 conserved hypothetical protein Comamonas testosteroni KF-1 ref|ZP_03542750.1| 61 158 2.0E-37 GMRHGY403GCHUL 293 hypothetical protein ebA3236 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158849.1| 87 128 2.0E-28 GMRHGY403FPJS7 131 hypothetical protein cce_1552 Cyanothece sp. ATCC 51142 ref|YP_001802968.1| 71 60.1 9.0E-08 GMRHGY403GKP3X 149 protein of unknown function DUF214 Thauera sp. MZ1T ref|YP_002355791.1| 74 71.6 3.0E-11 GMRHGY403GQJSD 275 hypothetical protein Spirs_0769 Spirochaeta smaragdinae DSM 11293 ref|YP_003802498.1| 51 61.2 4.0E-08 GMRHGY403GYXI0 386 hypothetical protein azo2561 Azoarcus sp. BH72 ref|YP_934065.1| 89 146 7.0E-34 GMRHGY403FZCX7 88 hypothetical protein azo1734 Azoarcus sp. BH72 ref|YP_933238.1| 92 51.2 4.0E-05 GMRHGY403FKURF 271 hypothetical protein Daro_1859 Dechloromonas aromatica RCB ref|YP_285075.1| 63 68.6 3.0E-10 GMRHGY403G01JD 135 conserved membrane protein of unknown function, UPF0005 Ralstonia solanacearum PSI07 ref|YP_003752806.1| 81 55.1 3.0E-06 GMRHGY403HD52G 223 pirin domain-containing protein Psychromonas ingrahamii 37 ref|YP_941973.1| 77 64.3 5.0E-09 GMRHGY403F7JLA 458 protein of unknown function DUF454 Comamonas testosteroni KF-1 ref|ZP_03544285.1| 58 64.3 5.0E-09 GMRHGY403GCFM2 163 hypothetical protein C1336_000600005 Campylobacter jejuni subsp. jejuni 1336 ref|ZP_06374539.1| 72 58.2 4.0E-07 GMRHGY403HB8DE 279 OsmC-like protein Ralstonia solanacearum UW551 ref|ZP_00943693.1| 72 91.7 3.0E-17 GMRHGY403G90BG 415 ErfK/YbiS/YcfS/YnhG family protein Thauera sp. MZ1T ref|YP_002889487.1| 80 110 6.0E-48 GMRHGY403GJK63 449 hypothetical protein Mpe_A2403 Methylibium petroleiphilum PM1 ref|YP_001021593.1| 98 295 2.0E-78 GMRHGY403G1YI9 265 hypothetical protein Sdel_2021 Sulfurospirillum deleyianum DSM ref|YP_003305070.1| 55 70.9 5.0E-11 GMRHGY403GBX1G 173 hypothetical protein RC1_0236 Rhodospirillum centenum SW ref|YP_002296498.1| 73 67.8 4.0E-10 GMRHGY403FN9D1 142 hypothetical protein CLOBOL_00335 Clostridium bolteae ATCC BAA-613 ref|ZP_02082822.1| 87 66.6 1.0E-09 GMRHGY403GX1UZ 421 hypothetical protein azo0579 Azoarcus sp. BH72 ref|YP_932083.1| 75 202 9.0E-51 GMRHGY403GQHF3 101 hypothetical protein azo3215 Azoarcus sp. BH72 ref|YP_934717.1| 80 50.8 6.0E-05 GMRHGY403FWMUB 222 hypothetical protein MCA0721 Methylococcus capsulatus str. Bath ref|YP_113231.1| 92 51.2 4.0E-05 GMRHGY403HGGCJ 446 hypothetical protein mma_1338 Janthinobacterium sp. Marseille ref|YP_001353028.1| 59 96.3 1.0E-18 GMRHGY403GCG3V 162 hypothetical protein uncultured Acidobacteria bacteriumHF4000_26D02 gb|ADI18657.1| 56 43.5 6.0E-05 GMRHGY403G6SF3 449 hypothetical protein Tmz1t_3864 Thauera sp. MZ1T ref|YP_002890830.1| 73 136 8.0E-31 GMRHGY403FJ8YK 315 hypothetical protein CAP2UW1_2113 Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003167338.1| 37 62.8 1.0E-08 GMRHGY403FYZL7 421 HPr kinase Dechloromonas aromatica RCB ref|YP_285650.1| 42 108 2.0E-22 GMRHGY403G5UIW 436 AE001886_6 hypothetical protein DR_0254 Deinococcus radiodurans R1 gb|AAF09840.1| 66 76.3 2.0E-25 GMRHGY403GO6ZH 545 hypothetical protein azo2152 Azoarcus sp. BH72 ref|YP_933656.1| 51 99.8 1.0E-19 GMRHGY403FUDQN 417 hypothetical protein MXAN_5553 Myxococcus xanthus DK 1622 ref|YP_633692.1| 44 61.2 4.0E-08 GMRHGY403G6KCS 223 hypothetical protein ebA5191 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159973.1| 79 61.6 3.0E-08 GMRHGY403GGUVW 407 hypothetical protein ebB151 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159552.1| 69 165 2.0E-39 GMRHGY403GHUH3 417 hypothetical protein BURPSS13_G0088 Burkholderia pseudomallei S13 ref|ZP_04903571.1| 65 62.8 1.0E-08 GMRHGY403GASCX 488 conserved hypothetical protein Capnocytophaga sputigena Capno ref|ZP_03391854.1| 42 67.8 3.0E-16 GMRHGY403GX3UX 260 hypothetical protein Tmz1t_2846 Thauera sp. MZ1T ref|YP_002889822.1| 74 126 8.0E-28 GMRHGY403GC2XR 461 conserved hypothetical protein Burkholderia sp. Ch1-1 ref|ZP_06846135.1| 45 105 2.0E-21 GMRHGY403GO9X1 479 hypothetical protein Tmz1t_1203 Thauera sp. MZ1T ref|YP_002354858.1| 71 163 6.0E-39 GMRHGY403G38OV 385 LOW QUALITY PROTEIN: cell wall-associated hydrolase Escherichia sp. 3_2_53FAA ref|ZP_04532936.1| 80 191 3.0E-47 GMRHGY403GTCHP 463 hypothetical protein PSR_56013 Salinibacter ruber M8 ref|YP_003566147.1| 40 115 2.0E-24 GMRHGY403FMUA6 141 hypothetical protein azo0392 Azoarcus sp. BH72 ref|YP_931896.1| 65 58.5 3.0E-07 GMRHGY403HDJYB 300 protein of unknown function DUF1355 Mesorhizobium opportunistum WSM2075 ref|ZP_05810774.1| 92 159 1.0E-37 GMRHGY403GXXEM 540 hypothetical protein CT0740 Chlorobium tepidum TLS ref|NP_661635.1| 45 48.1 2.0E-10 GMRHGY403FPUHH 504 hypothetical protein ebB92 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158715.1| 83 145 2.0E-34 GMRHGY403G9174 481 hypothetical protein ebA2421 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158384.1| 90 140 4.0E-32 GMRHGY403GUED4 97 hypothetical protein Tmz1t_0934 Thauera sp. MZ1T ref|YP_002354596.1| 96 61.2 4.0E-08 GMRHGY403GUXW3 178 hypothetical protein ebA6193 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160564.1| 88 98.2 3.0E-19 GMRHGY403F9012 505 hypothetical protein azo1748 Azoarcus sp. BH72 ref|YP_933252.1| 66 189 8.0E-47 GMRHGY403FOYH8 320 conserved hypothetical protein Alicycliphilus denitrificans BC ref|ZP_07024686.1| 100 111 4.0E-23 GMRHGY403F5QJG 566 hypothetical protein ANACOL_02699 Anaerotruncus colihominis DSM17241 ref|ZP_02443386.1| 68 209 1.0E-52 GMRHGY403F2JJI 504 hypothetical protein ebA5537 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160178.1| 83 154 5.0E-36 GMRHGY403GT044 464 hypothetical protein ebA4612 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159638.1| 61 127 8.0E-31
! ! 223!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403HD4GK 121 hypothetical protein Tmz1t_3636 Thauera sp. MZ1T ref|YP_002890606.1| 68 50.8 6.0E-05 GMRHGY403FZJXP 490 hypothetical protein Tbd_1238 Thiobacillus denitrificans ATCC 25259 ref|YP_314996.1| 40 73.9 6.0E-12 GMRHGY403F5MIC 483 hypothetical protein Sthe_1930 Sphaerobacter thermophilus DSM 20745 ref|YP_003320184.1| 28 58.5 3.0E-07 GMRHGY403F7GO9 492 hypothetical protein Tmz1t_1008 Thauera sp. MZ1T ref|YP_002354669.1| 100 245 2.0E-63 GMRHGY403GH2Y5 427 conserved hypothetical protein Bacteroides sp. 2_1_16 ref|ZP_06094661.1| 59 91.3 7.0E-21 GMRHGY403GX3DM 488 hypothetical protein ebA36 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157039.1| 48 131 3.0E-29 GMRHGY403G4HQO 383 hypothetical protein Arabidopsis thaliana dbj|BAF01964.1| 76 64.7 4.0E-22 GMRHGY403GDCHP 499 hypothetical protein ebA3532 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159014.1| 85 154 3.0E-36 GMRHGY403GXUHL 158 hypothetical protein CHLREDRAFT_155068 Chlamydomonas reinhardtii ref|XP_001698950.1| 93 74.7 4.0E-12 GMRHGY403FNX2Y 513 hypothetical protein ebB16 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157343.1| 53 76.3 1.0E-12 GMRHGY403HFQQB 536 conserved hypothetical protein Streptomyces violaceusniger Tu 4113 ref|ZP_07611793.1| 37 94.7 4.0E-18 GMRHGY403GUXBQ 524 hypothetical protein PMT1297 Prochlorococcus marinus str. MIT 9313 ref|NP_895125.1| 33 84.3 5.0E-15 GMRHGY403GVOT4 515 hypothetical protein ebA4287 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159452.1| 35 91.7 3.0E-17 GMRHGY403GE1OH 471 hypothetical protein DORLON_02997 Dorea longicatena DSM 13814 ref|ZP_01996967.1| 59 54.3 1.0E-15 GMRHGY403F7E4Q 499 conserved hypothetical protein gamma proteobacterium NOR51-B ref|ZP_04956697.1| 37 98.6 2.0E-11 GMRHGY403FQ9JV 530 hypothetical protein Daro_3742 Dechloromonas aromatica RCB ref|YP_286941.1| 74 87 1.0E-26 GMRHGY403G4Y0N 486 hypothetical protein Pnap_4502 Polaromonas naphthalenivorans CJ2 ref|YP_973517.1| 60 120 9.0E-49 GMRHGY403GJVYL 544 hypothetical protein CE1543 Corynebacterium efficiens YS-314 ref|NP_738153.1| 40 54.3 4.0E-16 GMRHGY403F3K4R 486 hypothetical protein BthaT_16239 Burkholderia thailandensis TXDOH ref|ZP_02372574.1| 28 69.7 1.0E-10 GMRHGY403G5KDG 482 hypothetical protein ebA2542 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158460.1| 82 212 1.0E-57 GMRHGY403FRRSY 463 hypothetical protein PSR_56013 Salinibacter ruber M8 ref|YP_003566147.1| 40 115 2.0E-24 GMRHGY403F9Y15 487 hypothetical protein plpp0123 Legionella pneumophila str. Paris ref|YP_122277.1| 58 89.4 1.0E-16 GMRHGY403FTB24 527 hypothetical protein Noca_1621 Nocardioides sp. JS614 ref|YP_922821.1| 49 144 4.0E-33 GMRHGY403F281B 529 hypothetical protein DR_1021 Deinococcus radiodurans R1 ref|NP_294745.1| 45 85.1 3.0E-15 GMRHGY403GCHEE 496 hypothetical protein ALIPUT_00519 Alistipes putredinis DSM 17216 ref|ZP_02424402.1| 63 103 6.0E-27 GMRHGY403GPRME 469 hypothetical protein azo1912 Azoarcus sp. BH72 ref|YP_933416.1| 61 174 4.0E-42 GMRHGY403F21FC 504 hypothetical protein FAEPRAM212_00540 Faecalibacterium prausnitzii M21/2 ref|ZP_02090300.1| 46 121 4.0E-26 GMRHGY403G386H 518 hypothetical protein NOC27_428 Nitrosococcus oceani AFC27 ref|ZP_05047005.1| 42 77 8.0E-13 GMRHGY403F9J6E 504 hypothetical protein uncultured delta proteobacterium HF0070_15B21 gb|ADI19064.1| 41 76.6 1.0E-12 GMRHGY403GBMYO 520 hypothetical protein PRU_1034 Prevotella ruminicola 23 ref|YP_003574364.1| 61 86.3 2.0E-27 GMRHGY403GAVDN 263 hypothetical protein ebA4705 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159683.1| 65 78.6 3.0E-13 GMRHGY403F3OV0 337 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 50 94.4 4.0E-18 GMRHGY403GCDSL 499 hypothetical protein ebA3532 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159014.1| 85 154 3.0E-36 GMRHGY403G3C5W 426 hypothetical protein azo0935 Azoarcus sp. BH72 ref|YP_932439.1| 78 89.4 1.0E-16 GMRHGY403G5IIP 507 hypothetical protein Rsph17025_4066 Rhodobacter sphaeroides ATCC 17025 ref|YP_001170223.1| 56 162 2.0E-38 GMRHGY403F4NNO 510 hypothetical protein Daro_1791 Dechloromonas aromatica RCB ref|YP_285008.1| 38 96.7 1.0E-18 GMRHGY403GXTUG 413 MORN variant repeat protein Fusobacterium sp. 4_1_13 ref|ZP_04572272.1| 49 56.6 1.0E-06 GMRHGY403HFJYP 508 probable aggregation factor core protein MAFp3, isoform C, putative Microscilla marina ATCC 23134 ref|ZP_01692644.1| 37 39.7 1.0E-07 GMRHGY403FXFEF 377 hypothetical protein HMPREF9010_01381 Bacteroides sp. 3_1_23 ref|ZP_07038995.1| 81 117 4.0E-25 GMRHGY403GMM3S 481 hypothetical protein p2A75 Azoarcus sp. EbN1 ref|YP_195591.1| 82 77 7.0E-13 GMRHGY403GVY5I 491 hypothetical protein ebA5821 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160330.1| 57 137 6.0E-31 GMRHGY403FY5AD 507 PKD domain protein Microscilla marina ATCC 23134 ref|ZP_01690911.1| 30 74.3 5.0E-12 GMRHGY403FYSV6 486 hypothetical protein BthaT_16239 Burkholderia thailandensis TXDOH ref|ZP_02372574.1| 28 69.7 1.0E-10 GMRHGY403FWX7B 507 hypothetical protein ebA1048 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157575.1| 71 177 4.0E-43 GMRHGY403GRJYQ 442 hypothetical protein ROP_24740 Rhodococcus opacus B4 ref|YP_002779666.1| 48 135 1.0E-30 GMRHGY403GKTUA 481 hypothetical protein ebA7066 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161034.1| 81 114 5.0E-24 GMRHGY403G5Z89 453 hypothetical protein Rmar_0356 Rhodothermus marinus DSM 4252 ref|YP_003289648.1| 33 71.2 4.0E-11 GMRHGY403GTZL3 450 hypothetical protein ELI_05935 Erythrobacter litoralis HTCC2594 ref|YP_458076.1| 62 149 1.0E-34 GMRHGY403F3R75 555 Conserved hypothetical protein Microscilla marina ATCC 23134 >ref|ZP_01691550.1| 38 70.1 1.0E-10 GMRHGY403HA575 111 hypothetical protein azo2810 Azoarcus sp. BH72 ref|YP_934313.1| 71 49.7 1.0E-04 GMRHGY403HBMMI 449 hypothetical protein Reut_B5581 Ralstonia eutropha JMP134 ref|YP_299770.1| 60 141 2.0E-37 GMRHGY403F457K 522 conserved hypothetical protein, membrane or secreted Dyadobacter fermentans DSM 18053 ref|YP_003085788.1| 31 60.1 1.0E-07 GMRHGY403F6D61 505 conserved hypothetical protein Bacteriovorax marinus SJ emb|CBW25287.1| 44 48.5 9.0E-07 GMRHGY403G9D57 150 hypothetical protein ebA4612 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159638.1| 56 54.7 4.0E-06 GMRHGY403FNCZZ 464 protein of unknown function DUF395 YeeE/YedE Bacillus coagulans 36D1 ref|ZP_04433323.1| 35 50.4 7.0E-05 GMRHGY403FJFEK 467 5'-Nucleotidase domain protein Chloroherpeton thalassium ATCC 35110 ref|YP_001995260.1| 54 70.9 5.0E-11 GMRHGY403GNCHC 535 hypothetical protein ebA1318 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157738.1| 63 231 4.0E-59 GMRHGY403F2I7Y 534 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 45 90.9 2.0E-17 GMRHGY403FMCCK 243 leucine rich protein Arachis hypogaea gb|ABH09321.1| 68 80.9 5.0E-14 GMRHGY403G5L1F 461 hypothetical protein NOC27_2103 Nitrosococcus oceani AFC27 ref|ZP_05048547.1| 73 222 1.0E-56 GMRHGY403FJL3Q 553 hypothetical protein PERMA_1141 Persephonella marina EX-H1 ref|YP_002730924.1| 64 63.9 8.0E-12 GMRHGY403GRXKY 457 putative regulator protein Azoarcus sp. BH72 ref|YP_934999.1| 83 249 1.0E-64 GMRHGY403FJSRT 474 hypothetical protein Mmc1_0294 Magnetococcus sp. MC-1 ref|YP_864227.1| 39 81.6 3.0E-14 GMRHGY403GZ9JN 568 hypothetical protein SCH_0249 Salmonella enterica subsp. enterica serovar Choleraesuis gb|AAX64155.1| 80 107 2.0E-30 GMRHGY403GUGV6 489 hypothetical protein ebA624 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157347.1| 68 208 2.0E-52 GMRHGY403G57VA 489 hypothetical protein BBta_2171 Bradyrhizobium sp. BTAi1 ref|YP_001238261.1| 51 108 4.0E-39 GMRHGY403FVJI9 482 hypothetical protein ebA3532 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159014.1| 85 154 3.0E-36 GMRHGY403HDMQ5 497 hypothetical protein ebD87 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159919.1| 90 142 2.0E-32 GMRHGY403HHHMC 464 hypothetical protein Dole_0812 Desulfococcus oleovorans Hxd3 ref|YP_001528699.1| 60 54.3 3.0E-06 GMRHGY403HB3IM 541 hypothetical protein uncultured Spirochaetales bacterium HF0500_06B09 gb|ADI19371.1| 54 82.4 1.0E-29 GMRHGY403FQ3B3 489 hypothetical protein RAZWK3B_14394 Roseobacter sp. AzwK-3b ref|ZP_01903320.1| 31 64.3 5.0E-09 GMRHGY403FJX6V 518 hypothetical protein Mpe_A2525 Methylibium petroleiphilum PM1 ref|YP_001021715.1| 50 100 5.0E-20 GMRHGY403FUNQO 517 hypothetical protein ebA3498 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158998.1| 81 103 1.0E-20 GMRHGY403F34TM 499 hypothetical protein Anae109_4221 Anaeromyxobacter sp. Fw109-5 ref|YP_001381383.1| 39 56.6 1.0E-06 GMRHGY403F35LH 502 hypothetical protein mlr6946 Mesorhizobium loti MAFF303099 ref|NP_107347.1| 58 118 3.0E-25 GMRHGY403FL7P8 475 protein of unknown function DUF21 Cyanothece sp. PCC 7424 ref|YP_002378853.1| 49 65.9 3.0E-24
! ! 224!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GPTSH 499 hypothetical protein Cpin_1611 Chitinophaga pinensis DSM 2588 ref|YP_003121308.1| 26 50.1 1.0E-04 GMRHGY403GUVKQ 491 hypothetical protein Daro_4140 Dechloromonas aromatica RCB ref|YP_287336.1| 68 97.8 1.0E-29 GMRHGY403GLM65 537 hypothetical protein uncultured Verrucomicrobiales bacterium HF0200_39L05 gb|ADI18136.1| 53 45.4 5.0E-08 GMRHGY403FW2SJ 509 hypothetical protein L8106_22119 Lyngbya sp. PCC 8106 ref|ZP_01623393.1| 44 88.2 3.0E-16 GMRHGY403GFK05 588 hypothetical protein BTH_I1579 Burkholderia thailandensis E264 ref|YP_442121.1| 63 51.6 4.0E-08 GMRHGY403GBIH8 595 hypothetical protein uncultured Spirochaetales bacterium HF0500_06B09 gb|ADI19371.1| 53 70.9 1.0E-23 GMRHGY403G6W94 494 hypothetical protein CtCNB1_2990 Comamonas testosteroni CNB-2 ref|YP_003279032.1| 53 67.8 4.0E-10 GMRHGY403HAY4H 551 hypothetical protein uncultured Verrucomicrobiales bacterium HF0010_05E02 gb|ADI16721.1| 72 144 5.0E-33 GMRHGY403FVG3U 484 hypothetical protein CAP2UW1_3344 Candidatus Accumulibacterphosphatis clade IIA str. UW-1 ref|YP_003168537.1| 57 169 1.0E-40 GMRHGY403F4PD6 518 hypothetical protein ebA6152 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160539.1| 77 273 8.0E-72 GMRHGY403F0TFQ 450 hypothetical protein MC7420_1313 Microcoleus chthonoplastes PCC 7420 gb|EDX75395.1| 25 58.9 2.0E-07 GMRHGY403GATXD 502 hypothetical protein Gbem_3443 Geobacter bemidjiensis Bem ref|YP_002140232.1| 32 91.7 3.0E-17 GMRHGY403G2DJC 564 conserved hypothetical protein, membrane or secreted Dyadobacter fermentans DSM 18053 ref|YP_003085788.1| 31 60.1 1.0E-07 GMRHGY403FTTEP 490 hypothetical protein Mpe_A3412 Methylibium petroleiphilum PM1 ref|YP_001022600.1| 61 116 1.0E-24 GMRHGY403HFEBZ 505 hypothetical protein azo2664 Azoarcus sp. BH72 ref|YP_934168.1| 57 81.3 4.0E-14 GMRHGY403FVX7A 532 hypothetical protein uncultured Desulfobacterales bacterium HF0200_07G10 gb|ADI17934.1| 70 79.3 2.0E-13 GMRHGY403FNZP0 517 hypothetical protein ebA3498 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158998.1| 81 103 1.0E-20 GMRHGY403FY7WP 271 hypothetical protein Lawsonia intracellularis PHE/MN1-00 emb|CAJ55203.1| 68 125 1.0E-27 GMRHGY403GHH8O 518 hypothetical protein ebA4705 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159683.1| 66 190 7.0E-47 GMRHGY403F6UPG 546 hypothetical protein uncultured Desulfobacterales bacterium HF0200_07G10 gb|ADI17933.1| 67 73.6 1.0E-11 GMRHGY403GCWYC 488 hypothetical protein Daro_1893 Dechloromonas aromatica RCB ref|YP_285109.1| 76 179 1.0E-43 GMRHGY403F9PQU 150 hypothetical protein ebA4612 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159638.1| 56 54.7 4.0E-06 GMRHGY403G8GRL 409 hypothetical protein GYMC52DRAFT_3608 Geobacillus sp. Y412MC52 ref|ZP_04394454.1| 57 60.8 3.0E-11 GMRHGY403HF687 504 hypothetical protein ebA6152 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160539.1| 83 206 7.0E-57 GMRHGY403F4ADI 468 hypothetical protein azo0185 Azoarcus sp. BH72 ref|YP_931690.1| 46 83.6 8.0E-15 GMRHGY403F61J4 490 hypothetical protein Swit_5335 Sphingomonas wittichii RW1 ref|YP_001260210.1| 64 77.8 6.0E-18 GMRHGY403GAJYW 518 hypothetical protein Daro_3465 Dechloromonas aromatica RCB ref|YP_286664.1| 35 70.1 1.0E-10 GMRHGY403FNMQ6 500 hypothetical protein ebA3686 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159103.1| 62 145 2.0E-33 GMRHGY403F5RIC 503 hypothetical protein ebA1048 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157575.1| 71 172 1.0E-41 GMRHGY403FPS0N 339 hypothetical protein PRABACTJOHN_04042 Parabacteroides johnsonii DSM 18315 ref|ZP_03478339.1| 45 85.9 2.0E-15 GMRHGY403GAZG3 508 hypothetical protein Lcho_4123 Leptothrix cholodnii SP-6 ref|YP_001793140.1| 52 107 3.0E-32 GMRHGY403GE3E7 491 putative GTP cyclohydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159536.1| 79 232 3.0E-62 GMRHGY403F5BYJ 509 hypothetical protein azo1092 Azoarcus sp. BH72 ref|YP_932596.1| 41 106 1.0E-23 GMRHGY403GXGE0 521 putative signal transduction protein with CBS domains Thauera sp.MZ1T ref|YP_002890513.1| 71 103 8.0E-21 GMRHGY403HDEZA 477 putative GTP cyclohydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159536.1| 81 210 2.0E-61 GMRHGY403FLXV5 493 hypothetical protein Acid_0976 Solibacter usitatus Ellin6076 ref|YP_822259.1| 64 70.9 4.0E-16 GMRHGY403FVWNN 499 hypothetical protein ebA3532 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159014.1| 85 154 3.0E-36 GMRHGY403FWIUH 486 hypothetical protein Tmz1t_1008 Thauera sp. MZ1T ref|YP_002354669.1| 100 245 2.0E-63 GMRHGY403GAAP6 508 hypothetical protein azo3263 Azoarcus sp. BH72 ref|YP_934765.1| 39 45.4 1.0E-08 GMRHGY403HA5LJ 488 hypothetical protein azo1294 Azoarcus sp. BH72 ref|YP_932798.1| 48 138 2.0E-31 GMRHGY403F9OW2 458 hypothetical protein azo0700 Azoarcus sp. BH72 ref|YP_932204.1| 100 39.7 2.0E-06 GMRHGY403GRN6A 360 conserved hypothetical protein Listeria monocytogenes J2818 ref|ZP_06684334.1| 70 57 1.0E-10 GMRHGY403GFPQJ 510 hypothetical protein Tmz1t_1203 Thauera sp. MZ1T ref|YP_002354858.1| 85 77.4 6.0E-13 GMRHGY403F4H32 501 hypothetical protein ebB218 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160534.1| 81 140 7.0E-32 GMRHGY403FLKK5 422 hypothetical protein BVU_3042 Bacteroides vulgatus ATCC 8482 ref|YP_001300301.1| 61 95.9 2.0E-18 GMRHGY403GNAZ5 481 Glycosidases Ruminococcus bromii L2-63 emb|CBL15040.1| 43 49.3 2.0E-04 GMRHGY403F1UYI 560 hypothetical protein BBR47_11460 Brevibacillus brevis NBRC 100599 ref|YP_002770627.1| 42 103 1.0E-20 GMRHGY403HE3VF 513 protein of unknown function DUF1156 Cyanothece sp. PCC 7425 ref|YP_002482827.1| 47 135 2.0E-30 GMRHGY403GMM10 329 hypothetical protein Cflav_PD2347 bacterium Ellin514 ref|ZP_03630218.1| 78 45.4 5.0E-05 GMRHGY403FXOF4 474 hypothetical protein Tmz1t_2121 Thauera sp. MZ1T ref|YP_002355757.1| 98 139 1.0E-31 GMRHGY403FRU8U 526 LOW QUALITY PROTEIN: conserved hypothetical protein Streptomyces ghanaensis ATCC 14672 ref|ZP_06578138.1| 34 50.8 7.0E-05 GMRHGY403GFABR 497 hypothetical protein azo2038 Azoarcus sp. BH72 ref|YP_933542.1| 60 141 2.0E-32 GMRHGY403GIZMK 590 hypothetical protein uncultured SAR406 cluster bacterium HF4000_22B16 gb|ADI18534.1| 38 73.6 1.0E-11 GMRHGY403F3PCZ 535 hypothetical protein Haur_3457 Herpetosiphon aurantiacus ATCC 23779 ref|YP_001546221.1| 40 128 3.0E-28 GMRHGY403HFY1V 509 hypothetical protein GobsU_23427 Gemmata obscuriglobus UQM 2246 ref|ZP_02734776.1| 54 55.5 2.0E-06 GMRHGY403GV0F5 566 hypothetical protein ANACOL_02699 Anaerotruncus colihominis DSM 17241 ref|ZP_02443386.1| 68 209 1.0E-52 GMRHGY403G6KC1 496 hypothetical protein bglu_1g25610 Burkholderia glumae BGR1 ref|YP_002912341.1| 52 126 8.0E-28 GMRHGY403G2OFK 584 hypothetical protein Bcep1808_6134 Burkholderia vietnamiensis G4 ref|YP_001115306.1| 58 75.5 3.0E-12 GMRHGY403F8S4F 430 hypothetical protein ebA948 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157529.1| 90 140 4.0E-32 GMRHGY403HCCYZ 520 hypothetical protein Daro_2101 Dechloromonas aromatica RCB ref|YP_285314.1| 48 137 7.0E-31 GMRHGY403GHNOS 523 hypothetical protein ebA7105 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161056.1| 72 187 6.0E-46 GMRHGY403FJ2B4 224 hypothetical protein bll2516 Bradyrhizobium japonicum USDA 110 ref|NP_769156.1| 50 47 1.0E-07 GMRHGY403FP2QB 492 hypothetical protein CtCNB1_2990 Comamonas testosteroni CNB-2 ref|YP_003279032.1| 53 67.8 4.0E-10 GMRHGY403FMERP 508 hypothetical protein sce6759 Sorangium cellulosum 'So ce 56' ref|YP_001617408.1| 43 65.5 2.0E-09 GMRHGY403FXUUP 536 hypothetical protein CDSM653_949 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092485.1| 50 115 2.0E-24 GMRHGY403HCLNJ 503 conserved hypothetical protein Lutiella nitroferrum 2002 ref|ZP_03697323.1| 45 120 6.0E-26 GMRHGY403GBSVK 523 hypothetical protein YPTB2512 Yersinia pseudotuberculosis IP 32953 ref|YP_071024.1| 40 102 2.0E-20 GMRHGY403FP4M4 485 hypothetical protein Mpe_A3412 Methylibium petroleiphilum PM1 ref|YP_001022600.1| 60 112 2.0E-23 GMRHGY403FOHP5 529 hypothetical protein CLOL250_00835 Clostridium sp. L2-50 ref|ZP_02074073.1| 81 80.5 2.0E-17 GMRHGY403GVT43 563 hypothetical protein FAEPRAM212_00540 Faecalibacterium prausnitzii M21/2 ref|ZP_02090300.1| 44 55.5 8.0E-18 GMRHGY403F7FAG 502 hypothetical protein azo2152 Azoarcus sp. BH72 ref|YP_933656.1| 35 103 6.0E-21 GMRHGY403HCQQR 451 hypothetical protein STH2967 Symbiobacterium thermophilum IAM 14863 ref|YP_076794.1| 45 53.9 7.0E-06 GMRHGY403F1VWX 483 hypothetical protein c1A25 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158314.1| 91 119 1.0E-25 GMRHGY403F8OUR 552 hypothetical protein MXAN_2876 Myxococcus xanthus DK 1622 ref|YP_631087.1| 32 61.2 6.0E-08 GMRHGY403F7XB9 468 hypothetical protein ebA3587 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159054.1| 50 146 1.0E-33 GMRHGY403FVWT6 479 hypothetical protein uncultured SAR406 cluster bacterium HF4000_22B16 gb|ADI18534.1| 29 57 8.0E-07
