ABSTRACT GRAY, STEVEN R. Functional Genomics Analysis Of

Home , FMN adenylyltransferase, Strain 121, Thermotoga elfii, Thermotoga hypogea, Thermotoga maritima, Thermotoga naphthophila

ABSTRACT

GRAY, STEVEN R. Functional Genomics Analysis of Carbohydrate Conversion to Biohydrogen by Pure and Mixed Cultures of Hyperthermophilic Thermotoga Species. (Under the direction of Dr. Robert Kelly).

The genus Thermotoga is comprised of fermentative anaerobes with optimal growth temperatures as high as 80°C. To understand the genetic and physiological diversity within this genus, the genome sequences of five Thermotoga species (T. maritima, T. neapolitana, T. sp. RQ2, T. petrophila, T. lettingae) were compared using bioinformatics tools. Except for T. lettingae, the genomes exhibited high degrees of homology and shared organizational traits. The in silico comparison was supported by genomic DNA cross-hybridization to a T. maritima cDNA microarray, where 83-94% of the probes in the three other Thermotoga species were recognized. These results indicated that the four Thermotoga species share a core genome (~1470 ORFs); ORFs unique to particular species likely reflect the influence of specific environmental or evolutionary factors. The significant homology among the four Thermotoga species facilitated development of a multi-species cDNA microarray for use in pure and mixed culture transcriptional response studies.

The Thermotoga multi-species cDNA microarray was used to examine pure and mixed culture transcriptomes for growth on glucose and on a polysaccharide mixture.

The multi-species array was used to estimate species composition of the mixed culture; composition varied from 6:1.5:1:1 for glucose batch culture to 2.3:2:2:1 in glucose continuous culture for T. sp. RQ2: T. maritima: T. petrophila: T. neapolitana, respectively. Composition in polysaccharide batch culture was similar to glucose continuous culture. Transcriptional response analysis provided clues to interspecies

interactions. In glucose mixed culture, the ORFs encoding a phage tail-like bacteriocin

(TM0785), lon proteases (TM1633, TM1869), E (TM1598), and a putative bacteriocin

(TM1300) related to subtilosin A from Bacillus subtilis, were up-regulated relative to pure cultures. Differential regulation of several ORFs encoding HicAB Toxin-Antitoxin pairs (TM1310a-1313, TM1320-21) was noted, suggesting a potential role in interspecies interactions. Comparisons of growth on glucose and polysaccharides revealed changes in both core and non-core ORF transcription. All cultures exhibited upregulation of core genome xylan (TM0056-61, TM0070-77) and β-mannan utilization operons (TM1218-

1223) on polysaccharide culture. An unclassified ABC transporter operon found only in

T. neapolitana and T. sp RQ2 (TRQ2_0970-75) and a β-linked exopolysaccharide operon found only in T. maritima (TM0622-30) were up-regulated on glucose. A β-mannan utilization operon (TM1746-51), found only in T. maritima and T. sp RQ2, was up- regulated on polysaccharide culture.

To investigate the potential of Thermotoga for biofuels production, biohydrogen generation through carbohydrate fermentation was examined for both pure and mixed

Thermotoga cultures. T. maritima showed that, unlike the hyperthermophilic archaeon,

Pyrococcus furiosus, which uses similar fermentative metabolism, sulfur had minimal effect on transcription. Furthermore, T. maritima preferred cellobiose over maltose, perhaps related to superior bioenergetics mediated by a cellobiose phosphorylase

-3 (TM1848) encoded in its genome. Volumetric H2 production rates (~1.2x10 mol H2 liter-1 hour-1) were similar for pure and mixed cultures, perhaps related to the function of metabolic pathways comprising the core genome.

This work demonstrates the usefulness of multi-species arrays for examining closely related Thermotoga. The results indicted that, while differences in transcription were noted among pure and mixed cultures, culture growth and H2 production levels are not affected by species, substrate, or competition.

Functional Genomics Analysis of Carbohydrate Conversion to Biohydrogen by Pure and Mixed Cultures of Hyperthermophilic Thermotoga Species

by Steven Randall Gray

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Chemical Engineering

Raleigh, NC

2009

APPROVED BY:

______Dr. Amy Grunden Dr. Balaji Rao

______Dr. Jason Haugh Dr. Robert Kelly Chair of Advisory Committee

DEDICATION

I would like to dedicate this thesis to my parents for their constant support.

BIOGRAPHY

Steven Randall Gray was born in Greenville, South Carolina, but moved to

Cincinnati, Ohio before the age of two. There he attended Hopewell Elementary School and Liberty Junior High School before moving on to St. Xavier High School. He attended the University of Virginia as an Echols Scholar, receiving his degree in Interdisciplinary

Studies with a concentration in Biochemistry. He then attended the UVA School of

Engineering for his Masters of Science in Chemical Engineering. After graduating he worked briefly at the University of Cincinnati before coming to North Carolina State

University for his Ph. D. work.

iii

ACKNOWLEDGEMENTS

I have to start by thanking my parents, my grandparents and the rest of my family, my friends and everyone else in my life that has supported me outside the lab through this long process.

So many incredible people I‟ve worked with here at NCSU have helped me get to where I am. So first I have to thank Clemente Montero for training me in the ways of

RNA and Keith Shockley for providing all sorts of microarray analysis consulting services. Also here in the Kelly lab, I have to thank the man himself, Dr. Robert Kelly, for providing me with the mentorship I needed to get through this process. Thanks to the rest of the grad students and post-docs in the Kelly Group: Matthew Johnson, Shannon

Conners, Kevin Epting, Donald Comfort, Joshua Michel, Sabrina Tachdjian, Hank Chou,

Morgan Harris, Derrick Lewis, Amy Vanfossen, Inci Ozdemir, Charlotte Cooper,

Andrew Frock, Jaspreet Notey, and Sara Blumer-Schuette.

There are also those outside the Chemical Engineering Department who have helped me get here. Thanks to the BIT program people who helped so much with my classes and material lab support: Dr. Susan Carson and Dr. John Chisnell. Last but not least among those at NCSU, huge thanks to the man who really keeps the Chem-E

Department running, Kit Yeung. Beyond North Carolina, I have to thank Dr. Camilla

Nesbø and her Norwegian-Canadian support team for providing us with more novel bacteria than we knew what to do with.

TABLE OF CONTENTS

LIST OF TABLES ...... vii

LIST OF FIGURES ...... ix

CHAPTER 1: Extremely thermophilic microorganisms ...... 1 ABSTRACT ...... 2 INTRODUCTION ...... 3 OBTAINING EXTREMELY THERMOPHILIC MICROORGANISMS FROM GEOTHERMAL SITES ...... 6 BIODIVERSITY OF EXTREME THERMOPHILES ...... 10 CULTIVATION METHODOLOGY FOR EXTREMELY THERMOPHILIC MICROORGANISMS ...... 23 NEW DEVELOPMENTS ...... 30 CONCLUDING REMARKS ...... 39 REFERENCES ...... 41

CHAPTER 2: Comparative genomic analysis of hyperthermophilic Thermotoga species ...... 79 ABSTRACT ...... 80 INTRODUCTION ...... 82 MATERIALS AND METHODS ...... 86 RESULTS AND DISCUSSION ...... 90 REFERENCES ...... 106

CHAPTER 3: Glucose and polysaccharide transcriptomes reveal physiological differences in hyperthermophilic Thermotoga species grown in pure and mixed cultures ...... 137 ABSTRACT ...... 138 INTRODUCTION ...... 140 MATERIALS AND METHODS ...... 142 RESULTS AND DISCUSSION ...... 150 REFERENCES ...... 167

CHAPTER 4: Biohydrogen production by Thermotoga species growing on simple and complex α- and β- linked carbohydrates ...... 197 ABSTRACT ...... 198 INTRODUCTION ...... 200

MATERIALS AND METHODS ...... 203 RESULTS ...... 211 DISCUSSION ...... 215 REFERENCES ...... 223

APPENDICES ...... 239 APPENDIX A: ORFs of the examined Thermotoga that are unique by 70% nucleotide identity...... 240 APPENDIX B: ORFs differentially transcribed in microarray experiments...... 255

LIST OF TABLES Page

Table 1.1. Representative extreme thermophiles ...... 75 Table 1.2. Thermotoga species and strains and their isolation sources ...... 77 Table 1.3. Biohydrogen yields of representative mesophiles and extreme thermophiles . 78

Table 2.1. Biogeography of Thermotoga isolation sites...... 124 Table 2.2. Genome properties of the four sequenced Thermotoga species examined in detail in this chapter...... 125 Table 2.3. 16S rRNA % identity in the of the sequenced Thermotoga...... 125 Table 2.4. The number of ORFs shared among the Thermotoga based on a 70% nucleotide identity threshold...... 126 Table 2.5. CRISPR related genomic inversions in T. petrophila and T. sp RQ2 compared to T. maritima...... 127 Table 2.6. Genomic DNA cross hybridization success rates for the sequenced Thermotoga species and the novel Thermotoga strain...... 127 Table 2.7. ORFs that failed to hybridize to the T. maritima array during homologous genomic DNA hybridization for T. neapolitana, T. petrophila, and T. sp RQ2...... 128