! ! 225!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403GN7BA 486 hypothetical protein Daro_1893 Dechloromonas aromatica RCB ref|YP_285109.1| 73 132 2.0E-38 GMRHGY403GBZUG 528 protein of unknown function DUF1555 Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003168131.1| 36 94.4 5.0E-18 GMRHGY403F2UVH 509 hypothetical protein GobsU_23427 Gemmata obscuriglobus UQM 2246 ref|ZP_02734776.1| 54 55.5 2.0E-06 GMRHGY403GCLU5 518 protein of unknown function DUF147 Sphaerobacter thermophilus DSM 20745 ref|YP_003319857.1| 78 176 1.0E-42 GMRHGY403FQSAJ 436 hypothetical protein ebA6169 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160550.1| 43 81.6 3.0E-14 GMRHGY403FOXS1 509 hypothetical protein azo0185 Azoarcus sp. BH72 ref|YP_931690.1| 57 122 2.0E-26 GMRHGY403G0ILL 579 hypothetical protein Ccel_0623 Clostridium cellulolyticum H10 ref|YP_002504984.1| 33 56.6 2.0E-06 GMRHGY403GNGT1 565 hypothetical protein CDSM653_949 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092485.1| 73 146 6.0E-46 GMRHGY403F28EQ 550 hypothetical protein CDSM653_949 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092485.1| 76 145 7.0E-46 GMRHGY403G5924 215 hypothetical protein DORLON_02997 Dorea longicatena DSM 13814 ref|ZP_01996967.1| 49 50.1 1.0E-04 GMRHGY403F4GVO 516 hypothetical protein ebA4870 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159794.1| 92 329 7.0E-89 GMRHGY403GIOQK 147 hypothetical protein Mpe_A2349 Methylibium petroleiphilum PM1 ref|YP_001021540.1| 96 57.8 2.0E-08 GMRHGY403GJ06V 555 hypothetical protein NA Lawsonia intracellularis PHE/MN1-00 emb|CAJ55201.1| 39 67 1.0E-09 GMRHGY403GHG3H 540 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 65 89.4 1.0E-34 GMRHGY403FT9A3 506 hypothetical protein HMPREF0675_5204 Propionibacterium acnes SK137 ref|YP_003582324.1| 91 222 1.0E-56 GMRHGY403GQC5K 549 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 65 90.9 2.0E-33 GMRHGY403GSWK5 556 conserved hypothetical protein, secreted Salinibacter ruber M8 ref|YP_003570527.1| 25 60.8 8.0E-08 GMRHGY403FKRVO 141 hypothetical protein CHLREDRAFT_155068 Chlamydomonas reinhardtii ref|XP_001698950.1| 46 42 1.0E-04 GMRHGY403G86FJ 171 hypothetical protein azo2317 Azoarcus sp. BH72 ref|YP_933821.1| 74 74.7 4.0E-12 GMRHGY403FLW3I 426 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 50 93.6 8.0E-18 GMRHGY403FJ0MC 166 hypothetical protein BBta_3525 Bradyrhizobium sp. BTAi1 ref|YP_001239524.1| 96 99.4 1.0E-19 GMRHGY403F0XF6 514 hypothetical protein ebA3370 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158930.1| 83 271 2.0E-71 GMRHGY403GEP10 522 hypothetical protein azo0985 Azoarcus sp. BH72 ref|YP_932489.1| 75 88.6 1.0E-38 GMRHGY403FJRGI 560 hypothetical protein TSTA_040370 Talaromyces stipitatus ATCC 10500 ref|XP_002484510.1| 70 97.1 8.0E-30 GMRHGY403FQLLC 456 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 47 83.6 4.0E-23 GMRHGY403GFDWN 236 hypothetical protein blr0930 Bradyrhizobium japonicum USDA 110 ref|NP_767570.1| 45 47.8 5.0E-04 GMRHGY403FJFM6 506 hypothetical protein sce6757 Sorangium cellulosum 'So ce 56' ref|YP_001617406.1| 38 95.5 2.0E-18 GMRHGY403FT6JA 509 hypothetical protein ebB140 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159279.1| 90 140 6.0E-32 GMRHGY403G6WDJ 179 hypothetical protein CTN_1800 Thermotoga neapolitana DSM 4359 ref|YP_002535342.1| 47 62 2.0E-08 GMRHGY403FNVQG 478 hypothetical protein ebA2408 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158371.1| 72 170 5.0E-41 GMRHGY403FT1NC 521 hypothetical protein ebA2648 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158516.1| 76 190 5.0E-47 GMRHGY403HC5Y4 478 hypothetical protein ebB149 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159502.1| 73 176 7.0E-43 GMRHGY403GSKMX 198 protein of unknown function DUF820 Nitrosococcus halophilus Nc4 ref|YP_003526903.1| 62 65.5 2.0E-09 GMRHGY403FW64L 498 hypothetical protein ebA6533 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160761.1| 86 158 2.0E-37 GMRHGY403G96ZA 508 hypothetical protein Bcep1808_7276 Burkholderia vietnamiensis G4 ref|YP_001109935.1| 41 67.4 6.0E-10 GMRHGY403F7F3Q 142 predicted protein Trichoplax adhaerens ref|XP_002118239.1| 91 47 1.0E-08 GMRHGY403FR18O 131 hypothetical protein Tmz1t_2873 Thauera sp. MZ1T ref|YP_002889849.1| 96 68.9 2.0E-10 GMRHGY403FMHSM 298 hypothetical protein azo2847 Azoarcus sp. BH72 ref|YP_934350.1| 55 53.9 3.0E-11 GMRHGY403G9LPB 490 putative pirin-like protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159227.1| 74 144 4.0E-33 GMRHGY403GOXOE 506 hypothetical protein azo1589 Azoarcus sp. BH72 ref|YP_933093.1| 80 174 1.0E-43 GMRHGY403F2Y5O 512 hypothetical protein HMPREF0189_01117 Burkholderiales bacterium 1_1_47 ref|ZP_07343585.1| 34 81.3 4.0E-14 GMRHGY403FS0NY 300 hypothetical protein Tmz1t_0975 Thauera sp. MZ1T ref|YP_002354637.1| 100 80.1 9.0E-14 GMRHGY403FQAGW 493 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 61 48.1 3.0E-10 GMRHGY403GJCWH 156 LOW QUALITY PROTEIN: conserved hypothetical protein Streptomyces ghanaensis ATCC 14672 ref|ZP_06580272.1| 60 48.5 3.0E-04 GMRHGY403FKWT4 496 hypothetical protein azo0750 Azoarcus sp. BH72 ref|YP_932254.1| 58 133 3.0E-45 GMRHGY403HFV4R 525 conserved hypothetical protein uncultured archaeon GZfos34A6 gb|AAU83825.1| 48 140 6.0E-32 GMRHGY403F9GIK 551 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955130.1| 72 52.8 2.0E-17 GMRHGY403HFQVU 492 hypothetical protein azo0700 Azoarcus sp. BH72 ref|YP_932204.1| 82 82.8 6.0E-16 GMRHGY403F2JXY 486 hypothetical protein bglu_1g20040 Burkholderia glumae BGR1 ref|YP_002911817.1| 61 70.1 5.0E-19 GMRHGY403GJQWT 480 hypothetical protein ebA6073 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160490.1| 80 94.4 2.0E-27 GMRHGY403HABNO 515 hypothetical protein pah_c222o002 Parachlamydia acanthamoebae str. Hall's coccus ref|ZP_06300713.1| 64 126 9.0E-28 GMRHGY403HHHLM 518 hypothetical protein BBR47_11460 Brevibacillus brevis NBRC 100599 ref|YP_002770627.1| 42 103 8.0E-21 GMRHGY403FJ9AC 292 hypothetical protein BCh11DRAFT_7065 Burkholderia sp. Ch1-1 ref|ZP_06845798.1| 57 79.3 1.0E-13 GMRHGY403FNQKE 502 hypothetical protein Tmz1t_0940 Thauera sp. MZ1T ref|YP_002354602.1| 71 170 5.0E-41 GMRHGY403G5JH9 519 hypothetical protein ALIPUT_00387 Alistipes putredinis DSM 17216 ref|ZP_02424272.1| 53 142 2.0E-43 GMRHGY403GW7YA 178 hypothetical protein BACDOR_01341 Bacteroides dorei DSM 17855 ref|ZP_03299974.1| 55 42.4 7.0E-07 GMRHGY403FTJKI 498 hypothetical protein ebA6152 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160539.1| 76 142 4.0E-34 GMRHGY403HAOV6 482 protein of unknown function DUF6 transmembrane Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06211284.1| 50 86.7 9.0E-16 GMRHGY403GNY6A 425 Exporter of the RND superfamily Shewanella piezotolerans WP3 ref|YP_002312125.1| 48 144 4.0E-33 GMRHGY403HESCD 497 hypothetical protein ebB218 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160534.1| 67 109 1.0E-22 GMRHGY403FON4G 510 hypothetical protein Tmz1t_0447 Thauera sp. MZ1T ref|YP_002354122.1| 81 84 6.0E-15 GMRHGY403F7XSH 169 hypothetical protein uncultured delta proteobacterium HF0070_15B21 gb|ADI19064.1| 57 54.7 4.0E-06 GMRHGY403FYOOO 487 hypothetical protein Daro_1893 Dechloromonas aromatica RCB ref|YP_285109.1| 76 179 1.0E-43 GMRHGY403FPML4 520 hypothetical protein Dehly_0289 Dehalogenimonas lykanthroporepellens BL-DC-9 ref|YP_003757933.1| 69 66.6 1.0E-09 GMRHGY403GXJCG 536 hypothetical protein Haur_3457 Herpetosiphon aurantiacus ATCC 23779 ref|YP_001546221.1| 37 97.1 8.0E-19 GMRHGY403F5ZGF 500 hypothetical protein ebA2531 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158454.1| 90 315 1.0E-84 GMRHGY403GBJVW 507 hypothetical protein azo1589 Azoarcus sp. BH72 ref|YP_933093.1| 65 64.3 5.0E-09 GMRHGY403GMPPU 197 hypothetical protein ebA6101 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160512.1| 80 112 2.0E-23 GMRHGY403G1RDY 462 hypothetical protein Tmz1t_0956 Thauera sp. MZ1T ref|YP_002354618.1| 72 83.2 1.0E-14 GMRHGY403GBXIS 518 hypothetical protein Tmz1t_2846 Thauera sp. MZ1T ref|YP_002889822.1| 58 169 1.0E-44 GMRHGY403G1JC3 493 Conserved protein Lactobacillus rhamnosus GG emb|CAR86202.1| 59 57.4 6.0E-07 GMRHGY403HBJ0N 367 hypothetical protein DR_0254 Deinococcus radiodurans R1 gb|AAF09840.1| 45 82.4 2.0E-14 GMRHGY403GJS0X 562 hypothetical protein BMULJ_05093 Burkholderia multivorans ATCC 17616 ref|YP_001949471.1| 62 97.4 2.0E-23 GMRHGY403F6BSK 410 hypothetical protein ebA2640 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158512.1| 63 140 5.0E-32 GMRHGY403GHSSQ 449 protein of unknown function DUF1555 Candidatus Accumulibacter phosphatis clade IIA str. UW-1 ref|YP_003168131.1| 37 51.6 2.0E-06
! ! 226!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403G4WGB 509 hypothetical protein ebB140 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159279.1| 89 142 2.0E-32 GMRHGY403GK3N1 202 LOW QUALITY PROTEIN: hypothetical protein SSOG_01100 Streptomyces hygroscopicus ATCC 53653 ref|ZP_07293019.1| 61 53.1 1.0E-05 GMRHGY403G1YRA 513 hypothetical protein ebA2758 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158576.1| 65 96.7 7.0E-37 GMRHGY403G4K1N 386 protein of unknown function DUF224 cysteine-rich region domain protein bacterium Ellin514 ref|ZP_03630641.1| 53 52 3.0E-05 GMRHGY403GKMD6 487 hypothetical protein ebA6688 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160849.1| 86 77.4 3.0E-29 GMRHGY403GFWWQ 214 conserved hypothetical protein Clostridium butyricum E4 str. BoNTE BL5262 ref|ZP_04526280.1| 69 63.9 6.0E-09 GMRHGY403HCZPC 152 unknown protein Streptococcus suis 98HAH33 gb|ABP91180.1| 76 58.2 4.0E-07 GMRHGY403G28YE 517 hypothetical protein Saut_1499 Sulfurimonas autotrophica DSM 16294 ref|YP_003892558.1| 31 53.5 7.0E-14 GMRHGY403FOTQT 474 hypothetical protein ebB139 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159256.1| 64 93.2 1.0E-17 GMRHGY403FNO66 257 hypothetical protein CPC_A0111 Clostridium perfringens C str.JGS1495 ref|ZP_02865941.1| 52 68.6 3.0E-10 GMRHGY403FKHF2 469 hypothetical protein UBAL3_49470002 Leptospirillum ferrodiazotrophum gb|EES53770.1| 44 109 1.0E-22 GMRHGY403GYL0B 527 hypothetical protein STHERM_c04540 Spirochaeta thermophila DSM6192 ref|YP_003873699.1| 56 177 5.0E-46 GMRHGY403G81J3 447 hypothetical protein GYMC52DRAFT_3608 Geobacillus sp. Y412MC52 ref|ZP_04394454.1| 48 54.3 1.0E-15 GMRHGY403HF0WR 520 hypothetical protein Sros_6496 Streptosporangium roseum DSM 43021 ref|YP_003341953.1| 66 181 7.0E-53 GMRHGY403GC2ZV 125 hypothetical protein CHLREDRAFT_155068 Chlamydomonas reinhardtii ref|XP_001698950.1| 93 72.4 2.0E-11 GMRHGY403GYRSK 528 hypothetical protein Rmar_0356 Rhodothermus marinus DSM 4252 ref|YP_003289648.1| 59 226 7.0E-58 GMRHGY403GNWPO 504 hypothetical protein ebA535 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157297.1| 61 203 7.0E-51 GMRHGY403F10D5 530 unknown protein Streptococcus suis 98HAH33 gb|ABP91180.1| 53 86.7 6.0E-30 GMRHGY403GCOJ3 527 hypothetical protein H16_A0916 Ralstonia eutropha H16 ref|YP_725430.1| 65 145 2.0E-33 GMRHGY403GMWHW 210 hypothetical protein ebA138 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157098.1| 75 101 3.0E-20 GMRHGY403FX7T1 474 hypothetical protein ebA4287 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159452.1| 54 79.3 1.0E-13 GMRHGY403FPRZO 352 hypothetical protein ebA138 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157098.1| 74 169 8.0E-41 GMRHGY403GSUW6 344 hypothetical protein Arnit_0197 Arcobacter nitrofigilis DSM 7299 ref|YP_003654370.1| 45 74.7 3.0E-20 GMRHGY403GP3VU 531 protein of unknown function UPF0150 Nitrosococcus halophilus Nc4 ref|YP_003526193.1| 59 122 2.0E-26 GMRHGY403FO33J 284 hypothetical protein COLSTE_02537 Collinsella stercoris DSM 13279 ref|ZP_03298598.1| 50 56.2 1.0E-06 GMRHGY403GE35I 512 hypothetical protein ebA5828 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160335.1| 82 277 4.0E-73 GMRHGY403G2OF5 298 hypothetical protein CV_0752 Chromobacterium violaceum ATCC 12472 ref|NP_900422.1| 65 63.2 1.0E-08 GMRHGY403G52BG 531 hypothetical protein ebA6138 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160532.1| 46 122 5.0E-30 GMRHGY403G4UNO 462 hypothetical protein HM1_3148 Heliobacterium modesticaldum Ice1 ref|YP_001680917.1| 62 134 5.0E-30 GMRHGY403F4LBX 484 hypothetical protein ebA5327 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160054.1| 42 125 2.0E-27 GMRHGY403F9AGR 512 hypothetical protein azo1748 Azoarcus sp. BH72 ref|YP_933252.1| 65 131 9.0E-30 GMRHGY403FI87T 478 hypothetical protein ebA3993 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159289.1| 56 157 6.0E-37 GMRHGY403FLP19 512 hypothetical protein azo2810 Azoarcus sp. BH72 ref|YP_934313.1| 59 170 5.0E-41 GMRHGY403FS5OT 505 hypothetical protein azo3277 Azoarcus sp. BH72 ref|YP_934779.1| 48 62.4 2.0E-08 GMRHGY403GDNFT 480 hypothetical protein Bphy_5959 Burkholderia phymatum STM815 ref|YP_001862069.1| 76 262 9.0E-69 GMRHGY403GJNL8 122 hypothetical protein BMULJ_05098 Burkholderia multivorans ATCC17616 ref|YP_001949473.1| 91 67 8.0E-10 GMRHGY403FJ5KO 103 hypothetical protein BMULJ_05098 Burkholderia multivorans ATCC 17616 ref|YP_001949473.1| 88 52.8 2.0E-05 GMRHGY403FJ7S2 81 unknown Brugia malayi gb|AAD44515.1| 87 48.9 2.0E-04 GMRHGY403G3BZF 85 LOW QUALITY PROTEIN: conserved hypothetical protein Brucella suis bv. 3 str. 686 ref|ZP_05997968.1| 100 50.1 1.0E-04 GMRHGY403GEX2P 389 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 46 53.5 5.0E-07 GMRHGY403G6U7C 405 conserved hypothetical protein Bacteroides sp. 3_1_33FAA ref|ZP_06090968.1| 77 92.4 2.0E-27 GMRHGY403GKQHJ 507 cell wall-associated hydrolase Burkholderia multivorans ATCC 17616 ref|YP_001949468.1| 64 214 3.0E-54 GMRHGY403GM9UO 471 conserved hypothetical protein Brucella sp. 83/13 ref|ZP_06097399.1| 96 191 3.0E-47 GMRHGY403FNHK6 243 conserved hypothetical protein Vibrio cholerae 1587 ref|ZP_01951366.1| 56 73.6 8.0E-12 GMRHGY403GM4N3 555 hypothetical protein CE1543 Corynebacterium efficiens YS-314 ref|NP_738153.1| 55 97.1 1.0E-27 GMRHGY403FNH00 546 hypothetical protein SULAZ_0908 Sulfurihydrogenibium azorense Az-Fu1 ref|YP_002728882.1| 82 68.6 6.0E-19 GMRHGY403GNULY 492 hypothetical protein CLM_0052 Clostridium botulinum A2 str. Kyoto ref|YP_002802349.1| 50 95.9 2.0E-35 GMRHGY403GZP19 532 conserved hypothetical protein Clostridium sp. 7_2_43FAA ref|ZP_05132927.1| 51 81.3 5.0E-14 GMRHGY403GDL7M 556 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 45 62 3.0E-08 GMRHGY403F3I1C 493 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 72 104 6.0E-25 GMRHGY403GT5O4 515 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 80 101 3.0E-46 GMRHGY403F5WI5 496 hypothetical protein ANACOL_02699 Anaerotruncus colihominis DSM 17241 ref|ZP_02443386.1| 68 181 2.0E-44 GMRHGY403HDD2H 568 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 58 192 2.0E-47 GMRHGY403G1YRV 459 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 76 102 7.0E-24 GMRHGY403HCUQI 378 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 67 73.2 1.0E-17 GMRHGY403GNO7A 474 conserved hypothetical protein Clostridium botulinum C str. Eklund ref|ZP_02863228.1| 48 89.4 7.0E-22 GMRHGY403FWY3K 555 hypothetical protein RUMOBE_01295 Ruminococcus obeum ATCC 29174 ref|ZP_01963577.1| 72 84.3 2.0E-32 GMRHGY403GZNPZ 458 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 81 61.6 8.0E-11 GMRHGY403GH24U 579 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 71 101 1.0E-40 GMRHGY403GOI0C 528 conserved hypothetical protein Aurantimonas manganoxydans SI85-9A1 ref|ZP_01227369.1| 75 177 4.0E-43 GMRHGY403G9X7Y 386 conserved hypothetical protein Aurantimonas manganoxydans SI85-9A1 ref|ZP_01227369.1| 70 102 4.0E-38 GMRHGY403GNJP4 434 hypothetical protein ebA1598 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157896.1| 42 81.6 3.0E-14 GMRHGY403GKJ3R 520 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 56 169 9.0E-41 GMRHGY403G1HCG 520 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31922.1| 85 57.8 2.0E-16 GMRHGY403G3E45 560 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31922.1| 84 150 1.0E-48 GMRHGY403GZFOY 579 hypothetical protein CDSM653_949 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092485.1| 70 86.3 3.0E-31 GMRHGY403FNY0O 511 hypothetical protein HM1_3149 Heliobacterium modesticaldum Ice1 ref|YP_001680918.1| 62 141 3.0E-32 GMRHGY403FVCI5 552 hypothetical protein BACCAP_03832 Bacteroides capillosus ATCC 29799 ref|ZP_02038208.1| 64 137 8.0E-31 GMRHGY403GXU1U 277 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31990.1| 64 58.5 3.0E-07 GMRHGY403F44PQ 526 cell wall-associated hydrolase Burkholderia multivorans ATCC 17616 ref|YP_001949468.1| 73 246 8.0E-64 GMRHGY403GM2S8 482 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 64 137 2.0E-44 GMRHGY403FQJUL 524 hypothetical protein GCWU000323_01826 Leptotrichia hofstadii F0254 ref|ZP_05901904.1| 62 72.8 2.0E-11 GMRHGY403GUO79 534 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 54 126 1.0E-27 GMRHGY403G8D3H 543 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 66 192 2.0E-47 GMRHGY403FY5RM 556 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 55 124 6.0E-27 GMRHGY403FI7BB 362 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 71 65.9 4.0E-25 GMRHGY403FK0AA 604 conserved hypothetical protein Sulfurihydrogenibium yellowstonense SS-5 ref|ZP_04583994.1| 67 87.4 2.0E-23 GMRHGY403GQ7YE 566 hypothetical protein BMULJ_05092 Burkholderia multivorans ATCC 17616 ref|YP_001949470.1| 60 132 3.0E-29
! ! 227!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY403FVTRY 564 Conserved protein Lactobacillus rhamnosus GG emb|CAR86202.1| 62 92.4 1.0E-19 GMRHGY403F56GK 561 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 60 143 1.0E-32 GMRHGY403F8INS 552 conserved hypothetical protein Clostridium butyricum E4 str. BoNTE BL5262 ref|ZP_04526280.1| 51 97.8 6.0E-19 GMRHGY403HHG4K 566 conserved hypothetical protein Listeria grayi DSM 20601 ref|ZP_07052907.1| 86 172 2.0E-41 GMRHGY403GL52O 485 hypothetical protein azo2038 Azoarcus sp. BH72 ref|YP_933542.1| 62 140 5.0E-32 GMRHGY403GI3VR 503 hypothetical protein uncultured Acidobacteria bacterium HF4000_26D02 gb|ADI18656.1| 61 77 7.0E-13 GMRHGY403G8Y9L 414 hypothetical protein CDSM653_956 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092491.1| 90 62.4 2.0E-08 GMRHGY403GXDBQ 339 conserved hypothetical protein Clostridium butyricum E4 str. BoNTE BL5262 ref|ZP_04526295.1| 67 82.8 1.0E-17 GMRHGY403GGGTU 492 Hypothetical protein COLAER_01802 Collinsella aerofaciens ATCC 25986 ref|ZP_01772785.1| 55 95.9 2.0E-18 GMRHGY403GSVLK 411 hypothetical protein ACTODO_00001 Actinomyces odontolyticus ATCC 17982 ref|ZP_02043164.1| 86 147 4.0E-34 GMRHGY403FR1L6 536 AE002057_8 hypothetical protein DR_2252 Deinococcus radiodurans R1 gb|AAF11800.1| 42 79 2.0E-13 GMRHGY403HC51W 565 Hypothetical protein COLAER_02246 Collinsella aerofaciens ATCC 25986 ref|ZP_01773214.1| 43 63.9 5.0E-20 GMRHGY403FU75X 521 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 55 120 9.0E-26 GMRHGY403GHG5P 524 LOW QUALITY PROTEIN: conserved hypothetical protein Streptomyces ghanaensis ATCC 14672 ref|ZP_06578095.1| 62 94.4 2.0E-27 GMRHGY403FYOTV 490 hypothetical protein ebA6294 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160626.1| 69 202 9.0E-51 GMRHGY403HHM5C 551 hypothetical protein uncultured Rhizobiales bacterium HF4000_32B18 gb|ADI18732.1| 66 68.2 5.0E-10 GMRHGY403G28RI 543 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 72 82.4 1.0E-29 GMRHGY403GUKGE 480 hypothetical protein HMPREF9553_00243 Escherichia coli MS 200-1 ref|ZP_07172160.1| 66 144 5.0E-33 GMRHGY403GL2NH 538 hypothetical protein RUMOBE_01293 Ruminococcus obeum ATCC 29174 ref|ZP_01963575.1| 46 137 6.0E-31 GMRHGY403F1X11 522 PREDICTED: hypothetical protein Oryctolagus cuniculus ref|XP_002723895.1| 68 52.8 8.0E-13 GMRHGY403G9HKB 559 hypothetical protein N47_G38930 uncultured Desulfobacterium sp. emb|CBX28569.1| 59 117 5.0E-25 GMRHGY403F4HW7 499 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 88 218 2.0E-55 GMRHGY403F2ARW 553 cell wall-associated hydrolase Burkholderia multivorans ATCC 17616 ref|YP_001949468.1| 52 171 4.0E-41 GMRHGY403GMWY1 555 hypothetical protein CLOLEP_01448 Clostridium leptum DSM 753 ref|ZP_02079996.1| 78 81.6 1.0E-27 GMRHGY403FVFN6 556 unknow protein Oryza sativa Japonica Group gb|AAV44205.1| 52 103 4.0E-21 GMRHGY403FOPM8 539 hypothetical protein CTC00065 Clostridium tetani E88 ref|NP_780783.1| 72 144 2.0E-37 GMRHGY403F6IYJ 555 hypothetical protein CLOHIR_02084 Clostridium hiranonis DSM 13275 ref|ZP_03294132.1| 66 171 5.0E-41 GMRHGY403FOBQJ 500 conserved hypothetical protein Streptomyces violaceusniger Tu 4113 ref|ZP_07611793.1| 67 68.6 2.0E-14 GMRHGY403HEL3F 455 hypothetical protein uncultured delta proteobacterium HF0500_03A04 gb|ADI19336.1| 50 57 8.0E-07 GMRHGY403F0T3K 566 ybl206 Escherichia coli BL21(DE3) emb|CAQ34357.1| 67 138 4.0E-31 GMRHGY403GYLXA 370 ybl206 Escherichia coli BL21(DE3) emb|CAQ34357.1| 84 74.7 5.0E-26 GMRHGY403GH1KU 478 hypothetical protein ebA3882 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159228.1| 60 135 2.0E-30
Section 2. Assembled sequences isotig00077 gene=isogroup00005 3301 rplB 50S ribosomal protein L2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159186.1| 92 517 e-144 isotig00085 gene=isogroup00011 488 Lon ATP-dependent protease La Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159856.1| 74 140 5.0E-32 isotig00086 gene=isogroup00012 630 sensory histidine kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160937.1| 63 241 6.0E-62 isotig00089 gene=isogroup00015 662 gltA citrate synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160848.1| 99 435 e-120 isotig00090 gene=isogroup00016 1295 atpH F1-ATP synthase, delta subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158719.1| 86 285 1.0E-74 AtpF F0F1 ATP synthase subunit B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158718.1| 67 184 2.0E-44 isotig00095 gene=isogroup00021 517 fabG 3-oxoacyl-(acyl-carrier-protein) reductase Thauera sp. MZ1T ref|YP_002355961.1| 83 282 1.0E-74 isotig00096 gene=isogroup00022 483 FtsA cell division protein FtsA Azoarcus sp. BH72 ref|YP_932392.1| 94 242 1.0E-62 isotig00097 gene=isogroup00023 544 cytC3 di-Heme cytochrome c, class IC Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157591.1| 76 276 1.0E-72 isotig00098 gene=isogroup00024 1193 nuoG NADH dehydrogenase subunit G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159771.1| 78 561 e-177 isotig00099 gene=isogroup00025 502 narK2 nitrate/nitrite antiporter Thauera sp. MZ1T ref|YP_002889615.1| 90 194 1.0E-58 isotig00100 gene=isogroup00026 495 dnr transcriptional regulator Dnr/Nnr type Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159949.1| 89 283 4.0E-75 isotig00101 gene=isogroup00027 566 putative iron-sulfur 4Fe-4S ferredoxin transmembrane protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159943.1| 84 340 4.0E-92 isotig00102 gene=isogroup00028 793 dhaL aldehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158262.1| 93 434 e-120 isotig00104 gene=isogroup00030 836 atpD F0F1 ATP synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158722.1| 98 280 2.0E-73 isotig00105 gene=isogroup00031 458 nuoI NADH dehydrogenase subunit I Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159773.1| 93 192 2.0E-47 isotig00107 gene=isogroup00033 476 ispE 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157787.1| 85 142 2.0E-32 isotig00109 gene=isogroup00035 635 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 97 226 2.0E-88 isotig00110 gene=isogroup00036 463 rpsN 30S ribosomal protein S14 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159196.1| 96 125 5.0E-40 isotig00111 gene=isogroup00037 661 RpsF 30S ribosomal protein S6 [] Azoarcus sp. BH72 ref|YP_932222.1| 93 222 2.0E-56 isotig00113 gene=isogroup00039 1288 rplE 50S ribosomal protein L5 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159195.1| 85 293 3.0E-77 isotig00114 gene=isogroup00040 495 LysC aspartate kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157354.1| 86 206 6.0E-52 isotig00115 gene=isogroup00041 492 rplS 50S ribosomal protein L19 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161091.1| 96 210 4.0E-53 isotig00121 gene=isogroup00047 470 tsf elongation factor Ts Azoarcus sp. BH72 ref|YP_933412.1| 87 212 8.0E-60 isotig00124 gene=isogroup00050 476 ilvH acetolactate synthase 3 regulatory subunit Azoarcus sp. BH72 ref|YP_934658.1| 92 252 1.0E-65 isotig00125 gene=isogroup00051 489 ribE riboflavin synthase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159044.1| 86 169 6.0E-67 isotig00126 gene=isogroup00052 445 slyD FKBP-type peptidyl-prolyl cis-trans isomerase (rotamase) protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160724.1| 88 198 2.0E-49 isotig00128 gene=isogroup00054 486 speE spermidine synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158219.1| 78 257 3.0E-67 isotig00131 gene=isogroup00057 488 rpoZ DNA-directed RNA polymerase omega chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158996.1| 97 132 2.0E-29 spoT guanosine-3',5'-bis(diphosphate) 3'-pyrophosphohydrolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158997.1| 67 93.6 3.0E-19 isotig00132 gene=isogroup00058 482 nuoJ NADH dehydrogenase I, chain J Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159774.1| 77 223 5.0E-57 isotig00134 gene=isogroup00060 479 aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit A Dechloromonas aromatica RCB ref|YP_283347.1| 71 229 1.0E-58 isotig00135 gene=isogroup00061 741 dioxygenase related to 2-nitropropane dioxygenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158504.1| 91 468 e-130 isotig00136 gene=isogroup00062 505 rpsA 30S ribosomal protein S1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157511.2| 97 309 9.0E-83 isotig00137 gene=isogroup00063 491 etfB electron transfer flavoprotein, beta-subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160746.1| 91 282 1.0E-74 isotig00138 gene=isogroup00064 466 nucleotidyl transferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157626.1| 82 149 1.0E-34 isotig00139 gene=isogroup00065 472 rpsB 30S ribosomal protein S2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160440.1| 67 185 1.0E-45 isotig00141 gene=isogroup00067 572 norC nitric-oxide reductase subunit C Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157125.1| 87 210 1.0E-59 isotig00142 gene=isogroup00068 543 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157126.1| 93 278 2.0E-89 isotig00143 gene=isogroup00069 684 putative pterin-4-alpha-carbinolamine dehydratase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157105.1| 63 128 5.0E-28
! ! 228!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value isotig00149 gene=isogroup00075 607 rplF 50S ribosomal protein L6 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159198.1| 91 325 2.0E-87 isotig00154 gene=isogroup00080 487 tufB elongation factor Tu Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159169.1| 100 75.1 3.0E-12 isotig00155 gene=isogroup00081 958 nuoF NADH dehydrogenase I, chain F Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159770.1| 92 613 e-173 isotig00156 gene=isogroup00082 519 rplU 50S ribosomal protein L21 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157466.1| 83 147 4.0E-34 rpmA 50S ribosomal protein L27 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157467.1| 100 143 7.0E-33 isotig00157 gene=isogroup00083 936 sdhA succinate dehydrogenase, flavoprotein subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160851.1| 88 234 9.0E-60 isotig00158 gene=isogroup00084 772 rpsL 30S ribosomal protein S12 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) emb|CAI08277.1| 97 268 5.0E-70 isotig00160 gene=isogroup00086 656 rpsA 30S ribosomal protein S1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157511.2| 97 384 e-105 isotig00162 gene=isogroup00088 477 nosZ nitrous-oxide reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160614.1| 88 272 6.0E-75 isotig00163 gene=isogroup00089 917 rpsM 30S ribosomal protein S13 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159205.1| 95 232 6.0E-59 isotig00164 gene=isogroup00090 1253 rpoA DNA-directed RNA polymerase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159208.1| 92 460 e-127 isotig00165 gene=isogroup00091 1044 exaA3 putative quinoprotein ethanol dehydrogenase Azoarcus sp. BH72 ref|YP_934478.1| 79 588 e-166 isotig00176 gene=isogroup00102 1257 ompC outer membrane protein (porin) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159426.1| 64 497 e-138 isotig00177 gene=isogroup00103 3364 rplA 50S ribosomal protein L1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159173.1| 82 366 1.0E-98 NusG transcription antitermination protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159171.1| 97 347 6.0E-93 isotig00178 gene=isogroup00104 606 nuoL NADH dehydrogenase I, chain L Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159776.1| 82 347 5.0E-94 isotig00179 gene=isogroup00105 596 quiA quinate dehydrogenase (PQQ) Ralstonia eutropha H16 ref|YP_840568.1| 86 166 1.0E-39 isotig00180 gene=isogroup00106 519 hflB cell division protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159756.1| 94 320 3.0E-86 isotig00182 gene=isogroup00108 790 translation elongation factor Tu Thauera sp. MZ1T ref|YP_002890312.1| 89 429 e-118 isotig00183 gene=isogroup00109 923 gyrA DNA gyrase subunit A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157516.1| 91 550 e-155 isotig00184 gene=isogroup00110 509 outer membrane protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157518.1| 82 275 2.0E-72 isotig00185 gene=isogroup00111 510 CarA carbamoyl phosphate synthase small subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159750.1| 85 285 1.0E-75 isotig00186 gene=isogroup00112 514 sensory histidine kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160937.1| 44 60.8 6.0E-08 isotig00187 gene=isogroup00113 656 atpA F0F1 ATP synthase subunit alpha Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158720.1| 94 283 1.0E-74 isotig00188 gene=isogroup00114 575 hypothetical protein azo2189 Azoarcus sp. BH72 ref|YP_933693.1| 86 134 5.0E-30 rpsT 30S ribosomal protein S20 Azoarcus sp. BH72 ref|YP_933694.1| 92 124 5.0E-27 isotig00189 gene=isogroup00115 650 atpD F0F1 ATP synthase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158722.1| 91 389 e-106 isotig00191 gene=isogroup00117 807 rhlE1 ATP-dependent RNA helicase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159040.1| 94 335 4.0E-90 isotig00192 gene=isogroup00118 490 infA1 translation initiation factor IF-1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160858.1| 98 152 1.0E-35 isotig00193 gene=isogroup00119 880 metal-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160126.1| 71 241 7.0E-62 rpmF 50S ribosomal protein L32 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160127.1| 98 122 8.0E-26 isotig00194 gene=isogroup00120 598 conserved hypothetical protein,predicted phasin family Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157742.1| 72 201 6.0E-50 isotig00195 gene=isogroup00121 1198 narK1 nitrate/proton symporter Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160623.1| 73 593 e-167 isotig00196 gene=isogroup00122 464 mdh malate dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160856.1| 90 277 3.0E-73 isotig00197 gene=isogroup00123 502 LuxE acyl-CoA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160750.1| 73 205 1.0E-51 isotig00199 gene=isogroup00125 497 phasin family protein Thauera sp. MZ1T ref|YP_002354538.1| 58 150 4.0E-35 isotig00201 gene=isogroup00127 644 rpsD 30S ribosomal protein S4 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159207.1| 94 330 1.0E-88 isotig00203 gene=isogroup00129 513 narG nitrate reductase, alpha chain Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160621.1| 87 312 1.0E-83 isotig00204 gene=isogroup00130 506 tpiA triosephosphate isomerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159763.1| 63 207 5.0E-52 isotig00206 gene=isogroup00132 603 fusA elongation factor G Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159180.1| 99 384 e-105 isotig00207 gene=isogroup00133 575 infB translation initiation factor IF-2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160344.1| 94 352 1.0E-95 isotig00208 gene=isogroup00134 501 LuxE acyl-CoA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160750.1| 89 332 8.0E-90 isotig00209 gene=isogroup00135 680 nuoC NADH dehydrogenase subunit C Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159767.1| 85 365 3.0E-99 isotig00210 gene=isogroup00136 856 rplM 50S ribosomal protein L13 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157525.1| 95 281 7.0E-74 rpsI 30S ribosomal protein S9 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157524.1| 97 251 1.0E-64 isotig00211 gene=isogroup00137 1127 translation initiation factor IF-3 Thauera sp. MZ1T ref|YP_002889999.1| 84 274 2.0E-71 isotig00212 gene=isogroup00138 512 nuoB NADH dehydrogenase subunit B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159766.1| 100 244 2.0E-63 isotig00214 gene=isogroup00140 838 nodG Short-chain dehydrogenase/reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159700.1| 90 271 e-124 isotig00215 gene=isogroup00141 501 IclR family transciptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159322.1| 89 266 7.0E-70 isotig00216 gene=isogroup00142 507 ppa inorganic pyrophosphatase Azoarcus sp. BH72 ref|YP_932862.1| 89 180 2.0E-83 isotig00217 gene=isogroup00143 696 petB cytochrome B subunit of cytochrome bc1 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157659.1| 96 386 e-105 isotig00108 gene=isogroup00034 722 GcdH glutaryl-CoA dehydrogenase Azoarcus sp. BH72 gb|ABM69268.1| 89 340 ##### isotig00133 gene=isogroup00059 835 bktB beta-ketothiolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159977.1| 88 483 e-134 isotig00213 gene=isogroup00139 506 3-hydroxyacyl-CoA dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158051.1| 76 251 ##### isotig00001 gene=isogroup00001 2426 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00003 gene=isogroup00001 2462 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00005 gene=isogroup00001 2464 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00008 gene=isogroup00001 2428 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00002 gene=isogroup00001 2598 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00007 gene=isogroup00001 2600 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00004 gene=isogroup00001 2634 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00006 gene=isogroup00001 2636 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 9.0E-55 isotig00009 gene=isogroup00001 2524 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 7.0E-59 isotig00010 gene=isogroup00001 2522 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 7.0E-59 isotig00011 gene=isogroup00001 2350 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 6.0E-59 isotig00012 gene=isogroup00001 2352 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 6.0E-59 isotig00013 gene=isogroup00001 1848 hypothetical protein uncultured Spirochaetales bacterium HF0500_06B09 gb|ADI19371.1| 62 140 6.0E-31 isotig00014 gene=isogroup00001 1601 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 5.0E-55 isotig00020 gene=isogroup00001 1599 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 5.0E-55 isotig00015 gene=isogroup00001 2460 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 2.0E-51 isotig00018 gene=isogroup00001 2462 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 2.0E-51 isotig00016 gene=isogroup00001 1850 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 50 136 9.0E-30 isotig00017 gene=isogroup00001 2020 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 6.0E-48 isotig00019 gene=isogroup00001 1635 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 5.0E-55 isotig00023 gene=isogroup00001 1637 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 5.0E-55 isotig00021 gene=isogroup00001 2634 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 2.0E-51 isotig00022 gene=isogroup00001 2632 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 2.0E-51 isotig00024 gene=isogroup00001 2022 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 7.0E-48 isotig00025 gene=isogroup00001 1523 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 4.0E-59 isotig00026 gene=isogroup00001 1525 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 4.0E-59
! ! 229!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bP) Gene name Product Organism NCBI Accesion number %identity Score E-value isotig00031 gene=isogroup00001 1665 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 4.0E-59 isotig00035 gene=isogroup00001 1663 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 125 4.0E-59 isotig00027 gene=isogroup00001 1739 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 6.0E-55 isotig00028 gene=isogroup00001 1741 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 6.0E-55 isotig00029 gene=isogroup00001 1777 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 6.0E-55 isotig00030 gene=isogroup00001 1775 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 60 117 6.0E-55 isotig00032 gene=isogroup00001 1635 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 1.0E-51 isotig00036 gene=isogroup00001 1633 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 1.0E-51 isotig00033 gene=isogroup00001 1023 hypothetical protein FAEPRAM212_00540 Faecalibacterium prausnitzii M21/2 ref|ZP_02090300.1| 67 89.4 1.0E-29 isotig00034 gene=isogroup00001 1021 hypothetical protein uncultured Spirochaetales bacterium HF0500_06B09 gb|ADI19371.1| 62 140 3.0E-31 isotig00037 gene=isogroup00001 1519 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 5.0E-48 isotig00038 gene=isogroup00001 1347 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 50 136 6.0E-30 isotig00043 gene=isogroup00001 1355 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 50 136 6.0E-30 isotig00039 gene=isogroup00001 1161 hypothetical protein uncultured Spirochaetales bacterium HF0500_06B09 gb|ADI19371.1| 62 140 3.0E-31 isotig00040 gene=isogroup00001 1773 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 1.0E-51 isotig00042 gene=isogroup00001 1775 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 1.0E-51 isotig00041 gene=isogroup00001 1163 hypothetical protein FAEPRAM212_00540 Faecalibacterium prausnitzii M21/2 ref|ZP_02090300.1| 67 89.4 1.0E-29 isotig00044 gene=isogroup00001 1527 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 5.0E-48 isotig00045 gene=isogroup00001 1404 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 4.0E-48 isotig00046 gene=isogroup00001 520 hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30046.1| 73 172 2.0E-21 isotig00047 gene=isogroup00001 1232 hypothetical protein BMULJ_05092 Burkholderia multivorans ATCC 17616 ref|YP_001949470.1| 78 184 2.0E-44 isotig00048 gene=isogroup00001 823 cell wall-associated hydrolase Burkholderia multivorans ATCC 17616 ref|YP_001949468.1| 56 232 5.0E-59 isotig00049 gene=isogroup00001 859 cell wall-associated hydrolase Burkholderia multivorans ATCC 17616 ref|YP_001949468.1| 55 222 5.0E-56 isotig00050 gene=isogroup00001 526 conserved hypothetical protein Escherichia sp. 3_2_53FAA ref|ZP_04532941.1| 69 92.4 2.0E-17 isotig00051 gene=isogroup00001 747 conserved hypothetical protein Clostridium perfringens C str. JGS1495 ref|ZP_02865662.1| 69 145 7.0E-52 isotig00052 gene=isogroup00001 660 hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30046.1| 73 70.9 3.0E-25 isotig00053 gene=isogroup00001 735 hypothetical protein PERMA_1142 Persephonella marina EX-H1 ref|YP_002730925.1| 53 72.4 2.0E-28 isotig00054 gene=isogroup00001 403 hypothetical protein BMULJ_05092 Burkholderia multivorans ATCC 17616 ref|YP_001949470.1| 78 184 3.0E-45 isotig00055 gene=isogroup00001 598 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 59 162 3.0E-38 isotig00056 gene=isogroup00002 1492 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 62 197 4.0E-48 isotig00057 gene=isogroup00002 1509 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 56 176 1.0E-41 isotig00058 gene=isogroup00002 1495 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 62 197 4.0E-48 isotig00059 gene=isogroup00002 1512 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 56 176 1.0E-41 isotig00060 gene=isogroup00002 1372 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 56 176 9.0E-42 isotig00061 gene=isogroup00002 1355 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 62 197 4.0E-48 isotig00064 gene=isogroup00002 1352 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_06337182.1| 61 192 1.0E-46 isotig00062 gene=isogroup00002 1400 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp.avenae ATCC 19860 ref|ZP_06213060.1| 74 207 4.0E-51 isotig00063 gene=isogroup00002 1369 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 56 176 9.0E-42 isotig00065 gene=isogroup00002 528 putative lipoprotein Clostridium botulinum C str. Eklund ref|ZP_02863168.1| 69 103 6.0E-26 isotig00066 gene=isogroup00002 1401 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp.avenae ATCC 19860 ref|ZP_06213060.1| 74 207 4.0E-51 isotig00067 gene=isogroup00002 377 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC ref|ZP_01772659.1| 51 77 7.0E-22 isotig00068 gene=isogroup00003 608 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 71 95.9 1.0E-46 isotig00069 gene=isogroup00003 608 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 72 90.5 2.0E-35 isotig00070 gene=isogroup00003 719 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 61 106 1.0E-43 isotig00071 gene=isogroup00003 609 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 61 102 5.0E-41 isotig00072 gene=isogroup00003 606 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 62 108 9.0E-43 isotig00073 gene=isogroup00003 405 pG1 protein Lactobacillus jensenii 269-3 ref|ZP_04645459.1| 67 86.7 5.0E-30 isotig00074 gene=isogroup00003 516 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 63 126 1.0E-27 isotig00075 gene=isogroup00004 762 hypothetical protein LCRIS_00063 Lactobacillus crispatus ST1 ref|YP_003600535.1| 52 125 4.0E-27 isotig00076 gene=isogroup00004 1086 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 52 107 4.0E-36 isotig00078 gene=isogroup00006 582 hypothetical protein LCRIS_00063 Lactobacillus crispatus ST1 ref|YP_003600535.1| 48 89 3.0E-16 isotig00079 gene=isogroup00006 695 conserved hypothetical protein Clostridium sp. 7_2_43FAA ref|ZP_05132927.1| 74 66.2 9.0E-19 isotig00080 gene=isogroup00007 570 hypothetical protein CDSM653_949 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092485.1| 55 92.8 2.0E-17 isotig00093 gene=isogroup00019 487 hypothetical protein ebA4747 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159710.1| 87 248 1.0E-66 isotig00112 gene=isogroup00038 605 hypothetical protein ebA6294 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160626.1| 77 238 4.0E-61 isotig00117 gene=isogroup00043 127 hypothetical protein TRIADDRAFT_51202 Trichoplax adhaerens ref|XP_002118259.1| 90 59.7 4.0E-09 isotig00122 gene=isogroup00048 555 hemerythrin HHE cation binding domain-containing protein Magnetococcus sp. MC-1 ref|YP_865563.1| 37 60.5 4.0E-08 isotig00123 gene=isogroup00049 447 hypothetical protein GYMC52DRAFT_3608 Geobacillus sp. Y412MC52 ref|ZP_04394454.1| 61 75.1 3.0E-12 isotig00145 gene=isogroup00071 768 hypothetical protein CP0987 Chlamydophila pneumoniae AR39 ref|NP_445524.1| 49 84.7 1.0E-26 isotig00146 gene=isogroup00072 612 Hypothetical protein COLAER_01673 Collinsella aerofaciens ATCC 25986 ref|ZP_01772660.1| 64 166 1.0E-39 isotig00147 gene=isogroup00073 558 hypothetical protein RUMTOR_02783 Ruminococcus torques ATCC 27756 ref|ZP_01969198.1| 43 70.5 2.0E-12 isotig00148 gene=isogroup00074 516 conserved hypothetical protein Clostridium botulinum C str. Eklund ref|ZP_02863189.1| 49 76.3 1.0E-12 isotig00150 gene=isogroup00076 533 conserved hypothetical protein Clostridium butyricum E4 str. BoNT E BL5262 ref|ZP_04526499.1| 54 110 7.0E-23 isotig00152 gene=isogroup00078 528 hypothetical protein LCRIS_00063 Lactobacillus crispatus ST1 ref|YP_003600535.1| 49 100 6.0E-20 isotig00153 gene=isogroup00079 675 hypothetical protein EUBDOL_00582 Eubacterium dolichum DSM 3991 ref|ZP_02076790.1| 51 40 7.0E-06 isotig00159 gene=isogroup00085 880 hypothetical protein Tmz1t_2873 Thauera sp. MZ1T ref|YP_002889849.1| 75 267 1.0E-69 isotig00166 gene=isogroup00092 690 conserved hypothetical protein Faecalibacterium prausnitzii A2-165 ref|ZP_05616140.1| 50 154 7.0E-36 isotig00167 gene=isogroup00093 834 hypothetical protein BIFADO_00015 Bifidobacterium adolescentis L2-32 ref|ZP_02027619.1| 80 88.2 4.0E-24 isotig00170 gene=isogroup00096 544 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 65 84.3 3.0E-31 isotig00172 gene=isogroup00098 807 hypothetical protein PERMA_1141 Persephonella marina EX-H1 ref|YP_002730924.1| 58 67.4 4.0E-15 isotig00173 gene=isogroup00099 537 hypothetical protein PTD2_04958 Pseudoalteromonas tunicata D2 ref|ZP_01136111.1| 65 79.3 2.0E-13 isotig00174 gene=isogroup00100 859 hypothetical protein Nhal_0246 Nitrosococcus halophilus Nc4 ref|YP_003525836.1| 33 74.7 1.0E-11 isotig00190 gene=isogroup00116 494 unknown Zea mays gb|ACR38454.1| 61 41.6 4.0E-09 isotig00202 gene=isogroup00128 531 predicted protein Nematostella vectensis ref|XP_001624697.1| 78 66.6 9.0E-21 isotig00205 gene=isogroup00131 482 hypothetical protein ebA5838 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160342.1| 88 254 3.0E-66 contig00101 gene=isogroup00001 560 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 59 99 3.0E-21
! ! 230!
Table H.5. Genes that were transcribed in cells of Cartwright Consolidated culture during growth on benzene.
Section 1. non-assembled (singleton) sequences NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY404I5FXJ 176 prkA protein kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159851.1| 94 68.9 4.00E-13 GMRHGY404H7CXV 323 rubrerythrin Heliobacterium modesticaldum Ice1 ref|YP_001679063.1| 58 102 2.00E-20 GMRHGY404IK2HM 280 flagellar basal-body rod protein FlgF Thermincola sp. JR ref|YP_003641615.1| 82 61.6 3.00E-08 GMRHGY404IRTL6 133 predicted protein Physcomitrella patens subsp. patens ref|XP_001785944.1| 95 45.8 2.00E-06 GMRHGY404HZ07X 337 sensory histidine kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160937.1| 60 91.3 3.00E-24 GMRHGY404JPUEN 335 tar1 Tar1p, tar1 Saccharomyces cerevisiae S288c ref|NP_690845.1| 80 60.1 8.00E-10 GMRHGY404JP0XC 191 iron-sulfur cluster insertion protein ErpA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157522.1| 83 98.2 3.00E-19 GMRHGY404IXYP9 482 phosphoesterase, PA-phosphatase related Methanoculleus marisnigri JR1 ref|YP_001046414.1| 34 48.9 4.00E-06 GMRHGY404IZUXL 396 rhodanese domain-containing protein Polaromonas naphthalenivorans CJ2 ref|YP_983057.1| 48 119 1.00E-25 GMRHGY404I9DII 147 norB nitric-oxide reductase subunit B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157126.1| 81 50.4 7.00E-05 GMRHGY404H0CYJ 455 dihydropteroate synthase DHPS Thermincola sp. JR ref|YP_003639926.1| 80 228 1.00E-58 GMRHGY404H3XTO 335 RND family efflux transporter MFP subunit Roseiflexus castenholzii DSM 13941 ref|YP_001431318.1| 45 63.5 8.00E-09 GMRHGY404ILP5A 397 petA1 ubiquinol-cytochrome c reductase iron-sulfur protein Azoarcus sp. BH72 ref|YP_932464.1| 64 121 3.00E-26 GMRHGY404JVO68 460 TPR repeat-containing transcriptional regulator LuxR Acaryochloris marina MBIC11017 ref|YP_001520336.1| 62 148 4.00E-43 GMRHGY404ICMVX 291 otsA Alpha,alpha-trehalose-phosphate synthase otsA Cupriavidus metallidurans CH34 ref|YP_582512.1| 65 120 4.00E-26 GMRHGY404H3ZFS 485 typA GTP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160670.1| 93 214 1.00E-69 GMRHGY404IIONW 423 cation-transporting P-type ATPase Gemmatimonas aurantiaca T-27 ref|YP_002762415.1| 36 50.1 1.00E-04 GMRHGY404I6L89 344 RpoD DNA-directed RNA polymerase sigma subunit RpoD Achromobacter piechaudii ATCC 43553 ref|ZP_06686961.1| 62 85.9 2.00E-15 GMRHGY404JGSEL 103 sucC succinyl-CoA synthetase subunit beta Azoarcus sp. BH72 ref|YP_934835.1| 93 62 2.00E-08 GMRHGY404IIMZF 265 ATP-dependent metalloprotease FtsH Thiomonas intermedia K12 ref|YP_003643425.1| 78 133 9.00E-30 GMRHGY404H4WAA 437 ATP-dependent chaperone ClpB Sphaerobacter thermophilus DSM 20745 ref|YP_003318702.1| 54 139 1.00E-31 GMRHGY404JN35O 440 transcriptional regulator, LysR family Pantoea sp. At-9b ref|ZP_05730898.1| 51 60.5 2.00E-18 GMRHGY404JQ6XI 140 hitA HIT (histidine triad) family protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157714.1| 83 50.1 9.00E-05 GMRHGY404IJKSL 355 Nitric-oxide reductase Geobacillus sp. C56-T3 ref|YP_003672310.1| 46 67.8 4.00E-10 GMRHGY404IJLH5 437 ATP-dependent chaperone ClpB Sphaerobacter thermophilus DSM 20745 ref|YP_003318702.1| 54 139 1.00E-31 GMRHGY404IKAQ6 476 M48 family peptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159979.1| 56 167 3.00E-40 GMRHGY404I5M28 502 nadB L-aspartate oxidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160135.1| 77 196 1.00E-57 GMRHGY404H3IZI 469 SUF system FeS assembly protein, NifU family Thauera sp. MZ1T ref|YP_002355542.1| 73 210 6.00E-53 GMRHGY404H2J1X 273 ribosomal protein L14 Burkholderia multivorans ATCC 17616 ref|YP_001578451.1| 56 61.6 3.00E-08 GMRHGY404IEYF9 464 amidinotransferase Phenylobacterium zucineum HLK1 ref|YP_002131948.1| 50 103 4.00E-21 GMRHGY404IUMUB 401 two-component system sensor kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160271.1| 74 184 3.00E-45 GMRHGY404JTX71 488 trmU tRNA 5-methylaminomethyl-2-thiouridylate-methyltransferase protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157545.1| 83 174 3.00E-44 GMRHGY404JJQ10 239 RibD riboflavin biosynthesis protein RibD Clostridium ljungdahlii DSM 13528 ref|YP_003780371.1| 47 69.3 2.00E-10 GMRHGY404JLXV4 462 phosphoesterase, PA-phosphatase related Methanoculleus marisnigri JR1 ref|YP_001046414.1| 34 48.9 4.00E-06 GMRHGY404JCFPN 393 nitroreductase Frankia sp. EuI1c ref|ZP_06239177.1| 62 155 1.00E-36 GMRHGY404I3P67 477 sigma-24 (FecI-like) Saccharophagus degradans 2-40 ref|YP_529362.1| 37 73.6 8.00E-12 GMRHGY404I5GDW 462 serine/threonine protein kinase with PASTA sensor(s) Thermincola sp. JR ref|YP_003640537.1| 52 55.1 3.00E-06 GMRHGY404ILWMP 457 MarR family transcriptional regulator Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159244.1| 73 172 1.00E-41 GMRHGY404I43J7 472 cysD sulfate adenylyltransferase subunit 2 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158501.1| 85 271 3.00E-71 GMRHGY404I0Y5A 465 formate dehydrogenase, alpha subunit Thermincola sp. JR ref|YP_003639512.1| 59 144 2.00E-35 GMRHGY404IZKM2 477 NADH-ubiquinone/plastoquinone oxidoreductase chain 3 Thermincola sp. JR ref|YP_003640784.1| 53 129 1.00E-28 GMRHGY404JTG4T 506 rplD 50S ribosomal protein L4 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159184.1| 89 230 3.00E-62 GMRHGY404IAPT3 465 small heat shock protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159480.1| 87 240 4.00E-62 GMRHGY404JEOXO 513 acpD acyl carrier protein phosphodiesterase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159226.1| 86 294 2.00E-78 GMRHGY404H0FXZ 179 DSBA oxidoreductase Nitrosospira multiformis ATCC 25196 ref|YP_413135.1| 60 58.9 2.00E-07 GMRHGY404H0T61 443 TonB-dependent receptor plug Chloroherpeton thalassium ATCC 35110 ref|YP_001995030.1| 45 61.6 3.00E-08 GMRHGY404IQXQL 523 extracellular solute-binding protein, family 1 Paenibacillus sp. oral taxon 786 str. D14 ref|ZP_04852525.1| 65 218 2.00E-55 GMRHGY404H4QK6 493 binding-protein-dependent transport systems inner membrane component Natranaerobius thermophilus JW/NM-WN-LF ref|YP_001916513.1| 65 193 7.00E-48 GMRHGY404I83W4 469 pchA P-hydroxybenzaldehyde dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158801.1| 58 137 4.00E-31 GMRHGY404JFV2Y 414 rumA 23S rRNA 5-methyluridine methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157421.1| 74 182 9.00E-46 GMRHGY404IS4QK 493 membrane-associated Zn-dependent protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160447.1| 74 114 5.00E-44 GMRHGY404JMPVU 453 folE GTP cyclohydrolase I alpha proteobacterium HTCC2255 ref|ZP_01449120.1| 59 157 3.00E-37 GMRHGY404JW5U4 207 beta-lactamase domain protein Dethiobacter alkaliphilus AHT 1 ref|ZP_03728742.1| 63 50.1 1.00E-07 GMRHGY404H17KJ 499 Nucleoside-diphosphate kinase Thauera sp. MZ1T ref|YP_002890365.1| 85 191 3.00E-47 GMRHGY404IUTAR 493 flagellin domain-containing protein Desulfotomaculum reducens MI-1 ref|YP_001113764.1| 83 201 3.00E-50 GMRHGY404ILXKN 96 Xylose isomerase domain protein TIM barrel bacterium Ellin514 ref|ZP_03628383.1| 80 50.8 6.00E-05 GMRHGY404IHCW0 533 purN phosphoribosylglycinamide formyltransferase purN Brachyspira pilosicoli 95/1000 ref|YP_003785687.1| 47 140 9.00E-32 GMRHGY404H2M1H 211 proton-translocating NADH-quinone oxidoreductase, chain M Thermosinus carboxydivorans Nor1 ref|ZP_01666195.1| 46 55.8 2.00E-06 GMRHGY404IVC2H 384 putative Helicase domain protein Ralstonia solanacearum emb|CBJ38305.1| 39 80.1 9.00E-14 GMRHGY404IFYB2 479 rhlE2 ATP-dependent RNA helicase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159102.1| 96 302 1.00E-80 GMRHGY404I7YXK 491 fumA fumarate hydratase Bordetella avium 197N ref|YP_785244.1| 67 157 5.00E-54 GMRHGY404IE03S 540 transposase and inactivated derivatives Pelotomaculum thermopropionicum SI ref|YP_001212945.1| 45 80.9 1.00E-19 GMRHGY404I9U4E 522 Zn dependent peptidase Azoarcus sp. BH72 ref|YP_932269.1| 70 114 6.00E-49 GMRHGY404HYMCF 199 DoxX family protein Bacillus cellulosilyticus DSM 2522 ref|ZP_06362025.1| 74 72.8 1.00E-11 GMRHGY404IBE9I 489 serine/threonine protein kinase with PASTA sensor(s) Thermincola sp. JR ref|YP_003640537.1| 41 35 3.00E-05 GMRHGY404JFNOH 529 SpoOM family protein Thermincola sp. JR ref|YP_003639917.1| 56 171 4.00E-46 GMRHGY404H6TTD 512 phasin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159885.1| 46 130 8.00E-29 GMRHGY404IUM9V 486 ClpB ClpB protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160835.1| 88 224 4.00E-57 GMRHGY404IUWLK 493 aspartate/glutamate/uridylate kinase Roseiflexus castenholzii DSM 13941 ref|YP_001434109.1| 51 82.8 1.00E-14 GMRHGY404JQY4G 497 transporter, hydrophobe/amphiphile efflux-1 (HAE1) family Geobacter lovleyi SZ ref|YP_001953658.1| 80 88.2 3.00E-16 GMRHGY404I0PCS 523 oxygen-independent coproporphyrinogen III oxidase Clostridium botulinum B str. Eklund 17B ref|YP_001885439.1| 51 165 2.00E-39 GMRHGY404JAPPP 281 MucD putative serine protease MucD [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158697.1| 55 52.4 2.00E-05 GMRHGY404II7GD 208 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 81 89.4 4.00E-30 GMRHGY404IRI4U 503 ABC-type branched-chain amino acid transporter Aurantimonas manganoxydans SI85-9A1 ref|ZP_01225788.1| 73 246 5.00E-64 GMRHGY404JV1CI 521 Mg2 transporter protein CorA family protein Clostridium papyrosolvens DSM 2782 ref|ZP_05497991.1| 41 129 1.00E-28