Table 3.1. Unique ORFs/probes used to determine the composition of the Thermotoga zoo...... 180 Table 3.2. Thermotoga species with full genome sequences ...... 183 Table 3.3. 16S rRNA phylogeny for genome-sequenced Thermotoga species ...... 183 Table 3.4. Hybridization of Thermotoga species genomic DNA to T. maritima cDNA microarray ...... 184 Table 3.5. Common ORFs in selected Thermotoga genomes based on 70% homology at the nucleotide level...... 185 Table 3.6. Non-core genome features in Thermotoga species ...... 186 Table 3.7. Estimation of Thermotoga zoo population composition ...... 187 Table 3.8. Differential transcription of Thermotoga spp. for growth on the polysaccharide mixture compared to glucose...... 188 Table 3.9. ORFs encoding sugar utilization proteins in Thermotoga genomes. Positive values indicate up-regulation in polysaccharides vs. glucose, shaded squares indicate not present in the genome...... 189 Table 3.10. Exopolysaccharides synthesis operon regulatation on glucose and polysacahrides...... 193 Table 3.11. ORFs differentially transcribed in the mixed culture compared to the pure cultures of each Thermotoga species growing on glucose...... 194

vii

Table 3.12. ORFs up- or down-regulated ≥2-fold in the Thermotoga zoo compared to pure cultures ...... 194 Table 3.13. Response of selected ORFs in Thermotoga species in mixed culture vs. pure culture...... 195 Table 3.14. Differentially transcribed ribosomal protein ORFs in mixed culture during growth on glucose...... 196

Table 4.1. ORFs differentially transcribed in T. maritima during culture with and without sulfur...... 233 Table 4.2. ORFs differentially transcribed between cellobiose and maltose during chemostat growth...... 234 Table 4.4. ORFs differentially transcribed upon transition from maltose culture to maltose and cellobiose culture and then back to maltose culture...... 236 Table 4.5. Growth and H2 production properties of 10 ml/min and 50 ml/min sparged batch cultures of T. maritima...... 238

Table A1. T. maritima unique ORFs by 70% nt identity ...... 241 Table A2. T. neapolitana unique ORFs by 70% nt identity ...... 243 Table A3. T. petrophila unique ORFs by 70% nt identity ...... 251 Table A4. T. sp RQ2 unique ORFs by 70% nt identity ...... 253

Table B1. Fold changes of core ORFs in dye flip experiments ...... 256 Table B2. LS means differences of non-core ORFs in dye flip experiments...... 269 Table B3. ORFs differentially transcribed in the T. zoo vs. T. maritima in polysaccharide culture...... 276 Table B4. ORFs differentially transcribed in the T. zoo vs. T. neapolitana in polysaccharide culture...... 283 Table B5. ORFs differentially transcribed in the T. zoo vs. T. petrophila in polysaccharide culture...... 299 Table B6. ORFs differentially transcribed in the T. zoo vs. T. sp RQ2 in polysaccharide culture...... 303 Table B7. ORFs differentially transcribed in the T. zoo vs. T. maritima in glucose culture...... 308 Table B8. ORFs differentially transcribed in the T. zoo vs. T. neapolitana in glucose culture...... 325 Table B9. ORFs differentially transcribed in the T. zoo vs. T.petrophila in glucose culture ...... 336 Table B10. ORFs differentially transcribed in the T. zoo vs. T. sp RQ2 in glucose culture...... 359

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LIST OF FIGURES Page

Figure 1.1 Phylogenetic tree based on 16s rRNA shows evolutionary relationships among microorganisms. From: Pace, N.R. Science, 1997. (181)...... 72 Figure 1.2 Co-culture of Thermotoga maritima (rods) and Methanococcus jannschii (cocci), both hyperthermophiles, exist in exoploysaccharide matrix at 80oC. From: Johnson, et al., Mol Microbiol, 2005. (120)...... 73 Figure 1.3 Nanoarchaeum equitans (small cells) and Ignicoccus hospitalis (large cell) have a parasitic/symbiotic relationship at high temperatures. From: Huber, H. et al., Nature, 2002. (105)...... 74

Figure 2.1 Map of Thermotoga species and strain isolation sites. Different color rings indicate different species and strains ...... 115 Figure 2.2 Fractions of unique and similar ORFs as a function of percent identity for the sequenced Thermotoga ...... 116 Figure 2.3 Venn diagram of the shared ORFs of the sequenced Thermotoga as defined by having 70% nucleotide identity...... 117 Figure 2.4 Fraction of ORFs unique and in common between T. maritima and T. lettingae above a percent identity theshold using nucleotide and amino acid comparisons ...... 118 Figure 2.5 Genome alignment dot plot of T. maritima vs. T. lettingae ...... 119 Figure 2.6 Dot plot genome alignment results of T. maritima vs. T. neapolitana ...... 120 Figure 2.7 Dot plot genome alignment results for T. maritima vs. T. petrophila ...... 121 Figure 2.8 Dot plot genome alignment results: T. maritima vs. T. sp RQ2 ...... 122 Figure 2.9 Cumulative G+C skew plots of T. neapolitana, T. petrophila, T. sp RQ2 ..... 123

Figure 3.1. Venn diagram of the shared ORFs of the selected Thermotoga species, based on 70% identity at the nucleotide level ...... 174 Figure 3.2. Loop design for growth on glucose and polysaccharides ...... 175 Figure 3.3. The loop experimental design for the Thermotoga zoo batch culture experiment...... 176 Figure 3.4. The loop experimental design for the Thermotoga zoo continuous culture experiment...... 176 Figure 3.5. Population composition for Thermotoga zoo growing on glucose-based medium for batch culture (A) and chemostat culture (B) and the polysaccharide mixture for the zoo (C)...... 177 Figure 3.6. Comparison of the TM_1299 through TM_1336 gene neighborhood across multiple Thermotoga spp...... 179

Figure 4.1. Experimental loop design for cellobiose-maltose batch experiment ...... 230 Figure 4.2. Experimental loop design for cellobiose-maltose chemostat experiment. ... 230 Figure 4.3. H2 production on a volumetric basis for both glucose and polysaccharide batch culture...... 231 Figure 4.4. Volumetric H2 production after accounting for differences in cell densities at time of measurement and RNA sampling...... 231 Figure 4.5. Full T. maritima growth curves for glucose culture with 10 and 50 ml/min sparge rates...... 232 Figure 4.6 H2 production rates for the full growth cycle of 10 and 50 ml/min sparged batch cultures of T. maritima on glucose...... 232

CHAPTER 1: Extremely thermophilic microorganisms

Steven R. Gray1, Michael W.W. Adams2 and Robert M. Kelly1

1Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905

2Department of Biochemistry and Molecular Biology University of Georgia Athens, GA, 30602

This chapter will be published in:

Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology

Michael C. Flickinger, Editor-in-Chief

John Wiley & Sons

ABSTRACT

Extremely thermophilic microorganisms (Topt ≥ 70°C) are found in geographically diverse marine and terrestrial environments and represent a wide range of growth physiologies. Extreme thermophiles thrive at high temperatures and, as such, different approaches must be taken to cultivate them in laboratory settings. Genome sequences of many extreme thermophiles have been completed and offer a glimpse into the basis for their high temperature life styles. A number of biotechnological applications have been envisioned that take strategic advantage of their thermophilicity, including the production of biohydrogen and recovery of base and precious metals from ores. As genetic systems are developed and implemented for extreme thermophiles, metabolic engineering approaches will be possible to tune the unique characteristics of these microorganisms for bioprocessing uses.

INTRODUCTION

It has been over 35 years since the first reports that microbial life was possible above 80°C (41) and in that time the number and diversity of known thermophilic, or

“heat loving,” microorganisms has expanded considerably. The so-called extreme thermophiles, defined here as organisms having an optimal growth temperature of at least

70°C, come from a broad spectrum of sources ranging from terrestrial hot springs to seeps associated with volcanic activity, deep sea hydrothermal vents (121, 156, 240), oil wells and reservoirs (251), and volcanic sediments (84). The most thermophilic of the extreme thermophiles are often referred to as hyperthermophiles (growth at temperatures of 90°C or above), some of which grow at temperatures of 100°C or higher (34, 145). At issue for some time has been the ultimate maximum temperature at which microbial life proceeds (25, 64). From evidence to date, microbial growth in pure laboratory culture ceases near temperatures commonly associated with autoclaves. Pyrolobus fumarii has a reported growth temperature optimum of 113oC (34), while another microbial isolate, referred to as Strain 121, has been reported to grow up to temperatures of 121°C (64).