! ! 231!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY404IW9N0 511 translation elongation factor Tu Thauera sp. MZ1T ref|YP_002890312.1| 86 286 5.00E-76 GMRHGY404IW9UX 472 3-phosphoshikimate 1-carboxyvinyltransferase Nitrosococcus oceani ATCC 19707 ref|YP_342240.1| 66 58.2 4.00E-07 GMRHGY404ISBJW 308 hemerythrin HHE cation binding domain-containing protein Magnetococcus sp. MC-1 ref|YP_865563.1| 39 50.4 7.00E-05 GMRHGY404I1PD9 489 short-chain dehydrogenase/reductase SDR Polaromonas sp. JS666 ref|YP_552018.1| 61 132 2.00E-29 GMRHGY404IYC9W 512 sdhC succinate dehydrogenase subunit C Cupriavidus metallidurans CH34 ref|YP_584632.1| 47 91.3 2.00E-27 GMRHGY404JO28I 185 cell wall associated biofilm protein Oceanicola batsensis HTCC2597 ref|ZP_00999898.1| 58 38.1 1.00E-05 GMRHGY404IK909 476 hrcA heat-inducible transcription repressor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159745.1| 94 280 3.00E-74 GMRHGY404I95JY 477 Radical SAM domain protein Chlorobium limicola DSM 245 ref|YP_001943924.1| 53 87 2.00E-20 GMRHGY404JM2C2 508 typA GTP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160670.1| 95 323 5.00E-87 GMRHGY404HZ3LC 427 SpoOM family protein Thermincola sp. JR ref|YP_003639917.1| 50 139 9.00E-32 GMRHGY404INMDH 502 gyrA DNA gyrase subunit A Herminiimonas arsenicoxydans ref|YP_001100824.1| 82 289 7.00E-77 GMRHGY404JE2IA 495 putative peptidase uncultured bacterium dbj|BAG55465.1| 53 93.6 8.00E-18 GMRHGY404ILOOE 414 ATP-dependent Clp protease, ATP-binding subunit clpA Azoarcus sp.BH2 ref|YP_932636.1| 71 97.8 2.00E-22 GMRHGY404H5ZF0 498 putative amino-acid-binding periplasmic (PBP) ABC transporter protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159033.1| 80 183 1.00E-58 GMRHGY404H9IQZ 506 Cystathionine gamma-synthase Sphaerobacter thermophilus DSM 20745 ref|YP_003320275.1| 63 132 2.00E-29 GMRHGY404IVVWA 494 ATPase associated with various cellular activities AAA_5 Anaerococcus prevotii DSM 20548 ref|YP_003152366.1| 29 62 2.00E-08 GMRHGY404H9W8V 487 PrkA putative serine protein kinase Thauera sp. MZ1T ref|YP_002889546.1| 72 242 1.00E-62 GMRHGY404JB3IC 441 transcriptional regulator, LysR family Burkholderia sp. CCGE1001 ref|ZP_06293310.1| 60 70.9 2.00E-25 GMRHGY404I1ETT 482 cobalamin B12-binding domain protein Desulfatibacillum alkenivorans AK-01 ref|YP_002431004.1| 42 90.9 5.00E-17 GMRHGY404JBET0 495 CO-dehydrogenase large chain [Oligotropha carboxidovorans OM5 ref|YP_015605.1| 70 167 4.00E-58 GMRHGY404I46XE 125 PQQ-dependent dehydrogenase, methanol/ethanol family Thauera sp. MZ1T ref|YP_002889793.1| 80 85.9 2.00E-15 GMRHGY404JVYAQ 498 transposase IS66 Alicycliphilus denitrificans BC ref|ZP_07024823.1| 82 166 7.00E-52 GMRHGY404JDXU7 486 Transcriptional regulatory protein LysR family Burkholderia dolosa AUO158 ref|ZP_04945349.1| 72 147 4.00E-34 GMRHGY404IY8C6 479 molecular chaperone GrpE Pelotomaculum thermopropionicum SI ref|YP_001211427.1| 52 141 3.00E-32 GMRHGY404JI4UI 507 (Isocitrate dehydrogenase (NADP(+))) kinase Thiomonas intermedia K12 ref|YP_003644402.1| 70 258 2.00E-67 GMRHGY404IG51L 493 GMC oxidoreductase Burkholderia thailandensis MSMB43 ref|ZP_02461782.1| 63 212 1.00E-53 GMRHGY404JUNC9 210 ahpC peroxiredoxin Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159522.1| 83 94.4 4.00E-18 GMRHGY404I9WTA 460 calcium-transporting ATPase Syntrophus aciditrophicus SB ref|YP_462855.1| 62 154 2.00E-36 GMRHGY404IZIIQ 419 CoA-binding domain protein Geobacter sp. M18 ref|ZP_05309381.1| 63 42.7 6.00E-05 GMRHGY404ID5ZC 525 phosphoribosyl-ATP diphosphatase Thermincola sp. JR ref|YP_003639610.1| 61 177 4.00E-43 GMRHGY404IXEF8 128 rplN 50S ribosomal protein L14 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159193.1| 95 79.3 1.00E-13 GMRHGY404ID8P1 512 rhlE2 ATP-dependent RNA helicase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159102.1| 76 195 2.00E-48 GMRHGY404IDIYA 532 Extracellular ligand-binding receptor Truepera radiovictrix DSM 17093 ref|YP_003704250.1| 69 246 9.00E-64 GMRHGY404IKFWJ 549 cell division initiation protein Pelotomaculum thermopropionicum SI ref|YP_001212374.1| 58 76.6 6.00E-24 GMRHGY404I7NY8 495 typA GTP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160670.1| 90 295 2.00E-78 GMRHGY404JNRNY 489 transcriptional regulator, GntR family with aminotransferase domain Burkholderia sp. H160 ref|ZP_03267685.1| 70 154 3.00E-36 GMRHGY404I2H0V 549 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 65 90.9 7.00E-30 GMRHGY404JN8ED 203 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 90 111 4.00E-23 GMRHGY404IL6RW 539 NirS NirS Methylomonas sp. 16a gb|ADB24711.1| 56 207 5.00E-52 GMRHGY404JENCB 490 ABC-type branched-chain amino acid transport systems, ATPase component Magnetospirillum magneticum AMB-1 ref|YP_422951.1| 84 187 4.00E-46 GMRHGY404ITU9M 511 aspartyl-tRNA synthetase Rubrobacter xylanophilus DSM 9941 ref|YP_644126.1| 48 116 1.00E-36 GMRHGY404JUO77 531 trypsin Propionibacterium acnes SK137 ref|YP_003582497.1| 73 127 3.00E-51 GMRHGY404JJUE0 535 cytochrome c biogenesis protein transmembrane region Alicycliphilus denitrificans BC ref|ZP_07024360.1| 44 130 3.00E-29 GMRHGY404ITW4K 491 ATP-dependent metalloprotease FtsH Chloroflexus aggregans DSM 9485 ref|YP_002463968.1| 64 137 4.00E-31 GMRHGY404IBZN8 497 ATP synthase F0, A subunit Thermincola sp. JR ref|YP_003641626.1| 70 214 4.00E-54 GMRHGY404HYGDF 521 TolB translocation protein TolB [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158517.1| 87 305 1.00E-81 GMRHGY404JR50I 497 putative cytosolic aminopeptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161094.1| 67 103 2.00E-40 GMRHGY404IDLCA 479 carbamoyl phosphate synthase large subunit Desulfotomaculum reducens MI-1 ref|YP_001113570.1| 76 243 6.00E-63 GMRHGY404JI1FT 496 TfoX domain protein Methylotenera mobilis JLW8 ref|YP_003047827.1| 77 122 2.00E-26 GMRHGY404JJAXH 511 sdhC succinate dehydrogenase subunit C Cupriavidus metallidurans CH34 ref|YP_584632.1| 43 91.3 2.00E-21 GMRHGY404IPG9Q 485 type IV pilus response regulator PilH Pseudomonas stutzeri A1501 ref|YP_001174417.1| 100 87.8 9.00E-30 GMRHGY404H2FZP 514 flgI flagellar basal body P-ring protein Azoarcus sp. BH72 ref|YP_934236.1| 61 206 1.00E-51 GMRHGY404JBLVD 499 methyl-accepting chemotaxis sensory transducer Thauera sp. MZ1T ref|YP_002354091.1| 54 163 6.00E-39 GMRHGY404ISFOA 507 acsA acetyl-CoA synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157120.1| 86 207 3.00E-52 GMRHGY404H5DSO 508 isocitrate dehydrogenase, NADP-dependent Anaeromyxobacter sp. Fw109-5 ref|YP_001379372.1| 70 132 6.00E-61 GMRHGY404I1VC4 552 Asparaginase/glutaminase Clostridium carboxidivorans P7 ref|ZP_05391056.1| 52 176 2.00E-44 GMRHGY404I7K76 479 nadA quinolinate synthetase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159361.1| 95 313 6.00E-84 GMRHGY404IZF00 492 CoB--CoM heterodisulfide reductase subunit C Acidithiobacillus caldus ATCC 51756 ref|ZP_05291796.1| 90 214 4.00E-54 GMRHGY404I618M 540 phosphoribosylglycinamide formyltransferase (GART) (GAR transformylase) Candidatus Cloacamonas acidaminovorans ref|YP_001741594.1| 62 133 1.00E-29 GMRHGY404IC5AJ 502 DNA gyrase subunit A Herminiimonas arsenicoxydans ref|YP_001100824.1| 82 289 7.00E-77 GMRHGY404IJQ4O 474 APHP domain-containing protein Cyanothece sp. PCC 7822 ref|YP_003899949.1| 43 109 9.00E-25 GMRHGY404I3JUW 452 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 86 252 1.00E-65 GMRHGY404JF5WC 404 hslU ATP-dependent protease ATP-binding subunit Azoarcus sp. BH72 ref|YP_931928.1| 42 74.3 5.00E-12 GMRHGY404IBV2G 487 YbaK/prolyl-tRNA synthetase associated region Polaromonas sp. JS666 ref|YP_551516.1| 69 209 1.00E-52 GMRHGY404IU0Q0 292 alkylhydroperoxidase like protein, AhpD family Ferroglobus placidus DSM 10642 ref|YP_003436176.1| 55 73.2 1.00E-11 GMRHGY404H5XCR 508 asparagine synthase (glutamine-hydrolyzing) Solibacter usitatus Ellin6076 ref|YP_825868.1| 36 67.4 6.00E-10 GMRHGY404JVXGA 96 phosphoglycerate mutase, 2,3-bisphosphoglycerate- independent Sphaerobacter thermophilus DSM 20745 ref|YP_003319920.1| 79 53.1 1.00E-05 GMRHGY404JJFA5 482 ggt putative sensory box/GGDEF family protein ggt Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158656.1| 40 67.8 4.00E-10 GMRHGY404JNHGS 488 gyrA DNA gyrase subunit A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157516.1| 90 288 1.00E-76 GMRHGY404JP0A5 298 ABC-type branched-chain amino acid transport systems, periplasmic component Pelotomaculum thermopropionicum SI ref|YP_001210627.1| 50 73.2 1.00E-11 GMRHGY404I60RB 442 LysR substrate-binding Methylobacterium extorquens PA1 ref|YP_001637974.1| 42 74.7 4.00E-21 GMRHGY404H0FXV 477 clpP ATP-dependent Clp protease proteolytic subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159854.1| 95 287 4.00E-76 GMRHGY404IHUW1 563 fructose-1,6-bisphosphate aldolase, class II Thermincola sp. JR ref|YP_003641650.1| 81 296 6.00E-79 GMRHGY404H31QS 535 cell division initiation protein Pelotomaculum thermopropionicum SI ref|YP_001212374.1| 35 90.5 6.00E-24 GMRHGY404JURH0 467 RNA polymerase, alpha subunit; RNA polymerase alpha chain family Ralstonia solanacearum PSI07 ref|YP_003751181.1| 90 166 1.00E-39 GMRHGY404H9HU0 489 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 82 275 2.00E-72 GMRHGY404I3BBW 508 norQ nitric oxide reductase chaperone Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157129.1| 96 338 2.00E-91
! ! 232!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY404I6KYF 513 SecY preprotein translocase subunit SecY Azoarcus sp. BH72 ref|YP_934899.1| 76 213 5.00E-54 GMRHGY404IY47O 518 S-layer domain protein Thermincola sp. JR ref|YP_003641604.1| 50 127 4.00E-28 GMRHGY404H9BD3 384 putative toluene tolerance protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159890.1| 64 96.7 8.00E-26 GMRHGY404H6OKF 561 putative multidrug resistance protein Clostridia bacterium enrichment culture clone BF gb|ADJ94000.1| 43 88.6 3.00E-16 GMRHGY404IGMCI 441 flagellin domain-containing protein Desulfotomaculum reducens MI-1 ref|YP_001113764.1| 68 100 4.00E-24 GMRHGY404JB3O4 500 SpoVR family protein Thauera sp. MZ1T ref|YP_002889548.1| 80 288 2.00E-76 GMRHGY404I2CTI 515 GroEL chaperonin GroEL Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157650.1| 96 244 4.00E-63 GMRHGY404H2MPF 369 ATPase Pelotomaculum thermopropionicum SI ref|YP_001210747.1| 42 60.5 7.00E-08 GMRHGY404II124 494 response regulator Pelotomaculum thermopropionicum SI ref|YP_001211753.1| 78 260 5.00E-68 GMRHGY404H1NJ9 496 ffH signal recognition particle protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159492.1| 97 313 4.00E-84 GMRHGY404ITX4M 524 pemT phosphatidylethanolamine N-methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160843.1| 88 199 1.00E-49 GMRHGY404I76T3 496 hflB cell division protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159756.1| 85 266 6.00E-70 GMRHGY404JTVIC 496 nitrile hydratase, alpha subunit Variovorax paradoxus S110 ref|YP_002943581.1| 66 219 1.00E-55 GMRHGY404IS4VK 485 RNA polymerase sigma H (sigma 32) factor Ralstonia solanacearumCFBP2957 ref|YP_003746874.1| 74 230 5.00E-59 GMRHGY404IR0AY 536 Heat shock protein Hsp90-like Clostridium cellulovorans 743B ref|YP_003842049.1| 75 230 6.00E-59 GMRHGY404JR67L 436 branched-chain amino acid ABC transporter Thermomicrobium roseumDSM 5159 ref|YP_002523916.1| 36 43.5 9.00E-05 GMRHGY404ITYAN 481 atoC response regulatory protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157040.1| 78 159 3.00E-38 GMRHGY404I2TYD 521 NirS NirS Methylomonas sp. 16a gb|ADB24711.1| 56 207 4.00E-52 GMRHGY404JBDBW 508 3-dehydroquinate synthase Clostridium papyrosolvens DSM 2782 ref|ZP_05497490.1| 54 184 5.00E-45 GMRHGY404JJUNL 444 LysR substrate-binding Methylobacterium extorquens PA1 ref|YP_001637974.1| 40 58.5 2.00E-16 GMRHGY404H2MFJ 493 int phage-related integrase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157144.1| 80 259 6.00E-68 GMRHGY404IKJS6 492 penicillin-binding protein 2 Thermosediminibacter oceani DSM16646 ref|YP_003825132.1| 37 92.4 2.00E-17 GMRHGY404JI38E 510 transposase Paenibacillus sp. oral taxon 786 str. D14 ref|ZP_04853302.1| 82 291 2.00E-77 GMRHGY404IOT2Z 489 S-layer domain-containing protein Meiothermus ruber DSM 1279 ref|YP_003508754.1| 30 56.2 1.00E-06 GMRHGY404I9443 493 type I restriction-modification system methylation subunit-like Desulfovibrio desulfuricans subsp. desulfuricans str.G20 ref|YP_389915.1| 91 196 8.00E-49 GMRHGY404I5SPV 156 OmpA/MotB domain-containing protein Acidovorax sp. JS42 ref|YP_986700.1| 82 89.4 1.00E-16 GMRHGY404IPCTR 470 3-isopropylmalate dehydrogenase Thermincola sp. JR ref|YP_003639153.1| 86 178 2.00E-43 GMRHGY404I338G 340 ATP synthase F0, A subunit Thermincola sp. JR ref|YP_003641626.1| 67 94 8.00E-27 GMRHGY404IQFGG 494 transport system ATP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159809.1| 86 212 1.00E-58 GMRHGY404JN5TP 515 putative peptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157428.1| 96 195 2.00E-48 GMRHGY404IVOUY 491 ClpB ClpB protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160835.1| 88 268 2.00E-70 GMRHGY404JAV4Q 502 FdxA ferredoxin--NADP(+) reductase Rubrivivax gelatinosus gb|AAW67232.1| 75 137 5.00E-31 GMRHGY404I8YJ0 510 putative branched chain amino acid ABC transporter, ATP-binding protein Alteromonadales bacterium TW-7 ref|ZP_01611909.1| 54 82 2.00E-14 GMRHGY404H1XMG 521 TolB translocation protein TolB [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158517.1| 87 305 1.00E-81 GMRHGY404ITFVL 524 putative nucleotide-binding protein Clostridia bacterium enrichment culture clone BF gb|ADJ93992.1| 87 187 6.00E-46 GMRHGY404IOL87 451 putative ABC transporter, substrate binding protein (oligopeptide) Thermomicrobium roseum DSM 5159 ref|YP_002521643.1| 55 50.1 1.00E-04 GMRHGY404I6UQU 485 ppk polyphosphate kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160756.1| 85 253 6.00E-66 GMRHGY404IVZ9R 496 ompR1 osmolarity response regulator Azoarcus sp. BH72 ref|YP_933213.1| 79 243 8.00E-63 GMRHGY404JFD7U 519 Lysine decarboxylase Thauera sp. MZ1T ref|YP_002355244.1| 88 196 3.00E-77 GMRHGY404IWDPZ 495 IS66 family transposase Burkholderia xenovorans LB400 ref|YP_558605.1| 75 246 7.00E-64 GMRHGY404ILDIE 532 nuoA NADH dehydrogenase I chain A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159765.1| 78 151 4.00E-35 GMRHGY404H9KNC 497 ribosomal protein L13 [Truepera radiovictrix DSM 17093] ref|YP_003704952.1| 72 220 5.00E-56 GMRHGY404IAEGE 521 rimM 16S rRNA processing protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161089.1| 77 257 3.00E-67 GMRHGY404H9YV9 521 TolB translocation protein TolB Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158517.1| 87 305 1.00E-81 GMRHGY404ISJLQ 523 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 90 148 2.00E-34 GMRHGY404IUE4A 498 hybrid cluster protein Thermincola sp. JR ref|YP_003641673.1| 80 273 5.00E-72 GMRHGY404I7RR6 500 tetratricopeptide TPR_4 Leptothrix cholodnii SP-6 ref|YP_001793145.1| 49 157 4.00E-37 GMRHGY404IW7DU 470 putative transcriptional regulatory protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159660.1| 96 313 6.00E-84 GMRHGY404IKEH2 496 Fe-S-cluster redox protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157702.1| 81 212 1.00E-53 GMRHGY404H4CCG 485 phnF transcription regulator protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160854.1| 89 268 1.00E-70 GMRHGY404JBZDK 490 tig trigger factor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159853.1| 87 270 6.00E-71 GMRHGY404IIMI5 535 putative KHG/KDPG aldolase Propionibacterium acnes SK187 ref|ZP_06426082.1| 100 349 7.00E-95 GMRHGY404I9675 505 sensory box sensor histidine kinase/response regulator Myxococcus xanthus DK 1622 ref|YP_632059.1| 50 72.8 1.00E-11 GMRHGY404IE110 229 putative cytosolic aminopeptidase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161094.1| 72 68.2 9.00E-12 GMRHGY404H0SYE 300 biotin/lipoyl attachment Moorella thermoacetica ATCC 39073 ref|YP_430841.1| 63 81.3 4.00E-14 GMRHGY404JVETF 505 iron-sulfur cluster insertion protein ErpA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157522.1| 94 209 1.00E-52 GMRHGY404HZK2T 497 UbiH 2-octaprenyl-6-methoxyphenol hydroxylase oxidoreductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157628.1| 68 186 9.00E-46 GMRHGY404I4OY1 526 PpiA peptidyl-prolyl cis-trans isomerase Laribacter hongkongensis HLHK9 ref|YP_002795231.1| 69 99.4 2.00E-42 GMRHGY404IWEZC 491 Rho transcription termination factor Rho Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159706.1| 86 266 9.00E-70 GMRHGY404IB807 396 helicase domain protein Cyanothece sp. PCC 7425 ref|YP_002482823.1| 76 109 9.00E-41 GMRHGY404IRGAE 488 cysI sulfite reductase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158498.1| 96 317 4.00E-85 GMRHGY404H594L 495 transport system ATP-binding protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159809.1| 86 186 9.00E-53 GMRHGY404JFJLH 492 electron transfer flavoprotein beta-subunit Roseiflexus sp. RS-1 ref|YP_001276628.1| 50 101 8.00E-23 GMRHGY404IQKOH 507 osmY periplasmic or secreted lipoprotein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160066.1| 80 175 2.00E-42 GMRHGY404JPK8S 511 purC phosphoribosylaminoimidazole-succinocarboxamide synthase Rhodospirillum centenum SW ref|YP_002299532.1| 87 127 1.00E-51 GMRHGY404HZZHL 498 gyrA DNA gyrase subunit A Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157516.1| 89 243 4.00E-73 GMRHGY404I4Q8Z 517 Nucleotidyl transferase Thermincola sp. JR ref|YP_003640927.1| 78 268 2.00E-70 GMRHGY404IFZPJ 503 polyphosphate--glucose phosphotransferase Frankia sp. EAN1pec ref|YP_001505612.1| 60 169 1.00E-40 GMRHGY404H1OIM 541 membrane-bound metal-dependent hydrolase Thermincola sp. JR ref|YP_003640916.1| 59 145 4.00E-37 GMRHGY404JMJ11 442 rpoE RNA polymerase sigma factor (sigma24) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159586.1| 55 59.3 2.00E-07 GMRHGY404IYCZE 479 dihydroxyacid dehydratase/phosphogluconate dehydratase Pelotomaculum thermopropionicum SI ref|YP_001211076.1| 74 164 1.00E-50 GMRHGY404JEOBL 509 heat shock protein 90 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159790.1| 80 260 4.00E-68 GMRHGY404HZ9MI 557 nosZ nitrous oxide reductase Hydrogenobacter thermophilus TK-6 ref|YP_003431831.1| 47 129 2.00E-28 GMRHGY404JS2MY 506 strongly similar to 1-deoxy-D-xylulose 5-phosphate synthase Candidatus Kuenenia stuttgartiensis emb|CAJ71099.1| 49 149 9.00E-35 GMRHGY404JKYLC 517 nfeD putative nodulation efficiency protein D Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158506.1| 78 211 2.00E-53 GMRHGY404I0N8T 525 RNA methyltransferase, TrmH family, group 3 Thermincola sp. JR ref|YP_003639061.1| 62 121 3.00E-26 GMRHGY404H6CHP 463 two component transcriptional regulator, LytTR family Geobacillus sp. Y4.1MC1 ref|ZP_05371797.1| 37 79.3 2.00E-18
! ! 233!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY404ITJ7C 502 RNA binding metal dependent phosphohydrolase Thermobaculum terrenum ATCC BAA-798 ref|YP_003323286.1| 64 223 5.00E-57 GMRHGY404JHRCW 494 30S ribosomal protein S1 Achromobacter piechaudii ATCC 43553 ref|ZP_06686585.1| 89 285 1.00E-75 GMRHGY404IMTYF 466 ribosomal protein L13 Truepera radiovictrix DSM 17093 ref|YP_003704952.1| 72 174 4.00E-50 GMRHGY404JGN39 310 OmpA/MotB domain-containing protein Acidovorax sp. JS42 ref|YP_986700.1| 82 89.4 2.00E-21 GMRHGY404H0IGP 496 pantetheine-phosphate adenylyltransferase Dehalogenimonas lykanthroporepellens BL-DC-9 ref|YP_003758763.1| 48 79 1.00E-23 GMRHGY404JVTEZ 514 prkA protein kinase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159851.1| 96 301 3.00E-84 GMRHGY404IQ04R 517 putative glucose/sorbosone dehydrogenase Leptothrix cholodnii SP-6 ref|YP_001792401.1| 77 240 4.00E-62 GMRHGY404H7JYR 522 peptidase M24 Thermincola sp. JR ref|YP_003639679.1| 45 99 3.00E-26 GMRHGY404JET44 255 cell wall-associated hydrolase Streptomyces sp. e14 ref|ZP_06708250.1| 65 101 3.00E-20 GMRHGY404H8AHG 471 Integral membrane protein TerC Thermincola sp. JR ref|YP_003640238.1| 59 175 2.00E-42 GMRHGY404JWHGO 490 DNA gyrase subunit A Janthinobacterium sp. Marseille ref|YP_001354365.1| 86 211 1.00E-67 GMRHGY404JIY6I 479 nirE uroporphyrin-III C-methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157634.1| 67 186 8.00E-46 GMRHGY404IC4K7 477 Acyl dehydratase Magnetospirillum magnetotacticum MS-1 ref|ZP_00055005.2| 62 134 4.00E-30 GMRHGY404IXEFX 500 Methyltransferase type 11 Nitrosococcus halophilus Nc4 ref|YP_003528664.1| 40 112 2.00E-23 GMRHGY404JPTQX 503 LacI family transcription regulator Caulobacter sp. K31 ref|YP_001682050.1| 42 119 1.00E-25 GMRHGY404IMXGZ 518 coxB2 cytochrome-c oxidase subunit II Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159093.1| 80 237 4.00E-61 GMRHGY404JOFH3 141 DNA-directed RNA polymerase, sigmasubunit Cardiobacterium hominis ATCC 15826 ref|ZP_05706205.1| 93 85.5 2.00E-15 GMRHGY404I88NB 435 non-specific protein-tyrosine kinase Roseiflexus sp. RS-1 ref|YP_001278336.1| 60 57 8.00E-09 GMRHGY404IXW9R 455 methyltransferase type 11 Solibacter usitatus Ellin6076 ref|YP_828629.1| 49 51.2 4.00E-05 GMRHGY404IZI5Q 496 sucA 2-oxoglutarate dehydrogenase E1 component Cupriavidus taiwanensis ref|YP_002005876.1| 83 194 3.00E-63 GMRHGY404JCYNH 327 glucan 1,4-alpha-glucosidase Nitrobacter hamburgensis X14 ref|YP_576305.1| 55 60.5 7.00E-08 GMRHGY404I0OZ5 494 zinc-containing alcohol dehydrogenase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158774.1| 93 294 3.00E-78 GMRHGY404IKQVD 478 lipoprotein signal peptide Dechloromonas aromatica RCB ref|YP_287118.1| 44 85.9 2.00E-15 GMRHGY404JPES3 471 MsrA peptide methionine sulfoxide reductase MsrA Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159406.1| 85 144 4.00E-61 GMRHGY404H1CQX 230 ornithine carbamoyltransferase Thermococcus onnurineus NA1 ref|YP_002306817.1| 67 76.6 1.00E-12 GMRHGY404IECVB 370 methyltransferase DNA modification enzyme Candidatus Methanoregula boonei 6A8 ref|YP_001405363.1| 54 94.7 4.00E-24 GMRHGY404IOEFT 349 ugd UDP-glucose 6-dehydrogenase Azoarcus sp. BH72 ref|YP_932580.1| 67 135 2.00E-30 GMRHGY404H8EXK 486 outer membrane protein (porin) Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159426.1| 27 53.5 9.00E-06 GMRHGY404JG8C1 305 aminotransferase AlaT Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159842.1| 94 157 4.00E-37 GMRHGY404JM2TH 196 putative DNA recombination protein, rmuC family Ralstonia solanacearum emb|CBJ36254.1| 72 44.3 1.00E-09 GMRHGY404H01F2 310 hrcA heat-inducible transcription repressor Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159745.1| 93 143 8.00E-33 GMRHGY404IH1NX 519 manB phosphoglucomutase/phosphomannomutase family protein uncultured Chloroflexi bacterium emb|CAI78536.1| 59 117 2.00E-33 GMRHGY404IHA5C 512 MutS2 family protein Chloroflexus aggregans DSM 9485 ref|YP_002463047.1| 51 102 2.00E-28 GMRHGY404JTEYU 544 rplM 50S ribosomal protein L13 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157525.1| 95 268 3.00E-70 GMRHGY404IB05B 527 phrB deoxyribodipyrimidine photolyase Escherichia albertii TW07627 ref|ZP_02903548.1| 70 154 3.00E-42 GMRHGY404I9J5Q 487 fabF beta-ketoacyl-(acyl-carrier-protein) synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160133.1| 80 256 9.00E-67 GMRHGY404I8123 463 etfA electron transfer flavoprotein alpha subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160745.1| 93 122 2.00E-26 GMRHGY404IWI63 476 response regulator receiver protein Sideroxydans lithotrophicus ES-1 ref|YP_003525439.1| 77 127 6.00E-28 GMRHGY404I4G1Z 486 rplN 50S ribosomal protein L14 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159193.1| 97 216 6.00E-55 GMRHGY404JGYGC 259 peptidoglycan-binding LysM Alkaliphilus oremlandii OhILAs ref|YP_001513478.1| 46 84 6.00E-15 GMRHGY404I87QD 445 MFS family major facilitator transporter, nitrate:nitrite antiporter Staphylococcus epidermidis W23144 ref|ZP_04798253.1| 100 158 3.00E-51 GMRHGY404IFI80 459 cyclopropane-fatty-acyl-phospholipid synthase-related protein Clostridium kluyveri DSM 555 ref|YP_001396222.1| 46 97.4 9.00E-35 GMRHGY404IX50G 400 Methyltransferase type 11 Thermincola sp. JR ref|YP_003641244.1| 38 67 2.00E-17 GMRHGY404JB21T 500 mreB rod shape-determining protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158736.1| 93 219 7.00E-56 GMRHGY404IVZE6 494 ABC transporter, substrate binding protein (dipeptide) alpha proteobacterium BAL199 ref|ZP_02192218.1| 44 112 3.00E-26 GMRHGY404I61Y5 421 GroEL chaperonin GroEL Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157650.1| 96 57 4.00E-67 GMRHGY404H5TL5 513 UbiG 3-demethylubiquinone-9 3-methyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157519.1| 77 229 3.00E-61 GMRHGY404H13Q5 308 IscA IscA protein involved in Fe-S cluster synthesis Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160686.1| 88 166 3.00E-40 GMRHGY404IW9TO 453 ribosomal protein S1 Burkholderiales bacterium 1_1_47 ref|ZP_07342851.1| 84 248 2.00E-64 GMRHGY404IVTD7 503 competence protein ComEA helix-hairpin-helix repeat protein Thermosediminibacter oceani DSM 16646 ref|YP_003825162.1| 32 57.4 6.00E-07 GMRHGY404IJORM 484 nitric oxide reductase cytochrome b subunit Geobacillus kaustophilus HTA426 ref|YP_146611.1| 41 117 2.00E-29 GMRHGY404JD8ET 496 bioD cobyrinic acid a,c-diamide synthase:dethiobiotin synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160457.1| 82 270 3.00E-71 GMRHGY404H1LTS 448 potH spermidine/putrescine ABC transporter Agrobacterium radiobacter K84 ref|YP_002545722.1| 61 125 2.00E-27 GMRHGY404I4OD6 404 tpiA triosephosphate isomerase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159763.1| 62 157 6.00E-37 GMRHGY404JVYSF 194 ctpA carboxy-terminal processing protease Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157576.1| 76 57.8 1.00E-15 GMRHGY404JMCF9 449 hemD uroporphyrinogen III synthase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157635.1| 65 80.9 5.00E-14 GMRHGY404JDEM2 419 putative HlyD-like secretion protein Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161054.1| 55 97.4 5.00E-19 GMRHGY404IQB09 482 cytochrome c oxidase cbb3-type, subunit III Dechloromonas aromatica RCB ref|YP_283942.1| 52 171 2.00E-41 GMRHGY404IMA4M 546 glycerol kinase Candidatus Liberibacter americanus gb|ACD87749.1| 84 220 5.00E-56 GMRHGY404IIK53 491 acetyl-CoA acetyltransferase Desulfotomaculum reducens MI-1 ref|YP_001113134.1| 62 192 1.00E-47 GMRHGY404JLMPT 474 bktB beta-ketothiolase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159977.1| 83 254 2.00E-66 GMRHGY404IQIYT 486 gamma-carboxygeranoyl-CoA hydratase Pseudomonas stutzeri A1501 ref|YP_001173693.1| 94 109 3.00E-31 GMRHGY404JBS4L 487 acetyl-CoA acetyltransferase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158306.1| 87 146 8.00E-40 GMRHGY404I2D1U 505 putative acyl-CoA dehydrogenase Clostridia bacterium enrichment culture clone BF gb|ADJ93965.1| 89 186 1.00E-61 GMRHGY404JAV7L 370 rhlE1 ATP-dependent RNA helicase Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159040.1| 50 62.8 4.00E-09 GMRHGY404IX0X1 423 IscR iron-sulphur cluster assembly transcription factor IscR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160690.1| 74 202 2.00E-50 GMRHGY404H15DE 421 IscR iron-sulphur cluster assembly transcription factor IscR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160690.1| 94 258 2.00E-67 GMRHGY404JPV1D 466 korC 2-ketoglutarate: NADP oxidoreductase, gamma subunit Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159622.1| 89 291 2.00E-77 GMRHGY404ID1L7 467 IscR iron-sulphur cluster assembly transcription factor IscR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160690.1| 94 262 9.00E-69 GMRHGY404JUVRU 472 IscR iron-sulphur cluster assembly transcription factor IscR Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160690.1| 88 176 4.00E-43 GMRHGY404JO336 483 korB 2-oxoglutarate ferredoxin oxidoreductase subunit beta Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159624.1| 90 141 4.00E-60 GMRHGY404JTRG1 441 activator of 2-hydroxyglutaryl-CoA dehydratase Pelotomaculum thermopropionicum SI ref|YP_001211118.1| 56 105 4.00E-25 GMRHGY404IRMB3 507 putative ABC transporter substrate binding protein Thermomicrobium roseum DSM 5159 ref|YP_002522602.1| 39 137 5.00E-31 GMRHGY404H7EKU 542 FAD-dependent pyridine nucleotide-disulphide oxidoreductase Syntrophobacter fumaroxidans MPOB ref|YP_845438.1| 56 73.2 1.00E-11 GMRHGY404JR6NR 216 hypothetical protein uncultured Rhizobiales bacterium HF4000_32B18 gb|ADI18733.1| 73 89.4 1.00E-16 GMRHGY404IKFI2 426 hypothetical protein BACCOP_03323 Bacteroides coprocola DSM 17136 ref|ZP_03011416.1| 43 83.2 7.00E-17