Some extreme thermophiles are capable of withstanding other extremes in addition to temperature. Thermococcus barophilus (Topt = 85°C) not only thrives, but grows preferentially, at pressures as high as 17.5 MPa (156). Thermococcus gammatolerans, the most radiotolerant extreme thermophile yet discovered, grows at 85°C while under exposure to 30 kGy of gamma radiation (121). An extreme thermoacidophile,

Metallosphaera sedula, grows optimally around 75°C and at pH values of 2 or less

(107). Table 1.1 lists selected extreme thermophiles, along with their growth characteristics.

Protocols for culturing these novel organisms in laboratory settings have been developed (210, 211), facilitating examination of their physiological and metabolic characteristics (62, 99, 133, 225) and production of numerous highly thermostable proteins in native and/or recombinant forms for biochemical, biophysical and biotechnological evaluation (5, 262). Genetic techniques are being developed with certain extreme thermophiles, which will eventually enable metabolic engineering efforts and give rise to cloning and expression systems that are functional at elevated temperatures

(157, 220, 237).

The driving force to study extremely thermophilic microorganisms arises from both their scientific and technological potential. The origin of life on earth has been proposed by some to connect to this group of microorganisms (271). Furthermore, prospects for extraterrestrial life have been fortified by the discovery of these organisms in increasingly extreme environments on this planet, which were long thought to be barren of life. If one accepts the premise that among this group are the most primitive of all known life forms (68, 69, 269, 270), the templates for many metabolic strategies and bioenergetic processes could provide a better understanding of more complex cellular phenomena. Fundamental insights into the basis for biomolecular stability, particularly proteins, have been sought through the study of extreme thermophiles (6, 81, 82, 255).

Encouraged by the incredible utility of DNA polymerases from extreme thermophiles in

4 the polymerase chain reaction (PCR) (175, 189), commercial interest in enzymes from these organisms has always been high. Not only has biocatalyst instability often been a major drawback in many existing bioprocessing applications, it also restricts the utility of enzymes in the development of new products. Extreme thermophiles represent a fertile source of robust biocatalysts that may change this perspective.

Biochemical and biomolecular engineering has proven to enable expertise in the study of extreme thermophiles and is central to the eventual use of these organisms or their products for commercial purposes. The earliest efforts in this field of inquiry were facilitated by the development of unusual bioreactors and cultivation protocols to provide biomass for characterization (44, 56, 131). Imaginative approaches for utilizing extreme thermophiles, ranging from mixed communities of viable cells to thermostable enzymes catalyzing multiple step reactions, are being considered. As such, bioprocess engineering and technology will have to accommodate the anticipated application of extreme thermophiles and their constituent biomolecules.

In this review, the metabolic characteristics of extremely thermophilic microorganisms will be discussed in light of bioprocessing issues that arise in their cultivation. The physiological and phylogenetic diversity of extreme thermophiles will also be covered in addition to efforts to define the untapped microbial content of hydrothermal locales for biotechnological purposes. In a separate review in this volume, the characteristics of enzymes from extreme thermophiles will be examined.

OBTAINING EXTREMELY THERMOPHILIC MICROORGANISMS FROM GEOTHERMAL SITES

A variety of natural biotopes associated with hydrothermal features have yielded extremely thermophilic microorganisms (26, 33, 62). Samples from these sites (in the form of hot water, sediments, solid rock or even from intestines of animals inhabiting these sites (50)), inoculated into various types of liquid growth media at temperatures up to approximately 120°C and over the pH range from 0 to 11, can contain one or more culturable species of extreme thermophile. In fact, it has been demonstrated that samples from geothermal sites can contain a wide diversity of microorganisms and the cultivation of any in a laboratory setting may be difficult at best (23, 107, 180, 252). Obtaining samples containing extreme thermophiles from hydrothermal biotopes can be as simple as sampling mud from the edge of a hot spring, as has been done at Yellowstone National

Park (41). At the other extreme, to reach more remote sites, researchers must dive in a submersible several kilometers into the ocean and then expertly manipulate external sampling equipment to procure material uncontaminated by the cold surrounding seawater (156).

Although no correlation has been found between the degree of difficulty in sampling or remoteness of location and the novelty of the isolates obtained, sites which have unique environmental or ecological characteristics are continually sought. As more thermal locales are investigated, it has become clear that similar organisms are sometimes isolated from locations that have very different physicochemical characteristics and are geographically distant. For example, members of hyperthermophilic genera

Thermococcus, which are classified with the archaea domain, and Thermotoga, which are bacteria, have been found in shallow marine settings (109), deep sea vents (193) and both terrestrial surface waters (126) and petroleum reservoirs of various depths (251). To illustrate this, Table 1.2 lists the known members of the Thermotoga species and their wide-ranging geographic locales. The ubiquity of certain extreme thermophiles may reflect evolution under similar environmental conditions or, alternatively, a common source that was subsequently geographically distributed through global volcanic activity

(172, 206).

Enrichment cultivation of samples taken from hydrothermal niches to isolate extreme thermophiles typically follows methodology developed for mesophilic microorganisms, such as serial dilution and plating. However, there are several key exceptions (26, 211) and novel techniques for isolation of high temperature organisms have been developed (107). The method of enrichment culture can have a significant impact on the results with some common techniques favoring single, more robust organisms. For example, two enrichments from the same starting material can produce different results, depending upon bioreactor type. A gas lift bioreactor generated species from multiple genera from an environmental sample, while batch cultivation yielded isolates from a single genus (194). Phylogenetic analysis based on 16S rRNA sequences

(106) is often used to differentiate among isolates. Furthermore, new approaches to isolate novel extreme thermophiles have been reported (130), including the use of optical tweezers to separate individual cells (18, 19).

For extreme thermoacidophiles, typically growing aerobically at temperatures of approximately 75°C and at pH below 3.0, iron pyrite and/or elemental sulfur may be added to the medium to support chemolithotrophy (41). Many of these organisms grow better in complex media containing yeast extract or tryptone, although the nutritional requirements satisfied are often unclear. Most hyperthermophiles are anaerobic and oxygen must be eliminated from growth media. Fortunately, the reduced solubility of oxygen at such high temperatures somewhat simplifies anaerobic technique to the extent that glove boxes are usually unnecessary. Enrichment media for hyperthermophiles typically contain an array of trace elements, basic salts, oligosaccharides, complex substrates (e.g., yeast extract or tryptone) and elemental sulfur (26). Tungsten must be added as a trace element for several hyperthermophiles, which incorporate this metal into specific oxidoreductases (140). For example, a three-fold stimulation of cell density levels of one hyperthermophile, Pyrococcus furiosus, was noted in the presence of trace amounts of tungsten (225). Although there have been reports of chemically-defined growth media for certain hyperthermophiles, such media often result in significantly lower growth yields (210). Thermolability of many medium components is always a concern (143), especially for amino acids, such as glutamine and asparagine (210), certain vitamins and some monosaccharides. For example, maltose and cellobiose serve as excellent carbon/energy sources for P. furiosus growing at 98°C, while glucose does not. (257) Continuous culture growth can be used to reduce the potential for glucose

8 thermolability, however it is not a perfect solution as P. furiosus did not grow on glucose in a chemostat, so other factors may be involved (32, 73).

Extreme thermophiles can be very difficult to establish in pure, laboratory culture, whether the inoculum is directly from isolation efforts or from stocks. This is especially true for some of the hyperthermophilic, sulfur-reducing chemolithotrophs, such as

Pyrodictium occultum (186), which grow to very low cell densities, and are highly sensitive both to shearing forces and the presence of various metals (Schicho and Kelly, unpublished observation). Cultures of extreme thermophiles can be stored in various ways. In some cases, these microorganisms will remain viable for years in liquid media.

Attempts to freeze dry cultures for long-term storage have met with mixed success

(Rinker and Kelly, unpublished data). Lyophilization has been used successfully for some hyperthermophilic archaea, although there are also reports of limited storage times for organisms stored in this way (61). Lyophilization is carried out prior to storage of some extremely thermophilic bacteria by the German Collection of Microorganisms and Cell

Cultures (DSMZ) and the American Type Culture Collection (ATCC). The appropriate lyophilization protocol is highly species- and media-specific (164). P. furiosus (84) can be stored in dimethyl sulphoxide (DMSO) in glass capillary tubes over liquid nitrogen, although plastic cryotubes were found to be too permeable to oxygen thus ineffective for extended storage (61).