! ! 234!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY404IW43M 250 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 61 48.1 3.00E-10 GMRHGY404H27IT 484 hypothetical protein COLSTE_02537 Collinsella stercoris DSM 13279 ref|ZP_03298598.1| 48 61.6 3.00E-08 GMRHGY404H3KR5 322 hypothetical protein DESPIG_03156 Desulfovibrio piger ATCC 29098 ref|ZP_03313211.1| 82 94.7 4.00E-22 GMRHGY404H991T 464 hypothetical protein Rleg2_6086 Rhizobium leguminosarum bv. trifolii WSM2304 ref|YP_002283868.1| 52 89 2.00E-16 GMRHGY404I69YX 422 hypothetical protein mma_0219 Janthinobacterium sp. Marseille ref|YP_001351909.1| 48 105 3.00E-21 GMRHGY404IJZOX 470 protein of unknown function DUF255 Conexibacter woesei DSM 14684 ref|YP_003396021.1| 52 159 9.00E-38 GMRHGY404JMEK2 393 hypothetical protein uncultured delta proteobacterium HF4000_08N17 gb|ADI18416.1| 45 52.4 9.00E-08 GMRHGY404IJBT1 455 conserved hypothetical protein Flavobacteria bacterium MS024-3C ref|ZP_03700455.1| 71 198 2.00E-49 GMRHGY404H9HLF 542 HutP family protein Thermincola sp. JR ref|YP_003640362.1| 87 150 4.00E-49 GMRHGY404IP0KS 428 hypothetical protein ML5DRAFT_6290 Micromonospora sp. L5 ref|ZP_06402403.1| 33 39.3 9.00E-05 GMRHGY404JBLJX 439 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 67 170 6.00E-41 GMRHGY404JNRRJ 381 predicted protein Populus trichocarpa ref|XP_002298568.1| 58 101 3.00E-20 GMRHGY404I8XGW 506 conserved hypothetical protein Clostridium thermocellum DSM 2360 ref|ZP_05428580.1| 57 130 3.00E-45 GMRHGY404I6LTD 203 hypothetical protein ebB218 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160534.1| 71 95.9 2.00E-18 GMRHGY404JFXSF 535 hypothetical protein Mvan_4728 Mycobacterium vanbaalenii PYR-1 ref|YP_955508.1| 52 147 7.00E-44 GMRHGY404JPTVT 488 3D domain protein Thermincola sp. JR ref|YP_003638896.1| 35 82 2.00E-14 GMRHGY404H65CL 394 conserved hypothetical protein Mollicutes bacterium D7 ref|ZP_04563066.1| 54 112 2.00E-23 GMRHGY404IK7IY 423 conserved hypothetical protein Clostridium thermocellum DSM 2360 ref|ZP_05428293.1| 50 44.7 1.00E-09 GMRHGY404JMYYW 507 BNR repeat-containing glycosyl hydrolase Candidatus Ruthia magnifica str. Cm ref|YP_903504.1| 34 80.5 7.00E-14 GMRHGY404IGFT6 486 hypothetical protein Mmc1_2522 Magnetococcus sp. MC-1 ref|YP_866428.1| 59 62.8 1.00E-08 GMRHGY404JE1FT 470 PREDICTED: similar to DNA-dependent protein kinase catalytic subunit Gallus gallus ref|XP_001236146.1| 62 84.3 8.00E-31 GMRHGY404IITXR 510 hypothetical protein Cagg_2302 Chloroflexus aggregans DSM 9485 ref|YP_002463617.1| 25 62.4 8.00E-10 GMRHGY404HZRKE 463 hypothetical protein Ctha_1406 Chloroherpeton thalassium ATCC 35110 ref|YP_001996316.1| 30 57 8.00E-07 GMRHGY404H0QOQ 496 hypothetical protein uncultured Verrucomicrobiales bacterium HF0200_39L05 gb|ADI18134.1| 61 95.9 2.00E-18 GMRHGY404IMC29 515 hypothetical protein ebA3069 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158756.1| 86 99.8 8.00E-38 GMRHGY404I32VT 473 hypothetical protein Tbd_2543 Thiobacillus denitrificans ATCC 25259 ref|YP_316301.1| 91 194 2.00E-62 GMRHGY404HYMCF 199 DoxX family protein Bacillus cellulosilyticus DSM 2522 ref|ZP_06362025.1| 74 72.8 1.00E-11 GMRHGY404JJG9K 523 hypothetical protein Cagg_2302 Chloroflexus aggregans DSM 9485 ref|YP_002463617.1| 25 75.9 2.00E-12 GMRHGY404IDJNH 519 hypothetical protein LCRIS_00063 Lactobacillus crispatus ST1 ref|YP_003600535.1| 48 103 1.00E-20 GMRHGY404IIP1D 521 hypothetical protein ebA5153 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159955.1| 66 84.3 5.00E-15 GMRHGY404JU31I 97 conserved hypothetical protein Coxiella burnetii RSA 334 ref|ZP_02220074.1| 86 52 2.00E-05 GMRHGY404I714B 403 hypothetical protein ebA4962 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159848.1| 71 129 1.00E-28 GMRHGY404H0J83 393 hypothetical protein PPA2220 Propionibacterium acnes KPA171202 ref|YP_056882.1| 59 134 5.00E-30 GMRHGY404JBK1X 525 conserved hypothetical protein Microscilla marina ATCC 23134 ref|ZP_01691550.1| 29 79 2.00E-13 GMRHGY404IYLCD 436 hypothetical protein GYMC52DRAFT_3608 Geobacillus sp. Y412MC52 ref|ZP_04394454.1| 37 58.9 2.00E-07 GMRHGY404IPH5C 466 oxidoreductase family protein Clostridium hathewayi DSM 13479 ref|ZP_06115466.1| 42 117 5.00E-25 GMRHGY404JM5Q6 499 hypothetical protein uncultured Acidobacteria bacterium HF4000_26D02 gb|ADI18657.1| 66 71.6 2.00E-26 GMRHGY404H2L7R 505 Conserved hypothetical protein Ochrobactrum intermedium LMG 3301 ref|ZP_04682983.1| 50 90.5 7.00E-17 GMRHGY404JXMKY 497 hypothetical protein LCRIS_00063 Lactobacillus crispatus ST1 ref|YP_003600535.1| 53 104 4.00E-22 GMRHGY404IJA93 510 conserved hypothetical protein Afipia sp. 1NLS2 ref|ZP_07027716.1| 62 219 1.00E-55 GMRHGY404IO7NQ 243 hypothetical protein uncultured Rhizobiales bacterium HF4000_32B18 gb|ADI18733.1| 65 35.8 4.00E-06 GMRHGY404I58JM 500 putative prepilin-like protein Deinococcus deserti VCD115 ref|YP_002785002.1| 40 51.2 4.00E-05 GMRHGY404JSVG1 380 conserved hypothetical protein Streptococcus sp. 2_1_36FAA ref|ZP_06061528.1| 60 92.4 2.00E-17 GMRHGY404JE70W 549 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 65 90.9 7.00E-30 GMRHGY404JF6QD 528 hypothetical protein TherJR_0201 Thermincola sp. JR ref|YP_003638990.1| 45 51.2 5.00E-05 GMRHGY404JFVKU 206 hypothetical protein EDWATA_04070 Edwardsiella tarda ATCC 23685 ref|ZP_06716716.1| 56 59.3 2.00E-07 GMRHGY404H20IM 527 hypothetical protein PERMA_1142 Persephonella marina EX-H1 ref|YP_002730925.1| 53 125 2.00E-27 GMRHGY404IM680 540 hypothetical protein ML5DRAFT_6290 Micromonospora sp. L5 ref|ZP_06402403.1| 43 102 3.00E-20 GMRHGY404JIP9B 451 hypothetical protein uncultured delta proteobacterium HF0130_19C20 gb|ADI17622.1| 31 43.5 6.00E-05 GMRHGY404H9PZ1 521 hypothetical protein VOLCADRAFT_59982 Volvox carteri f. nagariensis ref|XP_002950234.1| 32 58.2 7.00E-09 GMRHGY404I5ZZE 174 conserved hypothetical protein Brucella suis bv. 4 str. 40 ref|ZP_05839403.1| 80 79.3 1.00E-13 GMRHGY404H8AK8 149 hypothetical protein CLOBAR_02295 Clostridium bartlettii DSM 16795 ref|ZP_02212678.1| 66 55.5 2.00E-06 GMRHGY404ING1L 539 putative senescence-associated protein Pisum sativum dbj|BAB33421.1| 86 139 5.00E-39 GMRHGY404JR2QF 544 hypothetical protein uncultured delta proteobacterium HF0500_03A04 gb|ADI19336.1| 66 70.5 2.00E-17 GMRHGY404IZF7Z 497 hypothetical protein ebA3370 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158930.1| 80 114 5.00E-24 GMRHGY404I31L1 484 hypothetical protein TcasGA2_TC016328 Tribolium castaneum gb|EFA13105.1| 38 47 4.00E-10 GMRHGY404JTZZ2 534 hypothetical protein LMOh7858_2508 Listeria monocytogenes str. 4b H7858 ref|ZP_00230261.1| 66 43.9 9.00E-08 GMRHGY404H3ZS0 499 hypothetical protein PM8797T_15596 Planctomyces maris DSM 8797 ref|ZP_01856128.1| 58 85.9 2.00E-15 GMRHGY404JPKRR 473 hypothetical protein LMED105_11115 Limnobacter sp. MED105 ref|ZP_01916410.1| 60 195 2.00E-48 GMRHGY404IUZ4B 560 AE001886_6 hypothetical protein DR_0254 Deinococcus radiodurans R1 gb|AAF09840.1| 39 51.2 4.00E-10 GMRHGY404IFKJS 585 conserved hypothetical protein Legionella longbeachae D-4968 ref|ZP_06187877.1| 40 69.3 2.00E-10 GMRHGY404JE6H8 282 conserved hypothetical protein Clostridium botulinum C str. Eklund ref|ZP_02863189.1| 52 64.3 5.00E-09 GMRHGY404IBIZB 513 hypothetical protein Tmz1t_2873 Thauera sp. MZ1T ref|YP_002889849.1| 75 240 5.00E-62 GMRHGY404IS98D 495 hypothetical protein Caur_3465 Chloroflexus aurantiacus J-10-fl ref|YP_001637039.1| 49 150 5.00E-35 GMRHGY404JT8TM 532 S-layer domain protein Desulfotomaculum acetoxidans DSM 771 ref|YP_003192837.1| 43 126 1.00E-27 GMRHGY404IYXD5 556 hypothetical protein LA_3533 Leptospira interrogans serovar Lai str. 56601 ref|NP_713713.1| 42 63.9 1.00E-16 GMRHGY404IC82F 530 hypothetical protein Daro_2884 Dechloromonas aromatica RCB ref|YP_286084.1| 59 110 9.00E-23 GMRHGY404I04FP 497 diacylglycerol kinase catalytic region Ktedonobacter racemifer DSM 44963 ref|ZP_06967118.1| 28 63.2 1.00E-08 GMRHGY404IXNQN 502 hypothetical protein GobsU_24171 Gemmata obscuriglobus UQM 2246 ref|ZP_02734922.1| 34 60.1 9.00E-08 GMRHGY404IXSXK 508 Cna B domain-containing protein Roseiflexus sp. RS-1 ref|YP_001276083.1| 50 121 3.00E-26 GMRHGY404JUCNI 503 hypothetical protein CLJU_c23640 Clostridium ljungdahlii DSM 13528 ref|YP_003780524.1| 33 60.5 2.00E-14 GMRHGY404JGEDE 218 hypothetical protein ebA6294 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_160626.1| 71 93.6 7.00E-18 GMRHGY404JL9YJ 477 hypothetical protein hCG2031198 Homo sapiens gb|EAW48781.1| 73 102 9.00E-26 GMRHGY404I77HM 502 hypothetical protein C1336_000600005 Campylobacter jejuni subsp. jejuni 1336 ref|ZP_06374539.1| 85 76.6 1.00E-12 GMRHGY404JWZV9 515 hypothetical protein GK2137 Geobacillus kaustophilus HTA426 ref|YP_147990.1| 63 78.6 1.00E-36 GMRHGY404I749M 407 hypothetical protein Nham_3119 Nitrobacter hamburgensis X14 ref|YP_578315.1| 48 112 2.00E-23 GMRHGY404IX9M3 134 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 71 57 8.00E-07
! ! 235!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY404IKDNQ 468 hypothetical protein azo3671 Azoarcus sp. BH72 ref|YP_935173.1| 71 126 8.00E-28 GMRHGY404INDXV 204 hypothetical protein uncultured Verrucomicrobiales bacterium HF0200_39L05 gb|ADI18134.1| 66 68.6 3.00E-10 GMRHGY404JNTWL 430 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 57 76.6 1.00E-12 GMRHGY404ICHPB 423 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 43 87 7.00E-16 GMRHGY404IWZTN 539 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 62 82.4 8.00E-30 GMRHGY404JQUQS 243 hypothetical protein C1336_000600005 Campylobacter jejuni subsp. jejuni 1336 ref|ZP_06374539.1| 78 73.9 6.00E-12 GMRHGY404JDF9P 549 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31922.1| 77 121 5.00E-33 GMRHGY404IO61V 243 LOW QUALITY PROTEIN: conserved hypothetical protein Streptomyces ghanaensis ATCC 14672 ref|ZP_06580272.1| 62 77 7.00E-13 GMRHGY404H3EKZ 551 hypothetical protein EUBVEN_00517 Eubacterium ventriosum ATCC 27560 ref|ZP_02025274.1| 49 59.3 2.00E-07 GMRHGY404IF3CD 268 conserved hypothetical protein Clostridium thermocellum JW20 ref|ZP_06250441.1| 48 53.9 7.00E-06 GMRHGY404JBC77 271 hypothetical protein uncultured delta proteobacterium HF0200_39N20 gb|ADI18196.1| 47 56.2 1.00E-06 GMRHGY404JUUTI 552 conserved hypothetical protein Legionella longbeachae D-4968 ref|ZP_06187877.1| 53 122 2.00E-26 GMRHGY404I965C 530 hypothetical protein Dtox_1610 Desulfotomaculum acetoxidans DSM 771 ref|YP_003191091.1| 58 136 9.00E-31 GMRHGY404JA1UM 561 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 60 83.2 1.00E-15 GMRHGY404ILABY 538 hypothetical protein DSY0811 Desulfitobacterium hafniense Y51 ref|YP_517044.1| 34 75.9 2.00E-12 GMRHGY404I6FSF 502 protein of unknown function DUF124 Geobacter lovleyi SZ ref|YP_001951058.1| 74 185 1.00E-45 GMRHGY404H77HY 505 hypothetical protein Tmz1t_0937 Thauera sp. MZ1T ref|YP_002354599.1| 94 284 3.00E-75 GMRHGY404JUGK7 430 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 40 62.8 1.00E-08 GMRHGY404IHKOD 522 hypothetical protein Flammeovirga yaeyamensis gb|ACA05077.1| 59 102 2.00E-20 GMRHGY404JLP81 525 protein of unknown function DUF853, NPT hydrolase putative Mycobacterium sp. JLS ref|YP_001071912.1| 68 238 2.00E-61 GMRHGY404HZ2PO 314 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 69 78.2 3.00E-14 GMRHGY404IHNRV 554 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 64 106 1.00E-34 GMRHGY404JM0ND 221 hypothetical protein ebA4104 Aromatoleum aromaticum EbN1 ref|YP_159350.1| 90 57.4 2.00E-08 GMRHGY404I2D8Q 507 hypothetical protein StAA4_38276 Streptomyces sp. AA4 ref|ZP_05484038.1| 55 73.6 9.00E-12 GMRHGY404IPP88 180 hypothetical protein CLOLEP_01442 Clostridium leptum DSM 753 ref|ZP_02079992.1| 57 55.5 2.00E-06 GMRHGY404IBMJM 317 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 67 48.5 5.00E-12 GMRHGY404H4CIV 500 conserved hypothetical protein Microscilla marina ATCC 23134 ref|ZP_01691550.1| 29 70.5 7.00E-11 GMRHGY404HZQF0 484 hypothetical protein Veis_3070 Verminephrobacter eiseniae EF01-2 ref|YP_997821.1| 63 142 1.00E-32 GMRHGY404I3BSO 101 hypothetical protein HM1_3150 Heliobacterium modesticaldum Ice1 ref|YP_001680919.1| 82 54.3 5.00E-06 GMRHGY404JG19E 456 hypothetical protein TherJR_2599 Thermincola sp. JR ref|YP_003641337.1| 36 102 2.00E-20 GMRHGY404H2KRG 514 hypothetical protein NB231_14818 Nitrococcus mobilis Nb-231 ref|ZP_01125619.1| 72 211 2.00E-53 GMRHGY404IUMAZ 451 hypothetical protein COLSTE_02537 Collinsella stercoris DSM 13279 ref|ZP_03298598.1| 58 84 6.00E-15 GMRHGY404I3L8G 218 hypothetical protein uncultured Rhizobiales bacterium HF4000_32B18 gb|ADI18733.1| 71 81.3 4.00E-14 GMRHGY404IFG6L 512 hypothetical protein sce8271 Sorangium cellulosum 'So ce 56' ref|YP_001618921.1| 29 79 2.00E-13 GMRHGY404JFW1J 445 hypothetical protein GYMC52DRAFT_3608 Geobacillus sp. Y412MC52 ref|ZP_04394454.1| 36 61.6 3.00E-08 GMRHGY404IDMAO 418 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 41 84 6.00E-15 GMRHGY404JFDBC 480 hypothetical protein ebA639 Aromatoleum aromaticum EbN1 ref|YP_157355.1| 95 162 1.00E-38 GMRHGY404H0PLE 551 hypothetical protein CIT292_08808 Citrobacter youngae ATCC 29220 ref|ZP_06571240.1| 44 83.6 1.00E-14 GMRHGY404H54PQ 319 hypothetical protein BthaT_07231 Burkholderia thailandensis TXDOH ref|ZP_02370785.1| 43 56.6 3.00E-07 GMRHGY404IYD6L 434 AE001886_6 hypothetical protein DR_0254 Deinococcus radiodurans R1 gb|AAF09840.1| 63 40.8 3.00E-07 GMRHGY404I4BDL 364 hypothetical protein NEMVEDRAFT_v1g155353 Nematostella vectensis ref|XP_001618200.1| 43 71.2 4.00E-11 GMRHGY404IRZ9C 490 hypothetical protein DSY3844 Desulfitobacterium hafniense Y51 ref|YP_520077.1| 81 220 1.00E-59 GMRHGY404H6IKT 474 hypothetical protein ebA7058 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_161030.1| 57 98.2 3.00E-19 GMRHGY404IY2I7 511 hypothetical protein ebA4963 [Aromatoleum aromaticum EbN1] Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_159849.1| 90 205 7.00E-75 GMRHGY404JB35S 504 hypothetical protein Tmz1t_0937 Thauera sp. MZ1T ref|YP_002354599.1| 98 261 3.00E-73 GMRHGY404JSRQ0 517 hypothetical protein ebA2092 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158181.1| 56 167 4.00E-40 GMRHGY404IVNQ2 467 protein of unknown function DUF224 cysteine-rich region domain protein Thioalkalivibrio sp. K90mix ref|YP_003459884.1| 81 213 7.00E-54 GMRHGY404JCI6S 472 hypothetical protein Clostridia bacterium enrichment culture clone BF gb|ADJ93989.1| 98 137 2.00E-37 GMRHGY404JFVXH 106 hypothetical protein CDSM653_974 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092508.1| 92 61.6 3.00E-08 GMRHGY404HZZKP 324 hypothetical protein ANACOL_00227 Anaerotruncus colihominis DSM 17241 ref|ZP_02440963.1| 69 115 2.00E-24 GMRHGY404IJ1LR 502 hypothetical protein BACOVA_00321 Bacteroides ovatus ATCC 8483 ref|ZP_02063376.1| 67 84.7 8.00E-24 GMRHGY404IF7FH 513 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 62 137 5.00E-31 GMRHGY404JT4R5 335 conserved hypothetical protein Vibrio mimicus VM603 ref|ZP_05720880.1| 98 166 9.00E-40 GMRHGY404IHQCV 439 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 63 67.4 1.00E-18 GMRHGY404H3OL7 337 conserved hypothetical protein Vibrio mimicus VM603 ref|ZP_05720880.1| 81 165 2.00E-39 GMRHGY404IG367 475 conserved hypothetical protein Faecalibacterium prausnitzii A2-165 ref|ZP_05616140.1| 50 70.1 1.00E-21 GMRHGY404H4C66 511 hypothetical protein HM1_3148 Heliobacterium modesticaldum Ice1 ref|YP_001680917.1| 63 132 1.00E-29 GMRHGY404H81F5 555 conserved hypothetical protein Clostridium perfringens C str. JGS1495 ref|ZP_02865662.1| 62 122 3.00E-49 GMRHGY404JH9LS 436 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 48 107 4.00E-22 GMRHGY404IHR97 513 hypothetical protein Flammeovirga yaeyamensis gb|ACA05077.1| 74 64.7 6.00E-17 GMRHGY404JLUCS 533 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 67 73.2 1.00E-17 GMRHGY404I6BBP 323 hypothetical protein SSAG_05517 Streptomyces sp. Mg1 ref|ZP_05001319.1| 68 47.8 5.00E-11 GMRHGY404JCP6Z 549 hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30046.1| 63 112 4.00E-28 GMRHGY404JK5K3 510 conserved hypothetical protein Aurantimonas manganoxydans SI85-9A1 ref|ZP_01227369.1| 76 139 4.00E-48 GMRHGY404JBEFZ 495 conserved hypothetical protein Brucella ceti M13/05/1 ref|ZP_05934035.1| 86 81.6 2.00E-29 GMRHGY404ILK6Z 496 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 67 73.2 4.00E-18 GMRHGY404JM36B 324 hypothetical protein ANACOL_00227 Anaerotruncus colihominis DSM 17241 ref|ZP_02440963.1| 69 115 2.00E-24 GMRHGY404IAR0P 560 conserved hypothetical protein Listeria monocytogenes J2818 ref|ZP_06684332.1| 59 58.9 1.00E-11 GMRHGY404IOZ2I 472 hypothetical protein HMPREF9553_00243 Escherichia coli MS 200-1 ref|ZP_07172160.1| 82 96.7 9.00E-21 GMRHGY404JVTMJ 554 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 63 161 2.00E-40 GMRHGY404I2VI9 585 conserved hypothetical protein Escherichia sp. 3_2_53FAA ref|ZP_04532941.1| 73 85.1 4.00E-16 GMRHGY404JGILM 373 hypothetical protein uncultured Verrucomicrobiales bacterium HF0010_05E02 gb|ADI16721.1| 51 83.6 8.00E-15 GMRHGY404JHRDR 547 hypothetical protein BMULJ_05092 Burkholderia multivorans ATCC 17616 ref|YP_001949470.1| 70 114 4.00E-24 GMRHGY404JBBNZ 576 Conserved protein Lactobacillus rhamnosus GG emb|CAR86202.1| 55 74.7 2.00E-14 GMRHGY404INQVF 491 hypothetical protein ANACAC_02713 Anaerostipes caccae DSM 14662 ref|ZP_02420107.1| 73 103 2.00E-26 GMRHGY404JG5T9 530 ORF58e Pinus koraiensis ref|YP_001152205.1| 78 100 1.00E-19 GMRHGY404JOYBF 232 LOW QUALITY PROTEIN: conserved hypothetical protein Brucella suis bv. 3 str. 686 ref|ZP_05997968.1| 95 47 6.00E-10
! ! 236!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value GMRHGY404IYZ7H 397 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 46 82.8 1.00E-19 GMRHGY404I420N 530 conserved hypothetical protein Brucella ceti M13/05/1 ref|ZP_05934035.1| 77 126 2.00E-37 GMRHGY404JDS57 491 conserved hypothetical protein Vibrio parahaemolyticus K5030 ref|ZP_07663463.1| 85 67 2.00E-20 GMRHGY404H06XU 331 hypothetical protein FAEPRAM212_00169 Faecalibacterium prausnitzii M21/2 ref|ZP_02089938.1| 56 103 1.00E-20 GMRHGY404H6IYC 434 conserved hypothetical protein Legionella longbeachae D-4968 ref|ZP_06187877.1| 54 57.4 4.00E-17 GMRHGY404JEH2K 451 unknown protein Streptococcus suis 98HAH33 gb|ABP91180.1| 70 70.9 9.00E-26 GMRHGY404H3BC4 561 hypothetical protein CLOLEP_01442 Clostridium leptum DSM 753 ref|ZP_02079992.1| 62 112 8.00E-28 GMRHGY404IF13K 546 hypothetical protein CLOSPI_01140 Clostridium spiroforme DSM 1552 ref|ZP_02867310.1| 69 110 9.00E-30 GMRHGY404H0III 537 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 76 142 5.00E-42 GMRHGY404ITEMJ 532 hypothetical protein CLOLEP_01442 Clostridium leptum DSM 753 ref|ZP_02079992.1| 64 114 2.00E-27 GMRHGY404JBXIH 552 hypothetical protein CLOBAR_02295 Clostridium bartlettii DSM 16795 ref|ZP_02212678.1| 89 170 9.00E-41 GMRHGY404IAI25 526 hypothetical protein CP0987 Chlamydophila pneumoniae AR39 ref|NP_445524.1| 60 102 3.00E-28 GMRHGY404I9WDW 302 hypothetical protein RUMOBE_01295 Ruminococcus obeum ATCC 29174 ref|ZP_01963577.1| 70 129 1.00E-28 GMRHGY404INE8R 511 AE002057_8 hypothetical protein DR_2252 Deinococcus radiodurans R1 gb|AAF11800.1| 55 90.5 7.00E-17 GMRHGY404IDDYG 532 hypothetical protein CTC00065 Clostridium tetani E88 ref|NP_780783.1| 72 144 4.00E-33 GMRHGY404JIS19 285 LOW QUALITY PROTEIN: conserved hypothetical protein Streptomyces ghanaensis ATCC 14672 ref|ZP_06580272.1| 53 89.7 1.00E-16 GMRHGY404JUAD5 479 unknown protein Streptococcus suis 98HAH33 gb|ABP91180.1| 51 107 4.00E-22 GMRHGY404JVCL9 585 hypothetical protein CLCAR_4307 Clostridium carboxidivorans P7 ref|ZP_06857157.1| 60 70.1 2.00E-33 GMRHGY404H7B1M 499 hypothetical protein CDSM653_949 Carboxydibrachium pacificum DSM 12653 ref|ZP_05092485.1| 58 151 3.00E-35 GMRHGY404I6P9F 539 conserved hypothetical protein Lactobacillus jensenii 208-1 ref|ZP_06337182.1| 61 134 4.00E-39 GMRHGY404JMD7T 560 hypothetical protein uncultured Desulfobacterales bacterium HF0200_07G10 gb|ADI17934.1| 55 47.4 1.00E-09 GMRHGY404IDO8Q 572 hypothetical protein uncultured Spirochaetales bacterium HF0500_06B09 gb|ADI19371.1| 55 116 1.00E-24 GMRHGY404I6XKY 519 conserved hypothetical protein Clostridium botulinum D str. 1873 ref|ZP_04861978.1| 61 174 4.00E-42 GMRHGY404H7ZAB 508 hypothetical protein ANACOL_02699 Anaerotruncus colihominis DSM 17241 ref|ZP_02443386.1| 63 165 2.00E-39 GMRHGY404I0R8P 98 Cell wall-associated hydrolase Roseobacter sp. AzwK-3b ref|ZP_01900972.1| 84 51.6 3.00E-05 GMRHGY404I3TGP 426 hypothetical protein uncultured delta proteobacterium HF0200_19J16 gb|ADI18019.1| 52 57.8 1.00E-13 GMRHGY404H5MGO 550 leucine rich protein Arachis hypogaea gb|ABH09321.1| 64 72 5.00E-16 GMRHGY404H60QO 508 hypothetical protein, NA Lawsonia intracellularis PHE/MN1-00 emb|CAJ55201.1| 43 52 2.00E-07 GMRHGY404H2UQI 507 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 48 119 1.00E-25 GMRHGY404JGUSV 325 leucine rich protein Arachis hypogaea gb|ABH09321.1| 83 63.5 3.00E-16 GMRHGY404IN3GV 337 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 80 93.2 1.00E-34 GMRHGY404JQ2VP 541 leucine rich protein Arachis hypogaea gb|ABH09321.1| 45 49.7 8.00E-13 Section 2. Assembled sequences isotig00059 gene=isogroup00010 537 cysM cysteine synthase B Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_157505.1| 92 337 5.00E-91 isotig00072 gene=isogroup00023 510 rpsL 30S ribosomal protein S12 Azoarcus sp. BH72 ref|YP_934924.1| 100 163 6.00E-39 Query= isotig00001 gene=isogroup00001 2168 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 4.00E-46 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 3.00E-36 Query= isotig00002 gene=isogroup00001 2024 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 3.00E-46 conserved hypothetical protein Clostridium botulinum D str. 1873 ref|ZP_04863280.1| 48 72.4 2.00E-10 Query= isotig00003 gene=isogroup00001 2012 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 9.00E-44 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 61 72 5.00E-25 Query= isotig00004 gene=isogroup00001 2156 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 1.00E-43 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 3.00E-36 Query= isotig00005 gene=isogroup00001 2023 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 3.00E-46 hypothetical protein CLOLEP_01448 Clostridium leptum DSM 753 ref|ZP_02079996.1| 60 65.1 2.00E-16 Query= isotig00006 gene=isogroup00001 2168 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 4.00E-46 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 3.00E-36 Query= isotig00007 gene=isogroup00001 2011 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 9.00E-44 conserved hypothetical protein Clostridium butyricum E4 str. BoNT E BL5262 ref|ZP_04526499.1| 48 60.8 7.00E-07 Query= isotig00008 gene=isogroup00001 2156 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 1.00E-43 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955128.1| 61 124 5.00E-36 Query= isotig00009 gene=isogroup00001 1281 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 61 72 3.00E-25 conserved hypothetical protein Clostridium botulinum D str. 1873 ref|ZP_04863280.1| 48 72.4 1.00E-10 Query= isotig00010 gene=isogroup00001 1425 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 2.00E-36 Query= isotig00011 gene=isogroup00001 1280 hypothetical protein uncultured alpha proteobacterium HF0070_14E07 gb|ADI17229.1| 61 72 3.00E-25 Query= isotig00012 gene=isogroup00001 1425 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 2.00E-36 Query= isotig00013 gene=isogroup00001 1618 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 3.00E-46 Query= isotig00014 gene=isogroup00001 693 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31922.1| 50 71.6 2.00E-24 Query= isotig00015 gene=isogroup00001 1606 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 7.00E-44 Query= isotig00016 gene=isogroup00001 837 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 8.00E-37 Query= isotig00017 gene=isogroup00001 692 hypothetical protein CLOLEP_01448 Clostridium leptum DSM 753 ref|ZP_02079996.1| 58 92.4 4.00E-25 Query= isotig00018 gene=isogroup00001 837 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 8.00E-37 Query= isotig00019 gene=isogroup00001 625 hypothetical protein AYWB_261 Aster yellows witches'-broom phytoplasma AYWB ref|YP_456457.1| 46 71.2 1.00E-16 Query= isotig00020 gene=isogroup00001 769 conserved hypothetical protein Clostridium botulinum Bf ref|ZP_02955261.1| 61 125 6.00E-37 Query= isotig00021 gene=isogroup00001 1054 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 1.00E-46 Query= isotig00022 gene=isogroup00001 875 hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30046.1| 59 131 9.00E-29 Query= isotig00023 gene=isogroup00001 1042 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 75 152 4.00E-44 Query= isotig00024 gene=isogroup00002 1313 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 59 148 2.00E-33 Query= isotig00025 gene=isogroup00002 1312 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 67 116 1.00E-41 Query= isotig00026 gene=isogroup00002 1307 hypothetical protein HMPREF9553_00243 Escherichia coli MS 200-1 ref|ZP_07172160.1| 77 194 2.00E-47 Query= isotig00027 gene=isogroup00002 1304 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 63 139 2.00E-37 Query= isotig00028 gene=isogroup00002 1211 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 63 141 2.00E-31 Query= isotig00029 gene=isogroup00002 534 hypothetical protein HM1_3149 Heliobacterium modesticaldum Ice1 ref|YP_001680918.1| 72 193 7.00E-49 Query= isotig00030 gene=isogroup00002 993 hypothetical protein FAEPRAM212_01166 Faecalibacterium prausnitzii M21/2 ref|ZP_02090904.1| 52 145 8.00E-33 Query= isotig00031 gene=isogroup00002 992 conserved hypothetical protein Brucella ceti M13/05/1 ref|ZP_05934035.1| 85 151 2.00E-38 Query= isotig00032 gene=isogroup00002 652 hypothetical protein AceceDRAFT_4092 Acetivibrio cellulolyticus CD2 ref|ZP_07328744.1| 57 131 7.00E-29 Query= isotig00033 gene=isogroup00002 586 Hypothetical protein COLAER_01671 Collinsella aerofaciens ATCC 25986 ref|ZP_01772659.1| 64 138 6.00E-38 Query= isotig00034 gene=isogroup00002 438 hypothetical protein HM1_3149 Heliobacterium modesticaldum Ice1 ref|YP_001680918.1| 51 79.7 1.00E-27
! ! 237!