Plating techniques have been used for other hyperthermophiles including

Thermotoga species (56, 101), Aquifex pyrophilus (109), P. furiosus (200), and

Sulfolobus strains (95). Growth on solid media can be challenging, given the impact of high temperatures on gelation processes. Geoglobus ahangari, an anaerobic hyperthermophile, was grown on solid media prepared by first separately preparing two solutions (127). One contained the media components at twice the final concentration along with several components designed to interact with the second solution, which contained the solidifying agent gellan gum, L-cysteine hydrochloride (0.5 mM), and

FeCl2 (2.6 mM). The first solution was then inoculated and an equal volume of the solidifying agent was then added. Among the reasons driving efforts to develop culture methods on solid media at high temperatures is the necessity for this method in performing genetic manipulations.

BIODIVERSITY OF EXTREME THERMOPHILES

There are now over 100 species of extreme thermophiles that can be grown in pure laboratory culture and these are classified into at least 28 genera and 11 orders (51,

62, 240) (see Table 1.1). 16S rRNA-based phylogeny has been widely used for distinguishing Archaea from Bacteria and Eucarya (270) (see below).

Phylogenetic diversity. Using the sequence of 16S rRNA as a chronometer, evolutionary trees showing the relationships among extreme thermophiles can be

10 developed and such sequenced-based maps of biodiversity have proved to be valuable tools in studying these microorganisms (181). Figure 1.1 shows one such representation.

With the recent development of whole genome sequences for numerous extreme thermophiles, similar approaches can now be pursued using entire genomes (158). The extreme thermophiles include two genera that group with the Bacteria: Aquifex and

Thermotoga, although most extreme thermophiles are found within one of two archaeal kingdoms: the Crenarchaeota and the Euryarchaeota. It should be pointed out that the

Archaea include members which are not thermophilic, such as certain methanogens, halophiles, recently discovered ammonia oxidizers (51, 254, 277), and, in contrast to thermophiles, the Archaea also include “cold-loving” organisms, or psychrophiles (52,

53).

It can be difficult to distinguish members within a given genus of extreme thermophiles based strictly on 16S RNA sequence. Within the genus Thermococcus, for example, only very small differences may exist between species, which have otherwise different physiological characteristics. The 16S RNA sequences of Thermococcus barossii and Thermococcus celer are 99.7% identical, although these two archaea have several distinguishing growth characteristics (75). It may be very difficult to explain such differences based on the genome sequences of these species. In addition to 16S rRNA sequences, phylogenetic differentiation of microorganisms has been based on

DNA polymerase restriction fragment length polymorphisms (RFLP) (211) and variations in 16S/23S rRNA spacer regions (70).

Whole genome sequence analysis is beginning to be used as a tool to examine biodiversity, although clear-cut conclusions may still be difficult to reach. For example,

Methanopyrus kandleri is a hyperthermophile whose 16S rRNA sequence places it in a very deep evolutionary branch, close to the root of the archaeal tree. A distinctive feature of M. kandleri is the paucity of proteins involved in signaling and regulation of gene expression. Also, M. kandleri appears to have fewer genes acquired via lateral transfer than other archaea. (236). These features are all consistent with the classification of M. kandleri as a primitive microorganism. However, genome comparisons using both gene content and ribosomal protein alignment indicate that, M. kandleri consistently groups with more recently branched archaeal methanogens. Thus, even with the benefit of genome sequence data, where M. kandleri fits into the microbial evolution scheme is not certain. Of course, the best approach to classification is to combine elements of molecular phylogeny with classical approaches to taxonomy and growth physiology.

Physiological diversity. Extreme thermophiles have either adapted to, or initially arose from, high temperature environments. They have also developed ways to grow on a wide variety of substrates in various concentrations, including what would be described as starvation conditions for many other organisms (250). Extreme thermophiles isolated to date include autotrophs and heterotrophs, aerobes and anaerobes, methanogens, acidophiles, and to a lesser extent thus far, nitrate reducers and sulfate reducers. Some details on these classifications and example organisms are provided below:

Fermentative anaerobes. Geothermal sites, both terrestrial and marine, have yielded an array of fermentative microorganisms that utilize a variety of organic carbon as energy sources. Most of these fermentative heterotrophs require elemental sulfur for growth, which is reduced to hydrogen sulfide. Notable exceptions include some species in the genera Pyrococcus, Thermococcus and Thermotoga. Of these fermentative anaerobes, species of the archaeal genus Pyrococcus and of the bacterial genus

Thermotoga have been the most extensively studied.

Pyrococcus furiosus, a hyperthermophilic archaeon. Members of the genus

Pyrococcus typically ferment peptides and saccharides to CO2, H2, and organic acids

(84). Alanine may also be produced in significant amounts in the absence of sulfur (135).

Hydrogen sulfide will be produced in copious amounts if a source of elemental sulfur, such as polysulfide, is present in the growth medium (36). P. furiosus, the type strain of this genus, was first isolated from geothermally-heated sediments near Vulcano Island,

Italy (84) and has been the subject of a number of physiological, enzymatic and bioenergetic studies (165, 199, 214, 225, 260). The genome of this organism has been sequenced (110), and full genome microarrays are available and have been used for metabolic studies (228, 230).

P. furiosus grows best in the laboratory on -linked polysaccharides, such as starch and glycogen (43), and the disaccharides maltose (225) and cellobiose (136).

Growth on -linked polysaccharides larger than cellobiose has not been reported.

Although P. furiosus will grow on pyruvate (224), it will not grow on glucose even though it has been shown to take up and metabolize this substrate (257). Defined media for this organism have been difficult to develop (35), however one such successful attempt has been reported (143). The significance of sulfur in the bioenergetics of this organism is not completely clear. Schicho et al. (225) showed that sulfur addition led to a two-fold improvement in maximum growth yield of P. furiosus grown in continuous culture on maltose. Ma et al. (154) showed that the hydrogenase from P. furiosus also catalyzes sulfide formation from polysulfide in vitro, although gene arrays showed that this is not a physiological reaction (154). It was later shown that P. furiosus produces significant amounts of alanine in the absence of sulfur (136), which is seemingly an energetically inefficient alternative to acetate formation. It remains to be seen why P. furiosus is capable of growth in the absence of sulfur, which is an unusual feature among hyperthermophilic heterotrophs.

There has been progress in elucidating the central metabolic pathways of P. furiosus, and this has served as a basis for examining other heterotrophic hyperthermophiles (133, 227). Studies on glycolysis have shown that this microorganism possesses a version of the Embden-Meyerhof-Parnas (EMP) pathway, which has some interesting differences compared to the classical pathway found in bacteria. These include ADP- rather than ATP-dependent kinases, glucokinase and phosphofructokinase

(134). Although ADP and ATP have similar thermodynamic properties in regard to the

14 free energy released upon hydrolysis, it is not clear why AMP-forming enzymes are involved (134). Another interesting feature in this modified pathway is the presence of a tungsten-based glyceraldehyde-3-phosphate ferredoxin oxidoreductase (166), which appears to oxidize glyceraldehyde-3-phosphate to 3-phosphoglycerate directly. Yet another apparent difference that appears to be present in all archaea is the direct conversion of acetyl-CoA to acetate by means of an acetyl-CoA synthetase (ADP- forming) (223). The reducing equivalents generated by glycolysis are passed to ferredoxin (15), which serves as an electron donor to a membrane-bound energy- conserving hydrogenase (15, 218). In sulfur grown cells, this hydrogenase system is not present and is replaced by an analogous membrane oxidoreductase system that channels electrons from reduced ferredoxin to a cytoplasmic sulfur reductase (229). The overall energetics of these metabolic processes have been reported (4, 14).

P. furiosus is an attractive candidate for enzymological studies because of its rapid growth rate (doubling time of ~40 minutes, in some cases), and its ability to grow in the absence of sulfur to high cell densities (over 109 cells/ml) (143). Yields of biomass of over a kilogram have been reported from 600 liter fermentations (6). Many enzymes in their native form have been purified and characterized from P. furiosus (7-9). Two other

Pyrococcus species, P. abyssi and P. horikoshii have sequenced genomes that have been compared to the better understood P. furiosus (147); several proteins from these species have been characterized (74, 137, 141, 165, 231). Thermococcus kodakaraensis is

15 another member of the Thermococcales with a sequenced genome and a metabolism similar to that of the Pyrococcus species (85, 114).

Thermotoga maritima and Thermotoga neapolitana, hyperthermophilic bacteria. The genus Thermotoga contains obligately anaerobic bacteria with optimum growth temperatures of up to about 80°C; some species are capable of growth up to 90°C

(108). Thermotoga species have been found in high salt, marine environments and low salt, terrestrial settings. A sheath-like outer structure envelopes these rod-like organisms and balloons over the ends, hence the designation “toga” (108). During stationary phase, cells may become spherical but remain within the “toga.” Nutritionally, Thermotoga species are very diverse and can use peptides, simple sugars and polysaccharides, such as starch (108), xylan (268) and galactomannan (76), as carbon and energy sources.

Thermotoga maritima (108), the type species, has been studied to the greatest extent in terms of its physiological and metabolic characteristics. Its genome sequence has been determined (169), a DNA microarray is available (197, 233), and a structural genomics project is underway to determine the structures of every protein in the organism. (183). The genome sequence of T. maritima revealed an unusually large number of carbohydrate metabolizing enzymes to process a wide range of sugars (170).

T. maritima ferments sugars to CO2, acetate, lactate and H2 by a standard EMP pathway

(2); hydrogen sulfide appears to replace H2 as a product in the presence of polysulfide.

Alanine is also produced from sugar fermentation (202). T. neapolitana (29) has also

16 been studied in some detail. For T. neapolitana, sugar transport was found to involve substrate level phosphorylation (86), which is presumably also the case for T. maritima and the other Thermotoga species. T. maritima and T. neapolitana are inhibited by H2 produced during fermentation; this inhibition is minimized or avoided in the presence of elemental sulfur or if they are co-cultured with an extremely thermophilic H2-utilizing methanogen (167). Both T. maritima and T. neapolitana are able to reduce thiosulfate to sulfide, which appears to improve growth rates and yields (202). T. maritima is motile and migrates at a speed that is proportional to temperature (88). While all Thermotoga species had long thought to be strictly anaerobic, recently there has been some limited evidence for microaerophily in T. neapolitana and another Thermotoga species, T. elfii

(259).

Methanogens. Biotic methane generation is characteristic of many anaerobic hydrothermal environments, even at the most extreme temperatures. Methanogenic microorganisms have been isolated from such locales. For example, Methanococcus jannaschii, isolated from the base of a deep sea “white smoker” chimney on the East

Pacific Rise, grows optimally at 85°C with a doubling time of 26 minutes (122). M. jannaschii has the distinction of being the first hyperthermophile and the first archaeon to have its genome sequenced (48). Subsequent work with this organism found it to be barophilic and elevated pressure promoted both increased growth rate and methane production (161). M. jannaschii grows well when co-cultured with hydrogen-producing

17 extreme thermophiles (38, 167) which may reflect its ecological role (Figure 1.2) (120).

The aforementioned M. kandleri represents a novel group of rod-like methanogens that have been isolated from marine geothermal systems. M. kandleri, the type species of the novel methanogenic genus, grows optimally at 98°C and up to 110°C. It has some unusual cell wall features, including a new type of pseudomurein-containing ornithine

(145). Both M. jannaschii and M. kandleri exhibit rapid growth rates under optimal conditions (less than 1 hour doubling times) relative to many mesophilic methanogens

(167). To date, only hydrogenotrophic methanogens have been found among the extreme thermophiles. The isolation of a rapidly growing, acetate-utilizing species would be interesting in view of attempts to accelerate mesophilic and moderately thermophilic anaerobic digestion processes. Finally, it should be noted that extremely thermophilic members of the genus Methanococcus have been proposed to be belong to a new genus,

Methanocaldococcus (93).

Extreme thermoacidophiles. Brock‟s pioneering work in investigating microbial life in hot springs in Yellowstone National Park revealed the existence of extreme thermoacidophiles that grew at temperatures in excess of 70°C and at pH under 2.0 (40).

Subsequently, a number of thermoacidophilic genera have been described, including the

Picrophilus genus, which can grow at pH values close to 0.0 (226). The metabolism of these organisms is complex and may involve the mixotrophic utilization of both organic carbon and CO2. Extreme thermoacidophiles typically require some complex organic

18 carbon source. Many extreme thermoacidophiles oxidize reduced metal and inorganic sulfur species and have, thus, been evaluated for use in the desulfurization of coal and the recovery of metals from ores (179).

Sulfolobus acidocaldarius was first isolated from Locomotive Spring (pH 2.4,

83°C) in Yellowstone National Park and subsequently found to be distributed widely in hot, acidic geothermal environments (41). Although its pH optimum for growth is very acidic, S. acidocaldarius was also found to grow at pH values up to 5.8. Genome sequences for three members of the Sulfolobus genus have been reported (54, 129, 232,

248). Another member of the order Sulfolobales, Metallosphaera sedula, grows optimally at pH 2.0 and 75°C (253). Unlike S. acidocaldarius, this organism does not use saccharides as carbon or energy sources, but grows on peptide-based media supplemented with ferrous iron or elemental sulfur, while rapidly solubilizing metals from ores (253).

Some further aspects of M. sedula‟s metabolism have been elucidated, including a whole pentose oxidation pathway (42) as well as both the semi-phosphorylative and non- phosphorylative Entner-Doudoroff pathway. (10) These studies have been supported by the characterization of several proteins from these pathways (12, 111). Respiratory gene clusters have been identified and their expression levels during various stages and modes of growth have been shown to vary depending on the substrate and other conditions.

(125),

The identification of an HSP60-like heat shock protein (designated TF55) from

Sulfolobus shibatae (253) has led to studies focusing on heat shock in the extreme

19 thermoacidophiles. Although survival rates for S. shibatae at supraoptimal temperatures increased by thermal acclimation (253), similar experiments with M. sedula revealed that thermotolerance was fleeting and no residual thermotolerance was observed once cultures were returned to normal growth temperature ranges (100). Thermal stress in M. sedula appears to result in damage to the proton pumping system or leakage of protons due to membrane damage. This caused the cytoplasmic pH to drop from around 6 to lethal values of 4 or below upon extended exposure to supraoptimal temperatures (188).

Other extremely thermophilic physiologies. Other growth physiologies have been identified in high temperature biotopes, which suggests that extreme thermophiles are represented in most classes previously studied in less thermophilic microorganisms.

Some of these growth physiologies may be unique to high temperature niches.

Sulfur-based metabolism. Many hyperthermophiles either oxidize or reduce sulfur as a key part of their metabolism. Archaeoglobus species carry out dissimilatory sulfate reduction and can also grow on sulfite and thiosulfate (1). In fact, Archaeoglobus species are commonly found in oil wells and are responsible for souring of oil reservoirs

(1, 238). The type species, Archaeoglobus fulgidus, isolated from a geothermal system on Vulcano Island, Italy, grows optimally at 83°C. Its genome has been sequenced (139) and a whole genome microarray is available (213). This organism is a facultative autotroph, growing heterotrophically on a variety of complex media, or on H2, CO2 and

20 thiosulfate. It also forms biofilms (146). Members of the hyperthermophilic archaeal genus Hyperthermus also reduce sulfur. The genome of the type species, H. butylicus, has been sequenced (45) and it grows by peptide fermentation. (278)

Nitrate reduction. Among the extreme thermophiles, only a few are known to reduce nitrate. They include Pyrobaculum aerophilum, which was isolated from a marine water hole in Ischia, Italy. It grows optimally at 100°C and pH 7.0 by dissimilatory nitrate reduction, forming dinitrogen as a metabolic product (263). This same organism also grows microaerophically on organic and inorganic compounds by aerobic respiration. In either growth mode, elemental sulfur was found to be inhibitory. Similarly, a strictly autotrophic hyperthermophilic bacterium, Aquifex pyrophilus, isolated from

Icelandic hot springs, grows by nitrate reduction or under low concentrations of oxygen.

Molecular hydrogen, thiosulfate and elemental sulfur can be used as electron donors, forming either sulfuric acid or H2S, depending on whether growth was aerobic or anaerobic. For A. pyrophilus, growth is optimal at 85°C with a doubling time of 75 minutes. A. pyrophilus represents the most deeply-rooted bacterial genus on the 16S rRNA phylogenetic tree (49, 109). As autotrophs, these organisms obtain all necessary carbon by fixing CO2 from the environment, while oxidizing H2 under micoraerobic conditions. A. pyrophilus uses a TCA cycle instead of the Calvin cycle for CO2 fixation and can grow anaerobically with nitrate as a final electron acceptor (28). Aquifex aeolicus is another member of this genus with similar metabolic properties, but unlike A.

21 pyrophilus, it cannot use nitrate as an electron acceptor. A. aeolicus is notable as it is the only organism of this genus that currently has a full genome sequence available (67).

Growth on carbon monoxide. Carboxydothermus hydrogenoformans is unique among hyperthermophiles in that it uses carbon monoxide as a sole carbon and energy source, producing H2 as an end product (103). Its genome sequence shows that several different carbon monoxide dehydrogenases are present (273). The combination of hydrogen production and growth on carbon monoxide has made C. hydrogenoformans an organism of interest for production of biohydrogen as a biofuel.

Hydrogen oxidation. The best studied of extremely thermophilic hydrogen oxidizers are the members of the Desulfurococcales. The most hyperthermophilic organisms yet discovered, the Desulfurococcales, include the Pyrolobus genus (34) and

Strain 121 (64), as well as the autotrophic Ignicoccus genus, which includes I. hospitalis which serves as a host to the parasitic Nanoarchaeaum equitans (Figure 1.3) (105, 185).