NCBI closest Gene products (Result of BlastX) Sequence ID Size (bp) Gene name Product Organism NCBI Accesion number %identity Score E-value Query= isotig00035 gene=isogroup00002 662 unknow protein Oryza sativa Japonica Group gb|AAV44205.1| 52 140 9.00E-32 Query= isotig00036 gene=isogroup00002 375 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31936.1| 92 77.4 4.00E-29 Query= isotig00037 gene=isogroup00003 724 Conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 80 180 6.00E-69 Query= isotig00038 gene=isogroup00003 738 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 56 214 4.00E-59 Query= isotig00039 gene=isogroup00003 721 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 79 135 9.00E-54 Query= isotig00040 gene=isogroup00003 735 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 49 166 2.00E-39 Query= isotig00041 gene=isogroup00003 647 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 55 145 3.00E-33 Query= isotig00042 gene=isogroup00003 649 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 50 107 1.00E-21 Query= isotig00043 gene=isogroup00003 635 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 55 87.4 2.00E-27 Query= isotig00044 gene=isogroup00003 633 hypothetical protein Curvibacter putative symbiont of Hydra magnipapillata emb|CBA31926.1| 55 123 9.00E-28 Query= isotig00045 gene=isogroup00004 949 conserved hypothetical protein Streptomyces sp. C ref|ZP_07287470.1| 75 73.2 3.00E-24 Query= isotig00046 gene=isogroup00004 797 leucine rich protein Arachis hypogaea gb|ABH09321.1| 60 112 3.00E-25 Query= isotig00047 gene=isogroup00004 1133 conserved hypothetical protein Clostridium sp. 7_2_43FAA ref|ZP_05132927.1| 74 66.2 2.00E-18 Query= isotig00048 gene=isogroup00004 632 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955130.1| 41 89.4 3.00E-16 Query= isotig00049 gene=isogroup00005 1446 hypothetical protein CTC00065 Clostridium tetani E88 ref|NP_780783.1| 87 180 3.00E-83 Query= isotig00050 gene=isogroup00005 1446 hypothetical protein CTC00065 Clostridium tetani E88 ref|NP_780783.1| 86 181 6.00E-83 Query= isotig00051 gene=isogroup00006 1327 conserved hypothetical protein Escherichia coli APEC O1 gb|ABJ03470.1| 54 166 1.00E-40 Query= isotig00052 gene=isogroup00006 1371 conserved hypothetical protein Clostridium botulinum D str. 1873 ref|ZP_04863280.1| 55 206 6.00E-51 Query= isotig00053 gene=isogroup00007 610 conserved hypothetical protein Clostridium botulinum NCTC 2916 ref|ZP_02955130.1| 44 79 3.00E-13 Query= isotig00054 gene=isogroup00007 1060 hypothetical protein uncultured delta proteobacterium HF0070_15B21 gb|ADI19064.1| 52 50.1 5.00E-11 Query= isotig00055 gene=isogroup00008 965 conserved hypothetical protein Faecalibacterium prausnitzii A2-165 ref|ZP_05616140.1| 63 150 1.00E-49 Query= isotig00056 gene=isogroup00008 562 conserved hypothetical protein Clostridium perfringens C str. JGS1495 ref|ZP_02865662.1| 62 153 9.00E-36 Query= isotig00057 gene=isogroup00009 1632 hypothetical protein AYWB_261 Aster yellows witches'-broom phytoplasma AYWB ref|YP_456457.1| 56 145 1.00E-43 Query= isotig00058 gene=isogroup00009 2063 conserved hypothetical protein Clostridium novyi NT gb|ABK60662.1| 78 164 5.00E-69 Query= isotig00060 gene=isogroup00011 221 hypothetical protein CLOSCI_02020 Clostridium scindens ATCC 35704 ref|ZP_02431788.1| 58 53.1 1.00E-05 Query= isotig00061 gene=isogroup00012 969 hypothetical protein CLOLEP_01448 Clostridium leptum DSM 753 ref|ZP_02079996.1| 52 84.7 1.00E-22 Query= isotig00062 gene=isogroup00013 485 hypothetical protein uncultured Acidobacteria bacterium HF4000_26D02 gb|ADI18657.1| 49 70.5 4.00E-23 Query= isotig00063 gene=isogroup00014 152 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 75 77.4 6.00E-13 Query= isotig00066 gene=isogroup00017 514 conserved hypothetical protein uncultured beta proteobacterium CBNPD1 BAC clone 578 gb|ABM53545.1| 64 97.1 3.00E-38 Query= isotig00067 gene=isogroup00018 480 hypothetical protein AcavDRAFT_4806 Acidovorax avenae subsp. avenae ATCC 19860 ref|ZP_06213060.1| 71 101 2.00E-26 Query= isotig00068 gene=isogroup00019 492 unknow protein Oryza sativa Japonica Group gb|AAV44205.1| 50 115 1.00E-24 Query= isotig00069 gene=isogroup00020 476 hypothetical protein RUMTOR_00910 Ruminococcus torques ATCC 27756 ref|ZP_01967363.1| 50 70.1 2.00E-13 Query= isotig00070 gene=isogroup00021 521 conserved unknown protein Ectocarpus siliculosus emb|CBJ34222.1| 73 103 6.00E-21 Query= isotig00073 gene=isogroup00024 513 hypothetical protein ebA3088 Aromatoleum aromaticum EbN1 (Azoarcus EbN1) ref|YP_158765.1| 57 156 8.00E-37 Query= isotig00075 gene=isogroup00026 517 hypothetical protein CaO19.6835 Candida albicans SC5314 ref|XP_710281.1| 56 69.7 4.00E-16 Query= isotig00076 gene=isogroup00027 546 hypothetical protein PERMA_1141 Persephonella marina EX-H1 ref|YP_002730924.1| 64 63.9 8.00E-11 Query= isotig00078 gene=isogroup00029 626 hypothetical protein RUMTOR_02824 Ruminococcus torques ATCC 27756 ref|ZP_01969239.1| 39 75.1 2.00E-12 Query= isotig00080 gene=isogroup00031 586 hypothetical protein TherJR_0197 Thermincola sp. JR ref|YP_003638986.1| 47 185 3.00E-45 Query= contig00007 gene=isogroup00001 508 conserved hypothetical protein Magnetospirillum gryphiswaldense emb|CAJ30045.1| 59 99 3.00E-21
! ! 238!
Nucleotide sequences of the genes that were transcribed in Cartwright Consolidated grown on benzoate GMRHGY403HDNLB (BclA/BzdA) ACTATACGAGTATTACAGCTACATGCTGCGCGACAGCCGCGCCCGTGCGCTGGTCGT GTCGTCGCTGCTGCTGCCCGCGTTCGACAGCGCGATCGAGGGAAGCCCGTTCGTCAA GAACGTCATCGTCTCGGGCGGAGACATCGGCACGCGCGGCGGCCACCTGGACTTCG CCGAGCTGATCGACGCGCCGCGTCCGCCGTTCGAAGCGGCGCTCACCTGTGCCGAC GACCCCTGTTTCTGGCTGTACTCGTCGGGCTCGACCGGCGCGCCGAAAGGCACCGTT CACGTGCATTCGAGCCTGATCACCACCGCGGAGCTGTACGCCAAACCCGTGCTGGG CGTGACCGAGGACGATGTCGTGTTCTCGGCCGCGAAGCTCTTCTTCGCCTACGGGCT CGGCAACGGGCTGACTTTCCCGCTCGCGACCGGCGCGACCGCGGTGCTGATGTCCG AGCGTCCGACGCCGGCGTCCGTGTCGAACGTGCTGCGCAAGCACAAGCCGACGA
GMRHGY403FX443 (BzdA) ACTATACGAGTGCGCGCGCACCGACGGACACGGGGCGACGACCGCAAGTTCGGAGC GGCGCAAGCCGGCCCGGAGAAATAAAAGCAGAAACAGGAGACGAGGAAGATGGCA GAACTCAGCAGCGCCGACCAAGGGACGACACCGCCGCGGATCACGATCCCGCGCGA TTACAACGCGGCCCACGACCTGATCGAGCGCAACCTGCGCGCCGAACGCGGCAGCA AGATCGCCGTCATCGACGACCGCGGCAGCTACACCTACGCACAGCTCGCCGAACGG GTCGACCGCTTCGCGCATGCGCTGTCGGAGCTGGGCGTCCGCATGGAGGAGCGGGT GCTGCTGTGCCTGCTCGACACCGTCGACTTCCCGACCGCCTTCCTCGGCTGCATCAA GGCCGGCGCCGTGCCGGTGCCGGTGAATACCCTGCTCACCGCCTCCGATTACAGCTA CATGCTGCGCGACAGCCGCGCCCCGTGCGCTGGTCGTGTCGTCGCTG
GMRHGY403GQNP9 (BcrA) ACTATACGAGTGTAACGGGGGCTCACGTTGAGCTTGGTCCCGATGGCTTCCTCGATC AGCTTGACCATCGCCTGGTTGCGGCTCACGCCGCCGGTGAAGGTCACTTCCGGGTTG ATGCCGACGCGCCGCGCGAGCGAGACGCAGCGCGCAGTGATCGCCTGGTGCACGCC CATCAGCACGTCTTCGGGAAGGATGCCCTTGGCCAGGTGGCTGATGATCTCCGATTC GGCGAACACCGTGCACGTGGTCGTGATGCGCACCGGGTTCTTCGAGCGCAGCGCCA TCGTGCCCAGCTCGGACAGCGGCAATTCGAGCGCGTACGACGCGGCGCCGAGGAAG CGCCCGGTGCCGGCGGCGCACTTGTCGTTCATCGTGAAGTCGTCGACCTGGCCATCG GGCTTCACCGAGATCGCCTTCGTGTCCTGCCCGCCGATGTCGATCACCGTGCGCGTG TTCGGGAACAGCGACACGGCGCCCTTCGCGTGACAGGTGA
GMRHGY403HDRMV (BzdO) ACTATACGAGTCTTCGCGACGTGCTGGTACCACTTGGAGTGCGAGCAGCAGATCTGC GTCTGGAACACGAAGTCGGCCTTCGGGAACTCGCCGCCGAACGCGTACTTGTTCAG GTGCATGCCGCCCCCAGTAGATCCGCATGTAGGAGCACAGGTCACGGGCGAAACCG TAGGCTTCCGCCGCGTCCATGCATTCCTTCGCGAACTTGCGGTCGTGCGACACCGCC GCGGCGTACGGCTCGCCGGTCAGCGAATAGACGTCGTTGCCGAGGCCGGCGGGCAG CGCGTCGAGCGCCCACGCCGAACCGGACCAGCGCAGACCGCCCTTCTCCTTGGCCC GCGCGTAGTTCATGTAGTACTGCTCGCGCAGTTCCTTTGCCTTGCCCCAGAGCTTGA GCGGCTCGGTCGGGTACTTGCTTGCCATTGCGTTCATCGTTCTCTCTCCGCAGTCAGT TCAGAACAGCTCTTCTTCGCCCAGCGTCTCGAGGAATGCCTCGATG
! ! 239!
GMRHGY403G2HMG (BzdO) ACTATACGAGTCGAAGAGATCGAGCAGCGCGTGCGCGAAGGCAAGGGTCCGATCAC GCCCGACGGCGAGATGACCGAGGAGAAATACCGCCTCATCGTCGAGGGGCCGCCCA ACTGGACCAGCTTCCGCGACTTCTGGAAGATGTTCTACGAGGAAGGCGCGGTGGTC GTCTCGTCCACCTACGCGAAGGTGGGCGGCCTGTACGACTTCGGCTTCCGCCACGAC CCGGATCATCCGCTCGAGTCGCTCGCCGAATACTGCCTTGGCTGCTACACCAACCTG AACCTGCCGTCGCGCGTCGACATGATCTGCAAGTACATCGAGGAGTACGAGGCCGA CGGCCTGCTGATCAACTCGATCAAGAGCTGCAACAGCTTTTCGGCGGGGCAGTTGCT GATCCTGCGCGAGGTGGAGAAGCGCACCGGCAAACCGGCCGCATTCATCGAGACCG ATCTCGTCGATCCGCGCTACTTCTCGGCGGCCAACGTAAAGAACCGGCTCGA
GMRHGY403GYJ3Y (BcrC/BzdN) ACTATACGAGTGTTCTCGCTGCGCTGCCGTCGCGCAAGATGGCCCGTCAGCAGGGCG TGCGCTTCATGACGATCGGTTCGGAAAACGACGACATCTCGTTCATGGCGATGGTGG AATCGGTCGGTGCGACGATCGTCGCGGACGACCAGTGCTCGGGCAGCCGCTACTTCT GGAACGCTTCGAAGCCGGAAGACGACGTGATCAAGGCGATCGCCGACCGCTACTGC GATCGTCCCGCCTGTCCGACGAAGGATTACCCGACCCATACCCGTTACGACCACGTT CTCGGCATGGCGAAGGACTTCAACGTCCAGGGCGCGATCTTCCTGCAGCAGAAGTT CTGCGATCCGCACGAGGGCGATTACCCGGATCTGAAGCGTCACCTGGAAGAAAACG GCATCCCGACGCTGTTCCTGGAATTCGACATCACGAACCCGATCGGCCCCTTCCGCA TCCGCATCGAGGCATTCCTCGAGACGCTGGGCGAAGAAGAGCTGTTCTGAACTGACT
GMRHGY403GJ00N (Dch/BzdW) ACTATACGAGTGAACCGGGAAATAACGGGGAACACGGGAGACACCATGACGCAAT CGCAATTCCAGTTCATCACCTACGGCGTCGCCGACGGCCTCGCAACGCTGACCATCA ACCGGCCGCCGTTCAACGTTCTCGACATCCCGACGATGGAAGAGGTCAATGCCGCG CTCGACCTGTGTCTCGCGGCCACCGACGTCAAGCTGCTCGTCATCACCGGCGCCGGC GACAAGGCGTTTTCGGCCGGTGTCGAGGTCGCGGACCACACGCCGGACAAGGTCGA ACGGATGATCGAGGTGTTCCACGGCATTTTCCGGCGCATGCAGGAGCTGCCGATCCC GACGCTCGCCGCGGTCAATGGCGCAGCACTCGGCGGCGGCATGGAAGTCGCGATCG CGTGCGACATGATCGTCGCGTCGGCGAACGCGAAGTTCGGCCAACCGGAGATCAAG CTCGCGGTGTTCCCGCCGATCGCCGCGGTGCTGCTGCCGCGCCTCGTGGCGCCGGCA CGCGCG
GMRHGY403HAJL4 (Dch/BzdW) ACATACGAGTGTCTTCTTCAGGGTGCCCGGCTCGGTCATCTGCCAGGTATCGATGAA TTGTGGAATCACGCTCGTCTCCAGTTATCTTCGTTCGGTCGTAGGTCGCGGCTCAGCA GTTGCGCCACACCGGCTTGCGCTTCTCGAGGAAGGCGGCTAGCCCTTCTTTCGCGTC CTCGGTCGCCATCAGCTCATTCAAGTAGATGTTCTCGGCCGCGTCGAGCGCGGTGCC GAAGGGCTTGCCCGCAGCGGCCCGGATCGTCTTGCGCGTCGAGGCGAGCGCGGCGC GCGACAGTGCGAGGTACGGGGCGACGAAGGCGGCGACCTCGTCGCGGAACGACTCC CTCGCGAATACGCGGTTCACCAGCCCGATCGCCTTCGCCTCGTCGGCGCCGATGTTC TCGCCGCCGAGCAGCAGCTCCATCGCGCGTGCCGGCGCCACGAGGCGCGGCAGCCA GCACCGCGGCGATCGGCGGGAACACCGCGAGCTTGATCTCCGGTT
! ! 240!
GMRHGY403GQ93M (BzdX) ACTATACGAGTCGGGCACCGTGATCGGTGGCGAAGCGTCGATGATCGGCAAGGAAG TCATCGTCCCGGCGGTGATCCCGTGCGGCGAGTGCGAGCTGTGCCACACCGGCCGC GGCAATCGCTGCCTGTCGCAGAAGATGCCGGGCAACTCGATGGGCATCTATGGCGG CTATTCGAGCCACATCGTCGCGCAGTCGAAGTACCTGTGCGTCGTCGAGAATCGCGG CGCGACGCCGCTCGAGCACCTGTCGGTCGTTGCCGACGCGGTGACGACGCCCTATCA GGCCGCCGTGCGCGCGGATCTGAAGAAGGACGACCTCGTGATCGTCGTCGGCGCGG CCGGCGGCGTCGGCAAGCTTGTGATCGTCGGCTACGGCACCGCCGAGACGACGTAC ATGCTGTCGAAGCTGATGGCCTTCGACGCCGAGATTATCGGCACCTGGGCTGCCCGC CGGACCGCTATGCGGCGGTGCGCGACATGT
GMRHGY403GSP1F (BzdX) ACTATACGAGTGTGGTCGTTCCGGCGGTGATGCCTTGTGGCGAATGCGAGGTTTGCT CGAGCGGTCGCGGCAACCGCTGCCTGTCGCAGCAGATGCCGGGCTACTCCATGGGG ATCTATGGCGGATTCTCGAGCCATATCGTGGTCCCGGCCAGGTTTCTCTGCGTTGTCG AACACCGGGGCGCAATCCCCCTGGAACACCTTTCGGTAGTCGCCGATGCGGTGACG ACGCCATACCAGGCCGCACTGCGCGCGCGTCTGCAACCCGGCGACCGGGCGATCGT GATCGGGGCGGCGGGCGGAGTCGGCTCTTTCATGACGCAGGTGGCCAAGGCGATGG GCTGCGCAGCCGTCGTCGGCATCGACATCAACGACGAAAAGCTGCAGGTGATGCGG GACTACGGCGCGGACTTCACGATCAATCCGCGCGGCCTCGCGCACAGCGACGTGAA GGGACTGCTCAAGACGTATTGCAAGGATCAGACTCGTAGAGT
GMRHGY403F9DOE (Had) ATATACGAGTCCCTTGGTCACGAGATCAGCGGACGCGTCGTCGAGGCGGGTCAGGG CGCCGAGAGCTGGCTCGGCAAGGCGGTGATCATCCCCGCCGTTCTGCCGTGCGGCG AGTGCGACCTGTGCAAGCGCGGCCTGGGCACGATCTGCCGCAAGCAGAAGATGCCG GGCAACGACATCCAGGGCGGTTTCGGCACGCACATCGTCGTGCCCGCGCGCGGCCT GTGCGCGGTCGACGAAGTGCGCCTCGAGCGCGCGGGCCTGAGCCTCGCCGAGGTGT CGATTGTCGCCGACGCGCTCACCACGCCCTTCCAGGCGGTGCGCCGCGCGGGCGTG AAGCCGGGCAGCCTCGCCGTGGTCGTCGGCGCGGGCGGTGTCGGCGGCGTACTGCG TGCAGATCGCCGCCGCTTTCGGCGCGAAGGTCGTCGCCATCGACGTCGACGACGCC AAGCTCGAGGCGATCGCCGCC
GMRHGY403GDGSR (BzdY) ACTATACGAGTCGCCCTGCTCGGTCACGAGCATTTCGGCACCGAGGCGCCCTCGGTC ATCTTCGAAAAGCGGCCGGTGACGGACCCGCAGGGCAAGCAGGTTCCGGGCCTGTA CTCCGCGTGGATCATTCTCAACAACCCGAAGCAGTACAACTCCTACACGACCGAAAT GGTCAAGGGCATCATCGCCGGTTTCCAGCGCGCCTCCGGTGACCGCACGGTCACGA CGGTGGTGTTCACCGCGATGGGCGACAAGGCGTTCTGCACCGGCGGCAACACGGAG GAATACGCGTCCTACTATGCCCGCCGCCCGAACGAGTACGGCGAGTACATGGATCT GTTCAACGCCATGGTCGACGGCATCCTGAACTGCAAGAAGCCGGTGATCTGCCGCG TCAATGGCATGCGCGTCGGCGGCGGCCAGGAGATCGGCATGGCGACGGACATCACG GTCACGTCGGACATGGCGGTGTTCGGCCAGGCCGGTCCGAAGCACGGCAGCGCGCC GGACGGCGGCTCCA
! ! 241!