The Desulfurococcales are all anaerobic archaea, except for Aeropyrum pernix, which is an aerobe (216). The metabolism of these organisms is based on the oxidation of hydrogen, but they can also convert thiosulfate to sulfate (115). P. aerophilum, mentioned above, carries out nitrate reduction in addition to hydrogen oxidation (263).

Thermoproteus tenax, another member of the Desulfurococcales, is an anaerobe that

22 chemoautolithotrophically oxidizes H2. T. tenax contains both semi- and non- phosphorylative Entner-Doudoroff pathways (10, 235).

CULTIVATION METHODOLOGY FOR EXTREMELY THERMOPHILIC MICROORGANISMS

The cultivation of extremely thermophilic microorganisms for physiological studies, or for generation of biomass, has relied on methodologies previously developed for mesophiles (132). Modifications have been made to equipment and protocols to accommodate the requirement of high temperature and other extreme conditions, such as pressure and pH. In some cases, specialized bioreactors and approaches have been developed to deal with the unusual bioprocessing conditions. For example, because most of these organisms either reduce (anaerobes) or oxidize (aerobes) sulfur, the highly corrosive conditions created by high levels of hydrogen sulfide or sulfuric acid make materials of construction an important issue. Gaseous products and substrates associated with these organisms also include H2 and CH4, so explosive conditions can exist.

Nevertheless, many extremely thermophilic microorganisms have been successfully cultivated, in some cases producing in excess of a kilogram of biomass (wet), from a single fermentation (211).

Batch cultivation. Batch cultivation of extreme thermophiles has been done in a variety of ways and on different scales. Hungate tubes or serum bottles are often used for

23 small-scale cultures, which can be made anaerobic if required. Aerobic thermoacidophiles are often grown at a pH of approximately 2.0, minimizing contamination. Many thermophiles grow well on complex media, and a few have been successfully grown on defined media (143, 210). Complex media usually support higher growth rates and cell densities, while defined media are more useful for examining certain metabolic conditions and for examining secreted molecules. Complex media typically contain yeast extract and tryptone, as well as essential salts, trace elements, and carbon sources (usually simple saccharides such as glucose, maltose, or cellobiose) (26).

It is best to avoid thermally labile media components wherever possible (208). Specific components for media have been reviewed and protocols for establishing cultures have been described elsewhere (24, 26, 209).

Fermentors with modifications for high temperature operation have been used to culture anaerobic extreme thermophiles. Glass vessels with working volumes up to approximately 20 liters (0.7 ft3) must also have provision for condensing large amounts of water that vaporize because of the high operating temperatures. Although mixing is important to suspend some sparingly soluble medium components (e.g., polysaccharides, elemental sulfur), many cultures are sensitive to even modest shear stresses. Glass fermentors that have heating mantels or baffle cages, which are metallic, can be susceptible to corrosion. In some cases, enough metal is leached from stainless steels to inhibit the growth of certain hyperthermophiles.

Growth at high pressure. Many extremely thermophilic archaea have been isolated from ocean depths of up to 3000 meters with pressures exceeding 200 atm, and from within temperature gradients that go from 350°C to 4°C over distances much less than one meter. Nonetheless, most isolates are enriched at atmospheric pressure or, for temperatures above 100oC, low hyperbaric pressure (less than 3 atm) to prevent boiling, and temperatures below 115oC for experimental convenience (211). It has been shown that there is a trend towards barotolerance and barophily at pressures greater than those encountered in the native environment (205), and this has spurred efforts to understand the interaction of pressure and temperature as parameters influential to microbial growth

(142). It should be noted that special precautions must be used at high pressures to ensure that changes in growth or gas production are coupled to increased pressure, and not a side effect related to other factors, such as protonation/de-protonation or gas solubility differences (30). High pressure culture conditions are most useful for certain barophilic species that grow significantly better at high pressures. Among the most extreme of these organisms is Thermococcus barophilus, which grows at pressures of at least 15.0-17.5

MPa and as high as 40MPa (156).

For hydrostatic pressure, a hydraulic fluid must be used to provide overpressures; often distilled water or growth media is sufficient for these purposes (205). Pressure is controlled manually during heating by adjusting hydraulic fluid volume. Hydrostatic pressure bioreactors may be as simple as a syringe or a metal canister. To ensure proper sampling and mixing within the hydrostatic chamber, more sophisticated systems have

25 been developed. A compressible “gold bag,” fitted with sampling ports and placed in an hydrostatic chamber within a heating mantel, has been used to culture extreme thermophiles (131). This system can be operated semi-continuously by adding and removing material through syringe pumps and rocked to promote mixing. Using such a system, two hyperthermophilic strains, showed tolerance to pressures up to 440 atm, and in one case, an improvement in growth rate was observed. In addition, cells grown under pressure appeared to have more uniform size and less irregular shape compared to the same cells grown at low pressure. These barophilic strains were designated only as AL1 and AL2. Strain AL2 was isolated from a location near the isolation site for

Thermococcus barossi, but did not share other characteristics with T. barossi (75, 205).

In a hyperbaric pressure reactor, pressure is transmitted to the culture by the gas phase. Clark and co-workers (160) developed a hyperbaric pressure reactor that was constructed from a transparent sapphire cell, allowing visual observation of the culture medium during growth experiments. The reactor and a re-circulating pump were both contained in an oven to facilitate experiments with extreme thermophiles. One version of this system could be operated at pressures of over 100 atm, with a 10 ml working volume.

A magnetically operated pump was used to sparge inert gases through the medium, providing some degree of agitation and promoting thermodynamic equilibrium between vapor and liquid phases. The culture medium was supplied directly from a glove box into the cell, and a gas chromatograph, used to measure volatile products of growth, was directly interfaced to the reactor. The sapphire cell could be replaced with a 167 ml

26 stainless steel vessel, which was then suitable for pressures up to 1000 atm (161). With the larger volume system, a more powerful pneumatic pump was necessary to achieve desired flow rates to the reactor. Growth of the extremely thermophilic methanogen M. jannaschii in the 10 ml hyperbaric bioreactor demonstrated that increases of pressure from 7.8 to 100 atm resulted in accelerated production of biomass and methane, and extended the maximum growth temperature from 90 to 92oC (160). In the stainless steel reactor, the high-temperature limit for methanogenesis was increased to 98oC by hyperbaric helium pressures up to 750 atm (161).

Continuous culture. In continuous culture systems used for the growth of extreme thermophiles, the same considerations must be taken as those described for batch systems. Internal parts are typically glass or teflon-coated to avoid corrosion. Media feed streams are heat-sterilized prior to use, or in some cases, in situ, given the normally high growth temperatures of these microorganisms. Inorganic feed streams are filtered through a 0.2 m porous filter to avoid contamination. Continuous culture systems have been described in some detail for M. sedula (100), P. furiosus (225) , A. fulgidus (97), and T. maritima (174, 233).

The “gas-lift” bioreactor is an alternative type of continuous culture, and has been demonstrated for the extreme thermophile P. furiosus (199, 200). As mentioned previously, some hyperthermophiles are sensitive to high shear rates during growth. Gas- lift systems characteristically have low shearing forces in conjunction with high rates of

27 mass transfer and are well-suited for cultivation of extreme thermophiles (199). Reactors of this cultivation of extreme thermophiles have been constructed of glass components, with a gas distribution tube for inert gas (filtered with 0.2 m) supply. The glass vessel can be heated through a heat transfer fluid-containing jacket; an oversized condenser is used to recover water vapor from the exhaust. Both pH and temperature can be controlled continuously. Experiments have shown that nitrogen is the preferred inert gas to use for sparging, allowing higher cell densities under otherwise equivalent conditions with other gases; the optimal dilution rate for cell mass production of P. furiosus was found to be 0.4 h-1, at which point the bioreactor generated over 1.5 g (wet weight) cells l-

1 (199). A defined medium was optimized for P. furiosus, resulting in reported cell densities of 1010 ml-1 (200).

Experiments using gas-lift reactors also have shown the importance of process engineering and reactor design in biotechnological applications. Changes in reactor size were noted to cause changes in growth and biochemistry due to differences in mass transfer through the reactor (155). The gas lift bioreactor produced evidence for species from multiple genera from an environmental sample, while batch cultivation only showed signs of containing one genus (194).

Dialysis membrane reactor. The impetus for using a membrane bioreactor is two-fold: carbon and energy sources can be supplied directly to the cells through the membrane, and inhibitory or toxic end products of fermentation can be removed through

28 the membrane. Taken together, these flows help to improve growth rate and cell yield.

The dialysis reactor described by Krahe et al. (143) is composed of two reactor chambers of unequal volumes, separated by a membrane. Both of these compartments can be sparged and agitated. The chamber, which houses the cells, is typically several times smaller than the chamber which contains the dialysing medium. With complex media in both chambers, a 100-fold increase (as compared to serum bottles) in cellular concentration of P. furiosus has been reported, along with a 10-fold higher cell density at stationary phase where cell lysis can be significant and lead to loss of whole cells for subsequent recovery steps.