GMRHGY403F9QCI (Putative benzoate transporter) ACTATACGAGTGCGGCGACGTGTCGAAGCGCGCCGAGCTCATCAAGGCGATGGAAG CGGCGACGATCGACAGCCCGCGCGGCAAATTCACGCTGTCGAAGGCGCACAACCCG GTGCAGGACATCTACCTGAGAAAGGTCGAGGGCAAGGAGAACAAGGTCGTTTCCGT TGCGGCCAAGGCGCTCGCGGATCCCGCGCGCGGCTGCCGCATGTAGCGCTCCGGTC CGGGGCTCGCGTCCCGGACTTTGCCTGACCACTCGTAGAGT
GMRHGY403HBVTC (Putative benzoate transporter) ACTATACGAGTGCGGCGACGTGTCGAAGCGCGCCGAGCTCATCAAGGCGATGGAAG CGGCGACGATCGACAGCCCGCGCGGCAAATTCACGCTGTCGAAGGCGCACAACCCG GTGCAGGACATCTACCTGAGAAAGGTCGAGGGCAAGGAGAACAAGGTCGTTTCCGT TGCGGCCAAGGCGCTCGCGGATCCCGCGCGCGGCTGCCGCATGTAGCGCTCCGGTC CGGGCTCGCGTCCCGGACTTTGCCTGACCACTCGTAGAGT
GMRHGY403GTHEE (Putative benzoate transporter) ACTATACGAGTGCGGCGACGTGTCGAAGCGCGCCGAGCTCATCAAGGCGATGGAAG CGGCGACGATCGACAGCCCGCGCGGCAAATTCACGCTGTCGAAGGCGCACAACCCG GTGCAGGACATCTACCTGAGAAAGGTCGAGGGCAAGGAGAACAAGGTCGTTTCCGT TGCGGCCAAGGCGCTCGCGGATCCCGCGCGCGGCTGCCGCATGTAGCGCTCCGGTC CGGGGCTCGCGTCCCGGACTTTGCCTGACCACTCGTAGAGT
GMRHGY403HBUV7 (Putative benzoate transporter) ACTATACGAGTCCGCGGTCGGCGGCGAGGCCCGCGGCGAGCAGCTGCGCCGAGTCG TAGCCCTGCACCGCATACACGTCCGGCTGCATCTTGAACGCCGACGCGTACGCGGCG CGGAACTCCTTGTCC
GMRHGY403FNO3H (Putative benzoate transporter) ACTATACGAGTCCGTAGGTGAGCAGTACCTGGTCGAGGTGGTCGCGCTTGTACAGCT TCGAGAAAAGCGCCCATTCGAGCAGGAAGCCGATTGCCGCGGACAGCGCGATCCCG AGCACGACGGCGACCAGCAGATTGCCCGTCAGCGACGTCAGCGAGAACGCGATATA CGCGCCCATCATGTAGAAGCTGCCGTGCGCGAGGTTGATGATGCCCATGATGCCGA AGATCAGCGTCAGGCCGCTGGCGACGAGGAACAGCAGCAGCCCGTACTGCAGCGCA TTCAGCAGCTGGATCAGGAAGGTCGTGAAATCCATGCGGACTCCCCTCCGGACGCG CTTGTCGGCGTCCGGATAATGTCGGTCAGGCAAAGTCCGGGACGCGAGCCCCGGAC CGGAGCGCTACATGCGGCAGCCGCGCGCGGGATCCGCGAGCGCCTTGGCCGCAACG GAAACGACCTTGTTCTCCTTGCCCTCGACCTTTCTC
GMRHGY403FQZTA (KorA) ACTATACGGAGTGCGAGCATCGATCCCGGCGGCTACCTCTTCTACGACTCGACGAAG CCGCTTCCGGCCTCGAAGTTCCGCGACGACATCACCGTCGTCGGCGTGCCGCTGACC GCCATCTGCAACAAGGAGTACACGGACGCGCGCCAGCGGCAGTTGTTCAAGAACGT GATGTACATCGGCGCGTTGATCGCGCTGCTCGACATGGACTTCACCGTCGCCGAGAC GCTGATCACCGACCGCTATCGAGGCAAGGACCAGCTGATCGAGGCGAACATTAACG CGCTGCGCCTCGGTTACGACTATGCGCGGACGAACCTGCCGTGTCCGATCGGACTGA CCGTCGCGCCCGCCGACGGCGTCGGCGACCGCATCTTCATCGACGGCAACAGCGCG TGCGGGCTCGGTGCGGTGTATGGCGGCGCGACCGTCGCCGGCTGGTATCCGATCACC CACTCGTAGAGT
! ! 242!
GMRHGY403FZA8J (KorA) ACTATACGAGTGTTGCAGATGGCGGTCAGCGGCACGCCGACGACGGTGATGTCGTC GCGGAACTTCGAGGCCGGAAGCGGCTTCGTCGAGTCGTAGAAGAGGTAGCCGCCGG GATCGATGCTCGCGATGTCGCGGTCCCAGGTCTGCGGATTCATCGCGACCATCATGT CGCAGCCGCCGCGCGCGCCGAGCCAGCCCACTTCGGACACGCGGACCTCGTACCAG GTCGGCAGCCCCTGGATGTTCGACGGGAAAATATTGCGCGGCGCGGCCGGGACGCC CATGCGCATCACCGCGCGAGCGAAGAGCTCGTTCGCCGACGACGAACCCGAGCCGT TGACGTTCGCAAACTTGATCACGAAGTCGTTGCAGCGCTGGATGGATTTTGCGTTCG TCATTTTCACTCCCCCTTCACGCCGGCCTGTGCGACGTCGATCCGGAATTTCTGCATG TCCCACGCGCCCGTCGGACAGCGTTCGGCGCACATGCCGCAGTGCAGA
GMRHGY403F5W7C (KorA) ACTATACGATCGGACGAACCTGCCGTGTCCGATCGGACTGACCGTCGCGCCCGCCG ACGGCGTCGGCGACCGCATCTTCATCGACGGCAACAGCGCGTGCGGGCTCGGTGCG GTGTATGGCGGCGCGACCGTCGCCGGCTGGTATCCGATCACCCCTTCGACGTCGGTC ATCGAGTCCTTCTCGAGCTACTGCCGGAAGTTTCGCACCGATCCCGACAACGGCCGC GCCCGCTACGCGATCGTTCAGGCCGAGGACGAGCTCGCGTCGATCGGCATGGTCAT CGGCGCCGGCTGGAACGGCGCGCGCGCCTTCACCGCGACGTCGGGCCCCGGCATCT CGCTGATGCAGGAGTTCTCGGTCTCGCGTACTTCGCGGAAATCCCGGCGGTGATCGT CGACATCCAGCGCGGTAGCCCGTCGACCGGGATGCCGACGAAGACGCAGCAGTCGG ACCTGCTGTCGTGCGCGTACGCGTCGCATGGCGATACGCGTCATGTGGTGCTGTT
GMRHGY403GIMYE (KorB) ACTATACGAGTCGAGAACTGGCCCTTGGTGAGTCCGTAGACGCCGTTGTTCTCGACG ATGTACGTCATGTCGACGCCGCGGCGCATCGCGTGCGCGAACTGGCCGAGTCCGAT CGACGCCGAGTCGCCGTCTCCCGAGACGCCGAGGTAGAGCAGCGTCCGGTTGGCGA GGTTGGCGCCGGTGAGCACCGACGGCATGCGCCCGTGCACCGAGTTGAAACCGTGC GAATGGCCGAGGAAATAGTCGGTGGTCTTCGACGAGCAGCCGATGCCGGAGAGCTT GGCCACGCGATGCGGCTCGACGTCGAGCGCCCAGCACGCCTGCACGATCGCCGCCG AGATCGAATCGTGGCCGCAACCGGCGCACAGCGTCGAGACGCGCCCTTCGTAATCG CGCCTCGTGTAGCCCAGGCCGTTCTTCGGCAGCGACGGGTGGTGCAGCACGGGTTTC GCGAGATAGGGTCATCGGGTCGCTCCGGAGTCGGTGCCACTCGTAGAGT
GMRHGY403HC7JI (KorB) ACTATACGAGTCGAGAACTGCCCTTTCGTCAGGCCATAGACGCCGTTGTTCTCGACG ATGTAGGTCATGTTCACGCCGCGTCGCATCGCGTGCGCGAACTGCCCGATGCCGATC GACGCCGAGTCGCCGTCGCCGGACACGCCGAGGTAGATCAGGTTGCGGTTCGCGAG CGCTGCACCGGTCAGCACCGACGGCATCCGCCCATGGACGCTGTTGAAGCCGTGCG AGTTGCCGAGGAAGTAATCCGGCGTCTTCGACGAGCAGCCGATGCCGGAGAGCTTC GCGACCTTGTGCGGCTCGACGTCGAGGTCGAAGCAGGCTTGCACGACCGACGCGCT GATCGAGTCGTGGCCGCAGCCGGCGCACAGCGTCGAGATCGCGCCTTCGTAGTCGC GCCTCGTGTAGCCGAGCGCATTGCGCGGCGCGTCGGCGGCGAGGAGCTTCGGTTTG GCGAGATAGGTCATAGGGCCACCTTGCGCAGCGGGCTG
! ! 243!
GMRHGY403FJCWF (KorB) ACTATACGAGTGCCGTCGCCGGTGACGACCCACACCGACAGCTCCGGGCGCGCCAC CTTCAGCCCGCTCGCGATCGCCGGAGCGCGGCCATGGATGCTGTGCATCCCGTAAGT GTCCATGTAGTACGGGAAGCGGCTCGAGCAGCCGATGCCGGAGATGAACGCAAAGT TCTCCTTCGGGATGCCGAGCGTCGGCAGCAGCTTCTGGATCTGCGCGAGCACCGCGT AGTCCCCCGCAGCCGGGGCACCAGCGCACGTCCTGGTTCGATTCGAAATCCTTTTTC GTCAGCGTGACGGGGAGCGTCATGTCGTTCATGGTGTTTTCCTTGTCGGTCTGGCAG CGGGACGGCGGCGGATGCGCGTCAGCTCAGTTCGAGGATGCGGTCGAGCACTTCGG CGACCTTGAACGGCTTGCCCTGCACCTTGTTCAGCGGCACGACGTCGCGCAGGAACT GCTCGCGCAGCAGCTTCTTGAGCTGGCCCATGTT
GMRHGY403GBLM7 (KorB) ACTATACGAGTGCCGTCGCCGGTGACGACCCACACCGACAGCTCCGGGCGCGCCAC CTTCAGCCCGCTCGCGATCGCCGGAGCGCGGCCATGGATGCTGTGCATCCCGTAAGT GTCCATGTAGTACGGGAAGCGGCTCGAGCAGCCGATGCCGGAGATGAACGCAAAGT TCTCCTTCGGGATGCCGAGCGTCGGCAGCAGCTTCTGGATCTGCGCGAGCACCGCGT AGTCCCCGCAGCCGGGGCACCAGCGCACGTCCTGGTTCGATTCGAAATCCTTTTTCG CAGCGTGACGGGGAGCGTCATGTCGTTCATGGTGTTTTCCTGTCGGTCTGGCAGCGG GACGGCGGCGGATGCGCGTCAGCTCAGTTCGAGGATGCGGTCGAGCACTTCGGCGA CCTTGAACGGCTTGCCCTGCACCTTGTTCAGCGGCACGACGTCGCGCAGGAACTGCT CGCGCAGCAGCTTCTTGAGCTGGCCCATGTT
GMRHGY403FJPQN (KorC) ACTATACGAGTCGAAGCGCGTCATCGTGCTCGGTGGCGGCAACACCGCGATGGACT GTTGCCGTTCTGCGCGACGGCTCGGCGGTGAGGACGTGCGGGTCGTCGTGCGCAGC GGCTTCGACGAGATGAAGGCGTCGCCGTGGGAAAAGGAGGACGCGCTCCACGAAG GCATCCCGATCCACAACTATCTGGTGCAGAAGGAATTCACGCACGACAACGGCCGC CTCACCGGCGTGCTGTTCGAGAAGGTGCGCGCCGAGTACGACGACAAGGGCGGGCG TTCGCTCGTGCCGACCGGCGAGCCCGACGTGCTGATGGAATGCGACGAAGTGCTGC TCGCGATCGGCCAGGAGAACGCGTTCCCGTGGATCGAGCGCGACATCGGCATCGAG TTCGACCGCTACGGAATGCCGGTGCTCGACGAGAAGTCGCTGCAATCGACGCTGCC GAACGTGTTCTTCGGCGGTGATGCGGCGACTCGTAGAGT
GMRHGY403FKB56 (KorC) ACTATACGAGTGTGAGCCAGAAGATGGGCATCCACGAGTGGAGCTACGACAACCAG GTGTCCGAGGAAGCGCGCCGCAAGGTGCCGGTGAAGACGCTCGAACTCGCGCTGAA GGACATCAAGCTCGAATTCGAACTCGGCTACGACCCCGTGGTTGCATGGGCGGAAG CTGAGCGCTGCCTGAACTGTGACATCGCCACCGTCTTCAAGCCGAAGCTCTGTATCG AATGCGACGCCTGTGTCGACATCTGTCCGACGCAGTGCATCACCTTCACCGGAAACG GCGAAGAGGACGAGCTGCGCGGTCGACTCAAGGCGCCCGCAGCCAACCTGGAGCA AGCGCTGTATCTCGCCGAAGGTCTCAAGACCGGCCGCGTGATGGTCAAGGACGAGA ATGTCTGTCTGCACTGCGGCATGTGCGCCGAACGCTGTCCGACGGGCGCGTGGGAC ATGCAGAAATTCCGGATCGACGTCGCACA
! ! 244!
GMRHGY403FMEA4 (KorC) ACTATACGAGTGCCGCCTCAAGCGCGTCGCCGCCGACTTCAAGGACGACATCGACG AACTGTTGCCGAAGGCGCCGACCGCCAAGAACGGCAAGCGCGTCGCCTGTGTCGGC GCGGGACCGGCATCGCTGACGGTCGCACGCGACCTCGCGGTGCTCGGCTACCAGGT CACCGTGTTCGACAGCGGCACGTCGGCCGGCGGCATGATGCGCAGCCAGATCCCGA AATTCCGCCTGCCCGACAGCGTGATCGACGAGGAGTGCGGCTACATCGCCGGGCTC GACGTCGAGATGCGGCAGAACACATGGATCGCGAGTTTGCGCGAACTCCTCGCCGG CAGCTGGGACGCGGTGTTCGTCGGTACGGGCGCGCCACGCGGGCGCGATGCCGACC TGCCCGGACGCAAGGAAGCGGCCGCGCATATCCACGTCGGCATCGACTGGCTGTCG AACGTCGCCTTCGGCCACGTCACGAGCATCGCGAAGCGCGTCATCGT
GMRHGY403G9FWL (KorC) ACTATACGAGTGGACGAGCTGCGCGGTCGACTCAAGGCGCCCGCAGCCAACCTGGA GCAAGCGCTGTATCTCGCCGAAGGTCTCAAGACCGGCCGCGTGATGGTCAAGGACG AGAATGTCTGTCTGCACTGCGGCATGTGCGCCGAACGCTGTCCGACGGGCGCGTGG GACATGCAGAAATTCCCGGATCGACGTCGCACTCGTAGAGT
GMRHGY403FWRN1 (KorC) ACTATACGAGTCGGCGCCCGAAGGTCCAACGAGGCGTCGAAGGAAGGTTACGGATT CCGCACCGGAATGCGCCGCCCAGCGAGGAGACGAGTGAAACCCACCGACATTGCCA ACCCCGACTATCTGCACAAGGTCGTGGATTGCCAGTGGGCCTGCCCGGCCCACA
GMRHGY403F6LH6 (BcrV/BzdV) ACTATACGAGTGCCAGGCGAGCCTCGCGAAGGTCCTCGGACCGCACCCGCACGTCT GCCTCACCTGTCCGCAGCGCGAAGGCTGTTCGCGCTCGCAGTGCTCGTACGGCAACC CGCAGGAGACGCGCTGCTGCACGATCTTCTCGAGCTGCGAACTGCGCAAGGTGTCA GATTTCATCGGCATCCCGAACACGACGCCGCCCTACAAGCCCGCGCAACTGCCGATC GTCCTCGACGAGCCGTTCTTCGACCGCGACTACAACCTCTGTATCGACTGCCGCCGT TGCCTCGTCGCCTGTAACGACGTGCGCGGCGTCGGCTGTCTCGAAGTGAAGGAAGTC GAGACGCCGCAAGGCAAGCGCACCTACGTCGGCACGATCGCGCCGACGCTGATCGA ATCGGGCTGCACCTTCTGCCAGGCCTGCGTGACCGTGTGCCCGACCGGCGCGCTGAT GGACCGCACGCTCGACCCCGCGCACCGCGAA
GMRHGY403G8ETO (BcrV/BzdV) ACTATACGAGTGTAGGTGATCTCCAGGCGCGACCCGTTGCGGCCCGGCTCGATCTTC TTCGGCGCGAGCAGGTAGCGGATATTCACGCCCTCGTCGAGGCAGCCGTCGAACTC GTCCTGACGGGCCGGCATTTCCTCGCGGGTGCGGCGATACACCATGTCGACATCGGC ACCGTAGCGGCGCGACGAGCGCGCGTTGTCGGTCGCGACGTTGCCGCCGCCGATGA CCGCGACCTTCTTGCCGGTCTGGATGCCGTCGGGTGTATTCACCAATCCCATCGTGA CCGCTTGCAGATAGGTCGGGCTGTCGATGACGCCGGGCAGGTCGTCGCCCGGAATC CCGAGCTTGTCGCCGCCCTGGCAGCCGATGCCGATGAACACGGCTTCGTAGCCGTCG GCGAAGAGGTCATCGACGTTCTCGATGCGCGTCTCGAAACGGAACTCGACCCCGAC GGCTGCGACTTCGGCGATCTCGTCGTCGATGACCTCGGCGGGCACGCGGTACGACG GGATGCCGTAACG
! ! 245!
GMRHGY403GM23D (BcrV/BzdV) ACTATACGAGTCCTCGACGAGGGCGTGAATATCCGCTACCTGCTCGCGCCGAAGAA GATCGAGCCGGGCCGCAACGGGTCGCGCCTGGAGATCACCTACGCGAAGATGAGCC TCGGCGAAGCGGATGCCTCCGGCCGCCGTCGTCCGATCGAGACCGGCGAGGAAATC ACCGAAGGCGTCGATCTCGTGATCTCGGCCGTCGGCCAGCATCCGAAGCGGTTCGA AGGATTCGGCGTCGCGACCGACCGCAAGGGCCGCATCACGGTCCGCGAGGACTCGA TGCTGACGAGCCGGCCCGGCATCTACGCCGGCGGCGACTGCGTGCTCGGACCCTCG ACTCTGATCGAGTCGGTCGCCCAGGGCCGCGTCGCGGCTTCGGCGATGGACCTCGCG CTCGGCGGCGACGGCGACATCTCGGAAGTGCTGCTGCGCGACGGCTGGGAAACGAA CCCGTGGATCGGTCGCAAGGACGGCTTCAATAAGGTCCGCAAGTTCCACCCGATCCT GCTGGCG
GMRHGY403HGINX (BzdR) ACTATACGAGTATCTGGCTCAAGGCTTCGCCCGAAGAGCACATGTCCCGCGTCGTCG CGCAGGGAGACTATCGGCCGATCAAGGACAGCCGCGAAGCGATGGACGACCCTGA AGCGCAATCCCTCGCCGGGCGCATCGCGATGTACGACAGGGCGGATGCGATCGTGG GACACGTCGGGAAAAGACGGTCGACGAGAGCTTTGCCGAAATGCTCGAGGCGCTCC GCGGCTGAACGCGGCCGTTCTGTGGAACCCCGTAACCGGTAGCCAATTGCCCGGCT GATCAACCCCGGCGCGACTCGGTGGCCGTTCCCACT
GMRHGY403HC3VO (BzdS/BzdM) ACTATACGAGTCGACGCTGCCCTGTACCACCGCCTGCACGCCGGGCGCGATCAAGC ACTCCTGGTGAGGGACGTGACATGAACACGACGACAGGCGATACCGCAATGAGCAC GAAGAAGCTAATCCCGATCAAGCTCGCCGGCTCGACGACGCCGGAAGAGGATGTCG AAGAGCGTCTGCTGGGCCAGGGCTGGAAGCGCCTGACGACGATTGGCGAGCCGCGA CTGTCCGAGATCGCCGAAGCGTATCGCGGCATGGGTTTCGAAGTGCATGTCGAGGTC TGGAAGAGCGAAGGCGACGGCTGCAACACCTGCCTCGACGCGGACACCGAAATGG GCAAGATCCTGGGCACCGTCTATACCCGCCCGGGCAACGGCGCAGTCGTACGCGAG CGCGCGGGTCTTTTCCTTCTTGACCCAGGCGACTTCGTCGTCGTAGTCGTCGGTGATG ATTTCGAACATCTCGTGCGGACAGGCGGTCAGGCAGCCGCGATCGGCGCAC
GMRHGY403G8R6S (BzdU) ACTATACGAGTCGCCGCGCTGATGGCCAGCCCGTTGTCGCTGTCGCGGTTACCGCCG TTGTGCGTTTCAATCGCGGCAATTCCGCTCAGGTCGAGCAGCCCTTCGAGCAAGCTC GACACCCCGATTTCGCGAAACGGGTGGGCCGGGACGCAGATCCCGCCGAGCCCGTT AATGTGTTCGACGACCTCGTTGATCGGGAGATAGTTGTCGCGTCCCCAGATATTCCA GCCGTCATCCTCGACCCCGTACGCGAGCATGTGTCCGCGATCGGTGGCGATCTCGAC GCCGCGCAGCACCAGCAGCCCCTCATCGCGCCCGACCTGCTCGACCGGCTCGGACG CTTCGTACGAGTAGTGCTCGGTGATGCACACCGCGTCGAGCCCCGAGCGCCTTCGCC CGCCGGATCAGGTCGACCGGCTCGAGCCAGTTGTCGTACGAGTACTTCGTATGGCAA TGGCAGTCGATCCACATGGCGAAAGGCTCCCACTCGTAGAGT
! ! 246!
GMRHGY403HDNNY (BzdU) ACTATACGAGTCTGTCGGCGGTTTGTCCGCTCTGGTGCAGCCGCGCGTGGGGCGACT GCGTCTGTGGGTCTCGGCGGGGGATGTTCGTCCCGCCCTGGCCGGACTTCACTTCAT CAGTCCGGTCCGCGGCGGTCCCGCCGACGTTTTTCCTTACTTACGTTCAGTACTCAG GCGGCAAGGGCGGCCTGCATGTAGCCGGGGAAATAACTGCCTCGGCACGCGCCGGC CCTGACCGCCGCTATGAAACTCTTGATGTCGGTCACGGGCTGCAGAAATTCCGCCGC ACAGCGCCCGACCGCTTCCGTCTTGTGGCAATCGCTGCCGCCGAGCGACGGCAGCC GCATGTGCCCCGCCGCGCTGATGGCCAGCCCGTTGTCGCTGTCGCGGTTACCGCCGT TGTGCGTTTCAATCGCGGCAATTCCGCTCAGGTCGAGCAGCCCTTCGAGCAAGACTC GTAGAGT isotig00198 gene=isogroup00124 (BcrA/BzdQ; BcrD/BzdP) GCGCTGGCCGCGGCCGGTGTGACGCGTGACCAGGTGAAGTCGGTCTTCGCGACCGG TGCCGGgCGCGGCCAGGTAGGGTTCGCGACCGAAGGCATCACCGAGATGACGGCCG GCGCGAAGGGTGCGGTGTTCATGTTCCCGCAAGCTCGCACGATCGTCGACGTCGGTG CCGAGGAAGGCCGCGGCATCAAGACCGATCCGGATGGCAAGGCGATCGACTTCGCC GGTAACGAGAAGTGCGCCGCCGGTGCCGGTTCGTTCGCCGAGGCGATGAGCCGCGC GCTGCAGCTGACGCTGAAGGAATTCGGCGAGGCGAGCCTGAAGTCGGACAAGAGCA TCCCGATGAACGCGCAATGCACCGTGTTCGCGGAATCGGAAGTGGTGTCGCTGATCC ACTCCTCGACGCCGAAGGAAGACATCGCCAAGGCGGTGCTGGATGCGGTGGCCAGC CGGGTGTGCGCGATGGTGCGCCGGGTCGGTATCGAGGGCAACGTCGTGCTGATCGG CGGGATGGCTCACAACCCTGGTTTCGTCCAGTCGCTGAAGAACGCGATGGATGTCGA TCAGGTGACGCTGCCTGAACTGCCCGAGTACGTCAGTGCTCTCGGATGCGCGCTGAT TGCGGCCGAGCGCCTGCACTGAACCCACGCGATAGACACGGATAAAGGAGCGATCA AATGAATGCAGAAGTTGCTGCGGCTACCGCCCCcGAGAAGAAGGAATTCTGGCGCT GGCAGGAAAaCACGGTCTGGGACGAGAACAAGGACTGGCGTGATTCCAAGATCATC ACccTGCGGTATCGACGTCGGTTCGGTGTCGTCGCAAGCGGTGCTGGTCTGTGACGGC GAGCTCTACGGTTACAACAGCATGCGCACCGGCAACAACTCGCCGGACTCCGCGAG GAATGCGCTGCAGGGCATCATGGACAAGATCGGCATGAAGCTCGAGGACATCAACT ATGTCGTCGGCACCGGCTACGGCCGGGTGAACGTGCCCTTcGCCCACAAGGCGATCA CCGAGATCGCGTGCCACGCCCGTGGTGCCAACTACATGGGCGGCAACGCGGTGCGC ACGATTCTCGACATGGGCGGCCAGGACTGCAAGgCCATCCATTGCGACGAGAAGGG CAAGGTCACGAACTTCCTGATGAACGACAAGTGCGCGGCCGGTACCGGGCGCGGCA TGGAAGTCATCTCCGACCTGATGCAGATCCCCATCGCCGAACTCGGACCGCGCTCGT TCGATGTCGAAGTCGAGCCGGAAGCCGTGTCGTCGATCTGCGTCGTGTTCGCGAAGT CCGAAGCGCTGGGTCTGCTCAAGGCCGGCTACACGAAGAACATGGTTATCGCCGCG TACTGCCAGGCGATGGCCGAGCGTGTCGTGAGCCTGCTCGAGCGGATCGGTGTCGA GGAAGGCTTCTTCATCACGGGCGGCATCGCGAAGAACCCGGGCGTCGTCAAGCGGA TCGAGCGCATCCTCGGCATCAAGGCGGTCGATACGAAGATCGACAGCCAGATCGCC GGCGCGCTGGGTGCTGCGCTCTTCGGCTACACGCTGATGCAGAAGCAAGCGAAGGC CGCGTAAGTAGAACAGGCCgaaccaccggcggccgacccggatccgccccagggagtccctggggcaccgggcgg ccggtaggcggcgaactcatttttcggg
! ! 247! isotig00092 gene=isogroup00018 (BcrB/BzdO) CCTAGATTCTCGGTTATCCATCAGGCGGCTTGGCGGCGCAGCCCGAGCTGCTCCATG AACGCATCGACGCGAGCCTGGGTACGCACTTCGTCGAATTCGCGCTCGTCACCCATG TTtCCTTCGAAGGTCATGACCGGCACGCCGGCCTTGGCGATCGCCAGGCGGTTTTCCA TGATGCCCAGCGACAGGCCTTCGCATCCGCGGTTCAGGTGCAGCATCACGCCATCGA CACCCCACTCCTTAATGATGcGAAGCATCATTTCGCTCTTCAGCTGCGGGTTGTAGAA GtGCTGCCACTGCGGCTTCGACAGGTTCCAGTCGGCGTACAGGCGGATCGCGGTGTC GCGGTCGTTGATCTCGATGCCCTTGTTCCACGGCAGCGTGCGACCACCCCAGGAACC ATCCGGCTTGTCTTCCCAGATGCCTTCCAGCGCGAACGTGTAGAGCGAGCCGATCGA CACCGCACCGTAGGTCTCGAGGTAGCGGAAGATCTTCAGGAAGGACCAGGGCGGCT GCGTGTCCGACATCATGCGCACGCTCTCGTTGGGCACCGCCGCGATGCCGCGGGCG ACACGATCCTTCACTTCCTCGTAGAGCTCGTCCATGAAGTCCGCGCACCATTGCGAC GACTTCGACAGCGTGCAGAGCACGTACAGCGAGTACATCGTCTTCTCGTCCAGCGG AGCCGGCTTCGCCTTGTTCAGCGCGCAGATGTCGGCCCAGCGGGACGTCGAGCGCA TTTCGTTCTTGACGGCCCTGAT isotig00103 gene=isogroup00029 GCGACGGCAAGATAATGCAGggCAACCgCAAACAGCGGACACATCAGAGGAGATAG TTCGATGAGCGATGGTTTGTTCGACCAGTTCAAGACCTGGTACGAGAAGCGCCACgA TtACGCACGGgACTGGAAGGCGCGGACCGGCGGTCAAGTGGTGGCGACGATGTGTAC CTATACGGCGGAAGAGTTGTTGATCGCCGCcGGCATGCTGCCGGTTCGGGTGCTCGG CGCCCACGAGCCGCAGAACGTGACCGAGCCGCACATTTTCGGCATGTTcTGCCCGTT CTGCCGCGATTCCCTCGCGCAGGGCCTGCTCGGCCGCTTCGACTACGCCgAAGGCgT GACGCTCACCCAGTCGTGCATCCAGTACCGCCAGACCTTCGGTTCGTGGCGCCTGCA CGTGCCGACGGTGAAGTGGgACTACTACGTGCCGATGCCCAACGAGGTGCAGTCGC AGCACGCCCGCAAGGCGCACTACGAAGAGCTGAAGTCGTTCCGTACTTTCCTCGAG ACGCTGACCGGcAAGAAGATCACCGACGACATGCTGCGCGAGTCGCTCGCCGTGGT CGATGAGAACCGCCGTCTGCTGCGTGAGCTGTACGAATACCGCAAGGAAGACAATC CGAAAGTCACTGGCGTCGAGGCGCTGTACGCGTCGCTGACCGCGCAATTCACCGAC AAGCGCGAGCACAACGAGCAGCTGAAGAAGGTTCTCGCTGCGCTGCCGTCGCGCAA GATGGCCCGTCAGCAGGGCGTGCGCTTCATGACGATCGGTTCGGAAAaCGACGACAT CTCGTTCATGGCGATGGTGGAATCGGTCGGTGCGACGATCGTCGCGGACGACCAGT GCTCGGGCAGCCGCTACTTCTGGAACGCTTCGAAGCCGGAAGACGACGTGATCAAG GCGATCGCCGACCGCTACTGCGATCGTCCCGCCTGTCCGACGAAGGATTACCCGACC CATACCCGTTACGACCACGTTCTCGGCATGGCGAAGGACTTCAACGTCCAGGGCGC GATCTTCCTGCAGCAGAAGTTCTGCGATCCGCACGAGGGCGATTACCCGGATCTGAA GCGTCACCTGGAAGAAAAcg
! ! 248! isotig00087 gene=isogroup00013 (KorA) CCGCTACCTCGACGTCGACGGCGACGGCATCCCGTACCGGACGATTCCCGGCACGC ACCCGACGCGCGGCGCGTTCTTCACGCGCGGCACGACGCGCAACCCGTACGCAAAA TACAGCGAGTCGGGCGCCGACTACGTCTACAACATGGAGCGGCTGCGGAGGAAGTT CGACACGGCGAAGTCACTCGTGCCCGCGCCGGTGCTGCAATCCGCGTCTCAGCCGA CGCGCTACGGCGCGATCCACTTCGGCTCGACCGCTCCGGCGATGGCGGAAGCGAGC GCGCTGCTCGAAGCCGACGGCATCCACGTCGACACGCTGCGCGTGCGCGGCTTCCC GTTCTGCGACGAGATCCTGCAGTTCATCGCGGACCACGAGCAGGTCTTCGTCGTCGA GCAGAACGAGAGCGGCCAGCTCCGCTCGATGCTCATCAACGAAGGCGAAATCGATC CGAAGAGATTGATCCGCGTCCTCCATTACGACGggCTCGCCGATCACCGCGCGCTTC ATCGCCGGCCGGATCGCCGACGCCCTGTCAGACCGCAAGATCAGCCCGCTGCGCAA G isotig00140 gene=isogroup00066 (KorB) GGACAACGGTCGCGTCAGGACTGAAGGTCGTCGCTTCACGATCGCTTACTTCTGCGA ATCGACATCGAAGCGGCGACGGGCGAGGGTTCGCTCCGTCGCCGCTCCGTCAGGAA AAGCACGATCTAGCGCAGCAGCGCATTCAGCGCATCGAGCGCCTTGCTGCCCGGAC AGAGATACGCGTCACCAAGCTGGTTCAGCGGCTTTtCGACCGTGTTCAGATGCGCGT GCATGTCCTGCCCGCTGCTGTCGACGTACAGCAACCCGGTGACGATCTCGCCGAGCG CGTGGCGCTCCTGCAGATGgTTCATCGCGCCGATCCGATCGGACGGGTCGTAATCGA CCGCGAGCTTCCGCAAATGCAGGATCGAGCCGTCGTGCTGCACGATGCGCCGCAGC GAACCGGCGGGATACGAGGTTTCGATCGGGTCGCGCGGCAGggATCACTTCGACGCG GTTGACCGCCTCGTTGTGTTCGCGCACGTAGTCGTAGCgTCTTCGTCGAACCGCTGTG GTTGTTGAAGGTCACGCAGGGGCTGATGACGTCGATGAAGGCCGggCCGtGTGGCGC AGCGCGCCCTTGATCAACGGC isotig00129 gene=isogroup00055 (BzdX) ggtgaacaccaccgtcgtgaccgtgcggtcaccggaggcgcgctggaaaccggcgatgatgcccttgaccatttcggtcgtgtaggagtt gtactgcttcgggttgTTGAGAATGATCCACGCGGAGTACAGGCCCGGAACCTGCTTGCCCTG CGGGTCCGTCACCGGCcGCTTTTCGAAGATGACCGAGGGCGCCTCGGTGCCGAAATG CTCGTGACCGAGCAGGGCGTGATCCTTGGTTTCGTTGTCGCGGGGCAGCCAATCGAG TGCCATGTCGAGTCTCCTATCCTTTTAGTGcaTTGCGTTGGTGATGGTGCGCCGCCGG CCTCCCGTTCGGCCGGCGGTATGTATCGCGTTGCCGCGTCAAAAATCCGGCGTCAGG ATGACCCGCCGCTTGAGCTTGCCGTGGTGCGCCTCGTCGAACACCTGCTCGATCTGG CTCATCGGACGCGTCTCGACGAAAGGTCCGAGCTGGATCCGCCCGTCGAGGCACAT GTCGCGCACCGCCGCATAGCGGTCCGGCGGGCAGCCCCAGGTGCCGATAATCTCGG CGTCGAAGGCCATCAGCTTCGACAGCATGTACGTCGTCTCGGCGGTGCCGTAGCCGA CGATCACAAGCTTGCCGGTGAAGGACAGCAGCGACAGCGCCAGTTCCTGGCCCGGC TTGCTGCCGGTGACCTCGAAGATCTTCCAGCCGTAGTTCGACGGCAGGCCGCGCTCC TTGCAGAAGCCCTTGAAGAGCTCCTTGACTTCCTTCGCGCTCTTGTCCTTCGGGTTGA TGATGAAGTCGGCACCGTAGTCCTTCATCATCACGAGCTTTTCCTCGTTGATGTCGAT GCCGATGACGGCCTTCGCGCCCATGCCCTTCGCGGTCTGGACCATGAAGCTGCCGAC GCCGCCGGCCGCGCCGACGACGATCACGAGGTCGTCCTTCTTCAGATCCGcGCGCAC GGCGGCCTGATAGGGCGTCGTCACCGCGTCGGcAACGACCGACAGGTGCTCGAGCG GCGTCGCGCCGCGaTTCtCGACGACGCACAGGTACTTCGACTGCGCGACGATGTGGC TCGAATAGcGCCATAGATGCCCATCGAGTTGCCCGGCATCTTCTGCGACAGGCAGCG
! ! 249!