Co-culture of extreme thermophiles. Co-cultivation of extremely thermophilic bacteria and archaea is an important area of investigation since studies have shown that certain physiological behaviors can be elucidated or enhanced only when multiple species are grown together. Co-culture also more accurately represents the natural ecology of high temperature environments. In natural settings, no species grows in isolation; certainly many different hyperthermophile species are often found in the same location.

For example, several thermophilic organisms were originally isolated from the waters around Vulcano Island, Italy (1, 84, 108). In another co-culture system, the symbiotic/parasitic N. equitans was co-cultured with its host I. hospitalis. In this case, co-culture is the only possible growth method because I. hospitalis is physically attached to N. equitans (105, 264).

The most extensively studied co-culture of hyperthermophiles is M. jannaschii and T. maritima, which represent a hydrogenotrophic methanogen and an anaerobic heterotroph, respectively. Cell densities of T. maritima increased 5-fold in co-culture with

M. jannaschii at 85oC, and M. jannaschii was able to grow in the absence of externally supplied H2 and CO2. The higher cell densities were dependent on close proximity of individuals of the two species and an exopolysachharide biofilm was discovered to form between them. Metabolite mass transfer models were also generated for the system (167).

Substantial changes in the T. maritima transcriptome were noted as a response to the co- culture. (119)

NEW DEVELOPMENTS

In the past few years, there have been advances in the study and use of extremely thermophilic organisms. Many of these new developments have been based on the use of various “omics” tools, such as functional and structural genomics, as well as proteomics.

Genetic systems for extreme thermophiles are just beginning to be developed. In addition, extreme thermophile-based applications that go beyond the use of single biocatalysts are emerging.

Functional genomics. Functional genomics approaches, mostly based on cDNA and oligonucleotide microarrays, have been used to explore issues related to microbial physiology, biochemistry and ecology in extreme thermophiles. Heat shock response has been examined for P. furiosus (234), T. maritima (197), S. solfataricus (248), and A.

30 fulgidus (213). In addition to confirming the role of previously studied molecular chaperones (e.g., the thermosome and small heat shock proteins), these efforts were useful in pinpointing specific genes and operons that responded to temperature shifts, many of which are annotated as “hypothetical proteins”. Cold shock response has been examined in P. furiosus (266). Carbohydrate utilization was studied in P. furiosus (148,

163) and T. maritima (55, 63), revealing pathways and transporters involved in uptake and processing of mono- and polysaccharides.

Structural genomics. Structural genomics efforts seek to determine the three dimensional structures of all proteins encoded in the genome of a particular organism.

Recombinant versions of proteins are generated in a high throughput manner and used for structural efforts typically using crystallography (92, 124). Hyperthermophiles were among the first organisms considered for such studies because of a belief that thermophilic enzymes were easier to crystallize (203). Three major hyperthermophile- based structural genomics projects have been undertaken, focusing on T. maritima

(http://www.jcsg.org/scripts/prod/targets5.cgi) (183), P. furiosus (http://www.secsg.org)

(3) and P. horikoshii (http://www.riken.go.jp) (16). Among the challenges facing these projects are difficulties in producing and crystallizing membrane proteins (182, 191), and producing cofactor-requiring proteins (117). It is important to note that the cellular functions of many of the proteins whose structures were determined by these projects is

31 not known. As such, the information is provided only in protein databases and on the project websites, rather than in formal publications (118).

Proteomics. Genome-wide analysis of the proteome of extreme thermophiles has been done in a few cases (104). For S. solfataricus, 1399 proteins or 47% of the annotated ORFs in the genome, were identified (58). Follow-up proteomics studies on the organism revealed a significant number of proteins from what were previously termed hypothetical genes (27) as well as some involved in propanol metabolism (57).

Proteomics was also used to examine the symbiotic/parasitic pairing of two hyperthermophiles, Nanoarchaeum equitans and Ignicoccus hospitalis, revealing aspects of how N. equitans has adapted to its unique lifestyle (65). The relationship between N. equitans and its I. hostpitalis is still not well understood and the exact nature, symbiotic

(105) or parasitic (65), is still under debate. N. equitans has the smallest genome of any organism yet sequenced and its genome lacks many genes typically found in free-living microbes (264).

Genetic Systems. One of the long-standing difficulties in studying and using extreme thermophiles has been the lack of genetic systems. However, the past decade has seen some progress in this regard. Because of issues with high temperatures (and in many cases a requirement for anaerobic conditions), plating of extreme thermophiles, important for classical and modern genetic manipulations, is problematic. Despite these

32 challenges, a chloramphenicol resistant, spontaneous mutant of T. maritima was isolated using continuous exposure to this antibiotic; antibiotic resistance was traced to mutations in 23S rRNA genes and other factors evident from transcriptional response analysis

(163). Plasmids have been identified in members of the Solfalobales (17, 150) and in

Thermoproteus tenax (11). Evidence for a virus-like particle was found in the genome sequence of Pyrococcus abyssi (87). However, so far gene disruption has been accomplished in S. solfataricus (211) and Thermococcus kodakaraensis (221). For S. solfataricus, Blum and co-workers have developed methods for mutant isolation (246), targeted recombination (272), as well as deletion and promoter gene fusion construction

(246, 272). In T. kodakaraensis, gene disruption systems have been used to examine

DNA modification, sugar transport, and gluconeogenesis (21, 157, 222). Work continues on T. kodakaerensis to develop methods for multiple disruptions (220). Shuttle vectors have been created for S. sulfataricus (237) and for Pyrococcus abyssi (152). Genetic manipulations were used to create a deletion mutation in a nucleoside diphosphate kinase in Pyrobaculum aerophilum providing insights into substrate specificity (187). While no genetic systems have yet been developed for hyperthermophilic Methanococcus species, the development of such systems in mesophiles from this genus, Methanococcus voltae and Methanococcus maripaludis, provides a framework for such efforts (256).

Bacteriocins. Antimicrobial peptides produced by mesophilic microbes are well studied; however, it is only in the last few years that extensive work has been done in examining this phenomenon in extreme thermophiles. For example, the hyperthermophilic archaeon Sulfolobus islandicus was found to produce a 20 kDa bacteriocin, termed a sulfolobicin. This sulfolobicin was found to have activity against

S.islandicus and the closely related species S. solfataricus and S. shibatae, but not S. acidocaldarius (195). Unlike the halocins from halophiles, which are the best studied among the archaea (102, 177, 212), sulfolobicins are not secreted into the environment but rather attached to the cell surface. (195) The relationship between the sulfolobicins and the halocins to other antimicrobial peptides has yet to be determined (177, 195).

Molecular Phylogeny, Genomics, and Metagenomics. Extreme thermophiles have long been of interest due to their placement in the “tree of life” (72, 192, 269).

They occupy the deepest branches, suggesting that they played a key role in microbial evolution (240). Many extreme thermophiles appear to have evolved in deep-sea environments, completely without sunlight and entirely dependent on chemosynthesis

(240). Current classification schemes rely on 16S rRNA to identify an organism's taxonomic group, determine related groups, and estimate rates of species divergence

(107, 138). This has now been expanded to encompass whole genomes of not only species, but entire communities (173, 180, 207). These so-called metagenomics projects enable genomes of entire microbial ecosystems to be examined (107). One extensive

34 metagenomics study examined the ecology of an oil reservoir using several complementary techniques, including comparisons of sequences of individual genes, 16S rRNA, isolate culture, 14C and 35S radiolabeling for detection of methanogenesis and sulfate reduction, respectively, and targeted DNA microarrays. As an example of the complementary nature of metagenomics, this study (37) used enrichment techniques to discover fifteen species of extreme thermophiles representing seven genera that were spread through ecologically distinct regions of the oil field. These results were confirmed with the microarray. Evidence for several uncultured, possibly novel, species in three other genera was also found. Metagenomics information can also be used to monitor the response of an entire microbial community to specific stimuli, for example, the response of a hot spring ecosystem to changes in temperature, pH and sulfide levels (196).

Metagenomics has further shown promise for aiding in the isolation of novel species, as exemplified by one study which examined 16S rRNA evidence for mesophile contamination of extreme thermophile isolates and developed techniques for eliminating such contamination (138). Finally, metagenomics has been the basis for discovery of a number of high temperature enzymes (83). When sequences are available for closely related species, additional techniques for examining genome arrangement and shuffling can be productive. One such study examined the changes among three sequenced

Pyrococcus species (147). P. horikoshii and P. abyssi have genomes that differ in structure by only one reversion and one transposition event, indicating recent evolutionary divergence of these hyperthermophiles. In contrast, the genome of P.