ATTGCCGCGGCCGGTGTGGCACAGCTCGCACTCGCCGCacgggatcaccgcc isotig00151 gene=isogroup00077 (Oah/BzdY; BzdZ) gtttctgcttttaTTTCTCCGGGCCGGCTTGCGCCGCTCCGAACTTGCGGTCGTCGCCCCGTGT CCGTCGGTGCGCGCGCCGTTACAGCCACGCGCGCTGACGGCCGCCCCCGCGACGGC CCGAAAGCGCGGGGCCGCTTAAGCGGCCCcGCGTCGACTCCATGTCAGTTGATCGTC ATCCCCCCGTCGGCCGCGAGCACCTGACCGgTGACGTAGCTCGACGCGTCCGACGCG AAGAACACGAACACCGGGgTGATCTCCTCGGGCTCGGCCCAGCGTCCGAGCGGGAT GCGGTCCAGGTATTTttCCTTGAACCGATCGTCGGTGCGGATCGTCTCGGTCATCGGC GTTGCCGCCCCGGGCGCGATCGCGTTCGCGGTGATGTTGTAGCGCGCGAGCTCCTTC GCGGTGCTCTTCGTCATGCCGAGGATGCCGGCCTTcGCGGCGCTGTAATTGATCTGG CCGATCGTGCCGAGCACGCCCGCGGCCGATGTCACGTACACGATGCGGCCGTAGCG CCGTTCGATCATGCCGCCGACGACCGCCTGCAGACAGTTGAACGCGCCGTTGAGAT GCACGTTGAGCACCTGCTGCCATTGCTCCCCGGTCATCTTGTTGAGCATCGCGGTGC GCGTGATGCCCGCGTTGTTGACGAGAATGTCGATCCGGCCCCAGCGCTTTTCGACTT CGCCCGCCATCGCCTCGACCTGTTCGCGGTTCGAGACGTCGCAGGCGATGCCGATCG CCTCGCCGCCGCGTTCGACGATGTCCGCCGCGGTGCGCTGGGCGACGTCGAGGTTCA GATCGATGACGGCGACTTTCGCACCTTCGCGCGCATACGCcgCCGCGACCGCTGCGC CGATGCCTTGCCCGGCGCcGGTCACGATTGCGACCCTGTCCTTCAGTTGCATCTGTGG TCCCCTGTATCGGCGGGAAGCGGCCCGTCGAGACTCCGCGAGCCGCGCTCCTCGCTC TTCCCGAAATGGAAAAAAATGGGGTGAGTTAAGGACGGTGGTCCGCCCTCACCCCG GGAAGGGAGAGGCCTTGAAAACTACGATTTCGGTTTCGCGAGGACCTGCTCGGCGA AGTTGTCGTCGAACATGGCGCCTTCGGCGACGAGCTGGCGGAACTTGATGAAGTCG ATGACGTCCTTGCCGGTCGCCTTCTTGTTGTTGAAGGCGGTGAAGCCGAGGTACGCC TCGTGGTTCATGTTCGCCGCGAGCCAGTGACGGTTGGCGAGCTTCATCTGATCCCAG AAGAACTTCTTCTTGCCGCGGATGCCGTCAATCGACTTGATCAGGCACTGCGGGAAG AGGTTCGTGAACTTCCACACGAGCGCATTGACTTCCGCGTCGAGGCGCGAGAAATC GGTCGTGCATTGGCCGATCAGATCCTTCGCCGCCTGTACCTTGTCGGCCGCGACCGA CTCGCCGTAGACCAGTTCGCCGTCATCGACGTAGGCATCGGTGCGCACGAGCGGATT GCGGACCCATTCGCCGTCCTTGCTCTTCAGCACGGGCACGACCTTGGTGACGAGGTT CTTCGACTTCATCTTGTACGCGCTCCACGGCTCGCACGAGATGCAGTTGTACATCGC GTCTTCCATGTTGAGCATCCACGGCAGGAAGTCCGTGGAGCCGCCGTCCGGCGCGCT GctGTGCTTCGGACCGGCCTGGCCGAACACCGCCATGTCCGACGTGACCGTGATGTCC GTCGCCATGCCGATCTCCTGGCCGCCGCCGACGCgcatgccattgacgcggcagatcaccggcttcttgca gttcaggatgccgtcgaccatggcgttgaacagatccatgtactcgccg isotig00106 gene=isogroup00032 (BzdB) TTGACGATCTTGCCGCCGGCCTTCTCGAACGCTTCCTTGAAGCCCGCGACCGACTGC TCGCCAAACGAATATTTCCAGGAGAGCGTGACGACGTTCTTGTGCTTCTTCTCGGCC ATCACGTTGCCCATCGCGTAGGCGGGCTGCcACGCGGAGAACGACGTACGGAAGAT GTTCGGCGCGCACAGCGGACCGGTGATTTCGTCCGCACCGGCGTTCGGGACGATCA ACAGCGTCTTCGTCTCGCGCGCGACTCGCGTCATCGCGAGCGCGACGCCGGAATGC ACGGTGCCGACGAGGACATCGACGTTGTCGCGCTTGATGAGCTTGTTCGCGTTCTCC GGCGCCTTCGCGGCGTCGGATTCGTCGTCGACCGTGAAGTACTCGATCTCGCGGCCG CCGAGCTTGCCGCCGCCCTGCTCCACCGCGAGCTTGAAGCCGTTCGTGATCGCGTTG CCGAGCGCTGCGTACGTGCCCGTGTAGGGGAGCATCAGTCCGAC
! ! 250! contig00073 gene=isogroup00008 (BcrV/BzdV) cGACGccGTAGGTGATGAACTGGAATTGCGATTGCGTCATGGTGTCTCCCGTGTTCCC cGTTATTTCCCGGTTCTTCTCTCGGTTGATGGCTGGTGTTGATtGCTTCTCTTGCTTCCG TTCGGGCtCGCGCCCCGCGTCGGCCTTCGCTTCGTTCAGCCCGACcTGCGATCCGCCC TGGGGGGCCgTTGCAGGTCgATCCGAGCCgCGGGCgATGTCCGACGCGCCGCTcAATC CTGATCCCGGcgTTGCAGCCGGTACGGACACACCGCATCGCAGTCCGCGCAGCAGAC TTCGCCGTCGCAGCGGATATGCCACAGGTCCGCGCCGCAGTTCGTGCACCGGCACTT GGCATCCGCCGACAAGGCTGGCCGGACTTCCTCGGCCGACCAGAAATCGATGACCT GTCCCATGTCCGTTCCTCCTCAGAACAGGTCGTCGAGTTCGTCCGATCCGCCTCCCG GCATCTTTCcGTACTGCTGCAGATAGGCCTGGATCATCTGGCTTTCGCGCGAGGTGA ACATCGCCGCTTCCTCGAACACGAAGTTCGTCGCTTCGGCCCCGCGTTCGAGCTGCT CCGTCAAGCCTTCGCGCAGATTCTCGACACCCTTGATCATCAGCACGTTCTTGTCGTC GTCGAGCAGCTGGAACACGCCCGCCTCGGTCGGCACCGCGGCGATCGACGCCTCGT CGAACTTCAGCGACGTCTCCGGCGGCAGCACCGCGTCGGCGATCTTCGGCGCGAGG TTGCACTTGAAGCAGCGTGCAGCCTCGGCGCGGGCGGTCGCGTCGTCGAACGCGAG CTCGACTTCGTCCCAGCCGCTGCGCTTTTCGGGCGCCAGCAGGATCGGGTGGAACTT GCGGACCTTATTGAAGCCGTCCTTGCGACCGATCCACGGGTTCGTTTCCCAGCCGTC GCGCAgcagcacttccgagatgtcgccgtcgccgccgagcgcgaggtccatcgccgaagccgcgac isotig00094 gene=isogroup00020 (BzdR) GTATTCGCGAACTCCGCGCCCGTCGCGGCATGACCCGCAAGATTCTCGCCCGGCAAT CCGGCGTCTCCGAGCGCTATCTCGCACAACTCGAAACGGGACACGGAAACATCTCC ATCATATTGCTGCGCCAGATCGCCCACGGGCTCGGACTGCCGATCGTCGATCTGGTT CGCGAGGAGGCCGAGCAACCGGCCGAGCTCGCGCAACTGATCCAGTACCTGAACCG CTTGCCGCCGACGGCGTTGGTACGCGCCCGGCAACTGCTCGAAGCCGAaGTCGCGTC GGCCGGCGAAGCGTCGCGCCGCCAGCGGATCGCGCTGATCGGCCTGCGCGGCGCCG GCAAGACGACCCTCGGCACGATGCTTTCCGAGCATCTGGGCGTTCCGTTCATCGAGC TCGCGAAGAGAATAGAGCAGGACGCCGGTGCCGAACTGTCGGAGATTTTCTCGCTG TACGGGCAGGCGGCATATCGCCGTTACGAGCGGCGCAGCCTCGAGGCGATCGTCGA G
! ! 251! isotig00127 gene=isogroup00053 (BzdT; BzdS; Fdx/BzdM) atcagcatcaacacggacaagtgcatcGAGTGCGCCGATCGCGGCTGCCTGACCGCCTGTCCGCACG AGATGTTCGAAATCATCACCGACGACTACGACGACGAAGTCGCCTGGGTCAAGAAG GAAAAGACCCGCGCGCTCGCGTACGACTGCGCCGAATGCAAGCCGGCCGGCCACAC GACGCTGCCCTGTACCACCGCCTGCACGCCGGGCGCGATCAAGCACTCCTGGTGAG GGACGTGACATGAACACGACGACAGGCGATACCGCAaTGAgCACGAAGAAGCTAAT CCCGATCAAGCTCGCCGGCTCGACGACGCCGGAAGAGGATGTCGAAGAGCGTCTGC TGGGCCAGGGCTGGAAGCGCCTGACGACGATTGGCGAGCCGCGACTGTCCGAGATC GCCGAAGCGTATCGCGGCATGGGTTTCGAAGTGCATGTCGAGGTCTGGAAGAGCGA AGGCGACGGCTGCAACACCTGCCTCGACGCGGACACCGAAATGGGCAAGATCCTGG GCACCGTCTATACCCGCCCGGGCAACGGCCCGAAGAAAGACGACGAGCTTTTCTGA ACGACCGACGCGCTCCGAGCGCGGAAACGGGTGACACCTTAGGAGGAAGGCAAAT GGCGGACAAGAAGCGTGTAATGCGGGTGCTGAACAAGGACGACCTGAACGCGGTCG TCAGGATCGACGCGGCGGCTTCCAAGGAAGAGCGCCGTGAGTACTACCAGCGCAAG GTCGACGCGACCGTCAATCCGAAGCACAACATCAACGCGTCGCTCGTCTGTGAAAT CGACGGCAAGGCGGTCGGCTTCGTGATGGGTGAAGTGTATTTCGGCGAGTACGGCA TTCCCGAAACCACGGCGACGATCGACACGATCGGCGTCGATCCTGAATACCAGAAC CACGGTGTCGCGAGCGAGCTGCTCGACCAGTTCATCACGAACATGAAGGCAGCGGG CGTCAGCAAGATCTACACGCTGGTGAACTGGGACGACTTCGCGCTCGAGAAATTCTT CTCGCGGCAGAAGTTCACGCCGTCCAAGCGCATCAACCTCGAATACACCCTCGCCTG ATCACGGCGTCACCGGGAGCCTTCGCCATGTGGATCGACTGCCATTGCCATACGAAG TACTCGTACgacaactg isotig00091 gene=isogroup00017 (BzdU) ctGACCGCCGCTATGAAACTCTTGATGTCGGTCACGGGCTGCAGAAATTCCGCCGCAC AGCGCCCGACCGCTTCCGTCTTGTGGCAATCGCTGCCGCCGAGCGACGGCAGCCGC ATGTGCCCCGCCGCGCTGATGGCCAGCCCGTTGTCGCTGTCGCGGTTACCGCCGTTG TGCGTTTCAATCGCGGCAATTCCGCTCAGGTCGAGCAGCCCTTCGAGCAAGCTCGAC ACCCCGATTTCGCGAAACGGGTGGGCCGGGACGaagatcccgccgagcccgttaatgtgttcgacgacctc gttgatcgggagatagttgtcgcgtccccagatattccagccgtcatcctcgaccccgtacgcgagcatgtgtccgcgatcggtggcgatct cgacgccgcgcagcaccagcagcccctcatcgcgcccgacctgctcgaccggctcggacgcttcgtacgagtagtgctcggtgatgcac accgcgtcgag
! ! 252!
Nucleotide sequences of the genes that were transcribed in Cartwright Consolidated grown on benzene
GMRHGY404JC5OM (AbcA) ACTCGCGTCGTGTCAAAGCAAACCTTAGATGCCTTGCTCCAGGTTCTCTCTTCCATGT CAAGGAATGGGTGCAGCGGCACGCCGACCGTCTTGTCGAAGAAATGCGTTCCCCGT TTCGGATGCCATTTTGTGGCAAAACATGCAATAACCTGAGATAGATCCTGAACATCA ACATCCTCATCGCAAACATAAATCTGGTGAAGGAAAAGACCAGTATTCTTTTCACCG AAGATCAGCTTAGCTACCTTATTGGGAATACTGCTGTAAGGTTTCTTGATGGAAATG AATGCTGAGTGCATAACCATTTCCGTCGGCATCGATACATCAAGTACCGGTATACCG TTTTCTTCCAGAA
GMRHGY404IQFR7 (AbcA) ACTCGCGTCGTCTGCCAGTGCCGACAGCATATCCTGATCTTGGCCAAACTCCTTCCA GGTGCTGCAGACCTTTTCTATAACCCGTTCTGGGTAAATATCCCAGAAGCTGGAGCG GACCGGGACTTCCGCCGTAGGCGACCATTCGCTGGGCCAGGTGCAGTCAAAGCAAA CCTTAGATGCCTTGCTCCAGGTTCTCTCTTCCATGTCAAGGAATGGGTGCAGCGGCA CGCCGACCGTCTTGTCGAAGAAATGCGTTCCCCGTTTCGGATGCCATTTTGTGGCAA AAACATGCATAACCTGAGATAGATCCTGAACATCAACATCCTCATCGCAAAACATA AATCTGGTGAAGGAAAAGACCAGTATTCTTTTCACCGAAGATCAGCTTAGCTACCTT ATTGGGAATACTGCTGTAAGGTTTCTTGATGGAAATGAATGCTGAGTGCATAACCAT TTCCGTCGGCATCGATACATCAAGTACCGGTATACCGTTTTCTCCAGAACCCGCATC
GMRHGY404IQ0MN (AbcD) ACTCGCGTCGTGCGGGAATAATTTGTTTATTTAATTTAAAAAGCAAAGGAGGAGAG GTTTTAATGTACGAAAAACCGTCAAACCGTTTGGAAATCCATAAAAATTTTACTTTT GGACCTGATCCTGATGAATTGAAAAAGCAGGAAGGCCGGGAGGTTTACGATGTAAT CATCCTGTCGGACTCCATTGATGAGCTTACCAGTCAACAGAATCAGGAAATTACGCT AGCCATCAGGTTGCTTACTCCTAAAGACAGAAAGATGAGGTACTGCACTAGGACGG TCAGTGCGATGATCAGCAGCGATCCGACCAAATACCGTGATCGTTTGCATGTGCGGT TCCAGCGGGGGTTATTGCATCCGAAACCCTGGAGTATTGAAATTGTTAAAGAACTTG AGGGCTTGATGAGCTTGGAAGCCGATCACACCATAGCACGTTCTTAAATATTGATAG AAAGGAAAGGTGATGTCCAAGATGTATCGT
GMRHGY404IXLZN (AbcD) ACTCGCGTCGTAACGTTTTTGTATATGAACACAAAAGTAAACCGGGGGAATCGGAG GGGAGGTGAAAATAAAGAAAATTTTAAGACGATTGACAGGAAGAGGATAAGTTATC TAGGGTTTTGCTTCACACGTTGCGGGAATAATTTGTTTATTTAATTTAAAAGCAAAG GAGGAGAGGTTTTAATGTACGAAAAACCGTCAAACCGTTTGGAAATCCATAAAAAT TTTACTTTTGGACCTGATCCTGATGAATTGAAAAAGCAGGAAGGCCGGGAGGTTTAC GATGTAATCATCCTGTCGGACTCCATTGATGAGCTTACCAGTCAACAGAATCAGGAA ATTACGCTAGCCATCAGGTTGCTTACTCCTAAAGACAGAAAGATGAGGTACTGCACT AGGACGGTCAGTGCGATGATCAGCAGCGATCCGACCAAATACCGTGATCGTTTGCA TGTGCGGTTCCAGCGGGGGTTATTGCATCCGAAACCCTGGAGTATTGAAATTGTTAA AGAACTTGAGGGCTTGATGAGCTTGGAAGCCGATCACACGACGGGAGT
! ! 253!
GMRHGY404I47SF (ORF133) ACTCGCGTCGTGAGGAATTGTATTTGCTTCTTTCAGCAGGGCGCTGGCCAAGCTAAG ACCGCTCAAAGTCATGGCTGGTCTATAAAATTGTTATGGAATAAAGATAGGAGGGTT ACTAATGGAAATTTGTTGCTGATAAGTTAAGGGAAGGGTTAGAAATATTTCACAGCT TTCTTGATGCCAACCAGGATAAGATTAAAAATGGAGAGCCTTTTATGATGCTTGTCC TAGATCTGGACAACATTTGTGACAAAGTAGTGAAAGGAATTGTGGCCCAGAATAGC GACGACGGGAGT isotig00071 gene=isogroup00022 (ORF126) CTCAAAGCTGACCCGTGTTGGGATCATCTCCTTGGGCCAAGTGGGATCAAGACAAGC ATTGATATACAAGCGCCCTGTGCGCTCACCGTCGTTCGGATATTTGGCAGAACGCGT AATTGCCGCCACATCGTCCTTAGAGAAATGGAAGGCATGCACCGGCGAAGCCTTTTG GATAAAGTCCTGCATAACCCTAGCCAACTCTTCGGCGCCGAGGGCGTTATCAACCAG CATTAGTTTGTCAAACCAGTGGCTGTAAGCGAAAACAATACGGGAGAAGTGGAACT CAAACCCTGGGTAGAGGGCGTTCACGGCGACTATACCCATCCCCATACGCATAGAA ACAGGGCAATAGACCCAACGGGCAATTTTGGTCCCCCACCACCAGCTGTTCAAAAT CCGATAGATTTTAAACGAGTGGGCCCAAGAGCGGAGACTCATAGAATCACTACCCT TGATACCGGTAACGTCAaATGGTATAATAGGATTTGTGCGGTGGGAAATTGCCTTGA CGGTGAATATTGGTTGCCATGTTGCTTTCTCGATGCGGTAGAAATTCGGATATGGCC CTTCTAAGGCAGTTTCCCCCGGTATAGCAACACCCTCTAGGATTATTTCGCTGCTGGC CGGAACAAGCAGACCATTGGTTTGGGTTTttACCAGTTCAATCGGTTCGCCACCCAAA TATCCGGCAAGGTCCGCGGCAGGAACTTCACGTTTCAGTCCGCCCAGCCCCACTCTT TCCATGAACACGGCTGTCGCTAAAGGGATGCTGGAGTCTAC
GMRHGY404IZ92L (BamD) ACTCGCGTCGTTTTACACTGAATTCAGGATACTCGTTCTTAAAGGTATGGTAACAAT GAGGTGAATTAACAAGGATTCTCTTAACCCCGTTGTCAATAAAGGTTTGATGTTTTC TCTGGCCATGCGTTTGAATAATTCCTCATTGCCTGTTTTCCGGATGCTTTCGCCGCAG CAGTTTTCCTTGGCACCCAGGATTCCAAAGCCCACCCCGGCTTTTTGGAGGATGTTG GCTGTAGCCTGGGCCACCTTTTCAATCTTGAATCAAAGCTCAGGTAGCAGCCGGGAA AATAAAGGATTTCCATCCCTTCGGCAAAGGGCTTGACACCAAGGCCTTCGGCCCAG GCTGCCCTGTTGGCCCTGGCTTCACCAAAGGGGTTGCCTTCGGCGTTCAGACCTGCA CTAACTCCGCGGTACGGCTTGACGGCGGCAGGAAATACACCATAACCTGTGGCCAT TCTTCTCAGGCCGACCATGTTTTGAATCTGTTTAACATCCCGGGGGCACTTCTGGGG GCATTTACCACAGGTTGTACAGCGCCAGATCTCTTCATTTTCGATTTCGGTAAGGCCT AAA
! ! 254!
GMRHGY404IBD8N (BamE) ACTCGCGTCGTTTTATATCCTCTTTAGAGATATCCGGACCTACGGATATAATGAAGA TTTTTACCGGGAAGCGGCAAGTAAAGAAGTTAAGTTTATCAGATATACACCTGAAG ACAAACCGGAGGTAGAAGCCGTAAAAGAGGGCGGCAAATCCGTGCTCAGGGTAAC AGTGACCGATCCTGTTTTAGGTGCCAGGCTGGAGATAGATGCTGATAACATAGCTTT AGCGGCAGCCATCATTCCTGGTGAAGGGACCCGGGATGCCATTCAGCAGTTTAAGG TAACCGCGAGTCCTGACGGTTTCTTCAAAGAAGCCCACGTCAAATTAAGGCCTGTAG AGTTTGGTGTAGATGGAGTATATCTCTGCGGGCTGGCCCACTATCCCAAGTTTATCT CAGAAACAGTCAGCCAGGCCTATGGCGCAGCCGGGCGGGCCATCACTCTGCTCTCT CATGATACCGTAGTAGCGTCGGGCTCTGTTTGTGAAGTCAAAGAGATAAATTGTATG GGTTGTGGTGCCTGTG
GMRHGY404I8T7K (Dch/BzdW) ACTCGCGTCGTCGATCCCGACGCTCGCCGCGGTCAATGGCGCAGCACTCGGCGGCG GCATGGAAGTCGCGATCGCGTGCGACATGATCGTCGCGTCGGCGAACGCGAAGTTC GGCCAACCGGAGATCAAGCTCGCGGTGTTCCCGCCGATCGCCGCGGTGCTGCTGCC GCGCCTCGTGGCGCCGGCACGCGCGATGGAGCTGCTGCTCGGCGGCGAGAACATCG GCGCCGACGAGGCGAAGGCGATCGGGCTGGTGAACCGCGTATTCGCGAGGGAGTCG TTCCGCGACGAGGTCGCCGCCTTCGTCGCCCCGTACCTCGCACTGTCGCGCGCCGCG CTCGCCTCGACGCGCAAGACGATCCGGGCCGCTGCGGGCAAGCCCTTCGGCACCGC GCTCGACGCGGCCGAGAACATCTACTTGAATGAGCTGATGGCGACCGAGGACGCGA AAGAAGGGCTAGCCGCCTTCCTCGAGAAGCGCAAGCCGGTGTGGCGCAAC
GMRHGY404HZ0K7 (Dch/BzdW) ACTCGCGTCGTCGATCCCGACGCTCGCCGCGGTCAATGGCGCAGCACTCGGCGGCG GCATGGAAGTCGCGATCGCGTGCGACATGATCGTCGCGTCGGCGAACGCGAAGTTC GGCCAACCGGAGATCAAGCTCGCGGTGTTCCCGCCGATCGCCGCGGTGCTGCTGCC GCGCCTCGTGGCGCCGGCACGCGCGATGGAGCTGCTGCTCGGCGGCGAGAACATCG GCGCCGACGAGGCGAAGGCGATCGGGCTGGTGAACCGCGTATTCGCGAGGGAGTCG TTCCGCGACGAGGTCGCCGCCTTCGTCGCCCCGTACCTCGCACTGTCGCGCGCCGCG CTCGCCTCGACGCGCAAGACGATCCGGGCCGCTGCGGGCAAGCCCTTCGGCACCGC GCTCGACGCGGCCGAGAACATCTACTTGAATGAGCTGATGGCGACCGAGGACGCGA AAGAAGGGCTAGCCGCCTTCCTCGAGAAGCGCAAGCCGGTGTGGCGCAAC
! !
References
Imachi, H., Y. Sekiguchi, Y. Kamagata, S. Hanada, A. Ohashi and H. Harada (2002). "Pelotomaculum thermopropionicum gen. nov., sp. nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium." Int. J. Syst. Evol. Microbiol. 52: 1729-1735.
Imachi, H., Y. Sekiguchi, Y. Kamagata, A. Loy, Y. L. Qiu, P. Hugenholtz, N. Kimura, M. Wanger, A. Ohashi and H. Harada (2006). "Non-sulfate-reducing, syntrophic bacteria affiliated with Desulfotomaculum Cluster I are widely distributed in methanogenic environments." Appl. and Environ. Microbiol. 72(3): 2080-2091.
Klappenbach, J. A., P. R. Saxman, J. R. Cole and T. M. Schmidt (2000). "rrndb: the ribosomal RNA operon number database." Nucleic Acids Res. 29(1): 181-184.
McCarty, P. L. (1971). Energetics and Bacterial Growth, in Organic Compounds in Aquatic Environments, Marcel Dekker.
Qiu, Y. L., Y. Sekiguchi, S. Hanada, H. Imachi, I. Tseng, S. S. Cheng, A. Ohashi, H. Harada and Y. Kamagata (2006). "Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens." Arch. Microbiol. 185: 172-182.
Rabus, R. and F. Widdel (1995). "Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria." Arch. Microbiol. 163: 96-103.
Reinhold-Hurek, B., T. Hurek2, M. Gillis, B. Hoste, M. Vancanneyt, K. Kersters and J. D. Ley (1993). "Azoarcus gen. nov., nitrogen-fixing proteobacteria associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth), and description of two species, Azoarcus indigens sp. nov. and Azoarcus communis sp. nov." Int. J. Syst Bacteriol 43(3): 574-584.
Rittmann, B. E. and P. L. McCarty (2001). Environmental Biotechnology: Principles and Applications. New York, McGraw-Hill.
Ulrich, A. C. and E. A. Edwards (2003). "Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures." Environ. Microbiol. 5(2): 92-102.
Urich, T., A. Lanze´n, J. Qi, D. H. Huson, C. Schleper and S. C. Schuster (2008). "Simultaneous assessment of soil microbial community structure and function through analysis of the meta- transcriptome " PLoS One 3(6): e2527.
! 255!