35 furiosus is scrambled with many more indications of alteration, suggesting its separation from the other two species occurred much earlier (147). Gene mobility and genome shuffling has also been examined in the three sequenced Sulfolobus species. An unusually large number of insertion sequence elements and genome rearrangements were found in S. solfataricus and S. tokodaii compared to the genomes of other mesophilic and thermophilic organisms but there was a total lack of such phenomena in S. acidocaldericus (46).

Biotechnological uses of extremely thermophilic microorganisms. While thermostable and thermoactive enzymes from extreme thermophiles have generated considerable biotechnological interest (5, 31), there are also biotechnological opportunities to use the intact organisms. Two examples of this are the production of biohydrogen from fermentable sugars and recovery of base and precious metals from refractory ores.

Biohydrogen production. In recent years, hydrogen has been of interest as an energy source due to the lack of polluting emissions from its use, as well as its ability to replace fossil fuels for many uses (59, 98). Extreme thermophiles have been examined for biohydrogen production because several fermentative anaerobic thermophiles appear to have significantly greater yields of H2 per unit sugar consumed than any mesophile (94,

247). Close to the theoretical limit for hydrogen produced from a standard glucose

36 substrate has been reported (see Table 1.3) (134, 274). The highest H2 production levels have been observed using species of the Thermotoga genus, with most producing near the theoretical maximum of four H2 molecules per glucose (59, 247, 251, 259). Addition of oxygen to cultures of T. neapolitana, which has demonstrated microaerophily (167), has not resulted in decreased H2 production (78). Interest in hyperthermophiles for hydrogen production also arises from their production of H2 during growth on certain waste products, such as recycled paper sludge hydrolysate (123) and cow manure (276).

The most significant problem encountered with batch culture production of hydrogen by extreme thermophiles has been high partial pressures of hydrogen, which is self-limiting (258). High pressures of hydrogen cause inhibition of growth (247) and cause a change in the metabolic products from hydrogen to alanine (136) or lactate (47).

The most commonly used solution to reduce the partial pressure of hydrogen is sparging with nitrogen gas (162). Another method of avoiding hydrogen inhibition is to avoid using the whole organism and only use the hydrogenases from thermophiles (153, 184,

219, 261), especially in cases where lower sensitivity to oxygen exists (259). Finally, it should also be noted that co-culture with H2-metabolizing microbes has been observed to modify the growth and metabolism of extreme thermophiles (119).

Biomining. Extremely thermoacidophilic archaea are preferred for certain biomining and bioleaching processes for two main reasons: the occurrence of mineral of interest as a primary sulfide (241) and the presence of toxins that would make industrial

37 smelting a health hazard to humans (77). The extremely thermoacidophilic species used in biomining are most commonly of the genera Sulfolobus and Metallosphaera. Both are used mainly in copper and gold mining and refining, and Metallosphaera is also used in coal depyritization processes (60). Techniques have been adapted from ecological studies to examine natural bioleaching conditions and industrial bioleaching reactors (159), and assays are available for both for detecting species in natural environments and monitoring populations in controlled reactors (13).

There are three main types of extreme thermoacidophile-based bioleaching processes in use today: in situ leaching, heap leaching, and concentrate leaching (77). In- situ bioleaching involves using extreme thermoacidophiles on the actual mining sites without any pre-processing over a lengthy, low-yield process and consequently, is only used when other methods are impractical or uneconomical (39). Concentrate leaching functions much like many other standard (bio-) chemical reaction processes with complex chemical reaction arrangements (242) and strictly designed reactor systems (91).

It also requires pre-processing of ores into a form that can be used in an industrial plant style reaction system (77). Commercial systems developed for concentrate leaching include the BioCopTM (91, 242), BiocynTM, (77) and GEOCOAT® (77) systems, while a hybrid concentrate-heap process involving attaching heap material to an inert support has been adapted from the GEOCOAT® system for use in zinc mining (217).

Heap leaching is another biomining method primarily used for copper mining when the copper is in the form of chalchopyrite (CuFeS2) or other high sulfur content

38 conditions (241). In the case of copper leaching using extreme thermoacidophiles, the system chemistry relies on the conversion of sulfur to sulfate to generate the heat needed to support the required temperatures of the bioleachers and speed recovery rates (190).

Optimum reaction conditions have been determined for many heap leaching processes, including pH (always under 2.0) (71) and use of air, O2 and CO2 sparging to optimize gas concentrations, (151) with the optimal O2 concentration for most processes determined to be 1.5-4.1 mg/L (66). Concentrations of as yet unidentified trace metals (204) and mass transfer effects caused by particle size have also been shown to change reaction kinetics in some biomining systems (171).

Conversely, several inhibitory chemicals and their critical levels for inhibition of specific heap leaching processes have been noted, including chloride (0.5 g/L), nitrate

(1.5 g/L), and fluoride (1.5 g/L) for leaching by Sulfolobus rivotincti (89), and aluminum

(10 g/L) and sulfate (100 g/L) for Thiobacillus thiooxidans (243). High general osmotic potentials also have been observed to inhibit leaching reactions (243). Electrochemical effects of the bioleaching process can have both positive and negative effects on the overall leaching results (96).

CONCLUDING REMARKS

This review summarizes the progress that has been made in exploring and applying elements derived from microbial life at extremely high temperatures.

Bioprocessing has made important contributions to this effort, especially in developing

39 ways in which extreme thermophiles can be grown for purposes of biomass generation and physiological study. The discovery of new extreme thermophiles will likely continue but is now guided more by phylogenetic and taxonomic evaluations and is greatly aided by the advances in –omics tools. Those involved in biotechnology will no doubt continue to keep a close watch on developments in the study of organisms at the upper thermal limits of life.

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Figure 1.1 Phylogenetic tree based on 16s rRNA shows evolutionary relationships among microorganisms. From: Pace, N.R. Science, 1997. (181). .

Figure 1.2 Co-culture of Thermotoga maritima (rods) and Methanococcus jannschii (cocci), both hyperthermophiles, exist in exoploysaccharide matrix at 80oC. From: Johnson, et al., Mol Microbiol, 2005. (120).

Figure 1.3 Nanoarchaeum equitans (small cells) and Ignicoccus hospitalis (large cell) have a parasitic/symbiotic relationship at high temperatures. From: Huber, H. et al., Nature, 2002. (105).

Table 1.1. Representative Extreme Thermophiles auto – autotrophic; hetero – heterotrophic; Cbh – carbohydrates; Pep- peptides; aa- amino acids; YE- yeast extract

Order Genus DNA Topt Growth Carbon Genome Notes Ref. G+C (Tmax) Mode Source(s) sequenced BACTERIA Aquifex aeolicus 40 85 (95) auto (109) Aquificiales CO2 Yes tolerates 6% O2 Carboxydothermus Clostridiales hydrogenoformans 42 75 (80) auto/hetero CO, pyruvate Yes anaerobic (245) Hydrogenivirga Aquificales caldilitoris 49.2 75 (78) auto CO2 Yes nitrate reducer (168)

H2 inhibited, Thermoanaero- Thermoanaerobacter anaerobic, bacteriales tengcongensis 33 75 (80) hetero cbh Yes fermentative (275) Thermotogales Thermotoga maritima 46 80 (90) hetero cbh Yes fermentative (108)

Thermotoga neapolitana 41.3 77 (95) hetero cbh, pep Yes inhibited by sulfide (267) ARCHAEA aerobic, So not Desulfurococcaceae Aeropyrum pernix 67 90 (100) hetero cbh, aa Yes required (216) oxidizes lactate Archaeoglobales Archaeoglobus fulgidus 46 83 (95) auto/hetero cbh, pep, CO2 Yes completely to CO2 (239)

growth on H2, Fe Geoglobus ahangari 59 88 (90) auto cbh, aa No electron acceptor (127) Methanococcus Methanococcales jannaschii 31.4 85 (91) auto CO2 Yes requires selenium (122) o Methanopyrales Methanopyrus kandleri 60 98 (110) auto CO2 Yes S inhibitory (145)

Table 1.1 continued parasitic/ Nanoarchaeota Nanoarchaeum equitans 31.6 90 (98) hetero symbiotic Yes parasitic/symbiotic (105) Picrophlius torridus 36 60 (65) hetero YE, cbh No aerobe, acidophily (226) 100 dissimilatory Thermoproteales Pyrobaculum aerophilum 52 (104) auto/hetero CO2, pep, acetate Yes nitrate reduction (263) Thermococcales Pyrococcus abyssi 44.7 96 (105) hetero pep, aa Yes fermentative (265) 100 Pyrococcus furiosus 38 (103) hetero cbh, pep Yes So not required (84) So not required, Pyrococcus horikoshii 44 98 (105) hetero pep Yes Trp required (90) facultative aerobe, 106 So, Cbh, and Pyrodictiaceae Pyrolobus fumarii 53 (113) auto CO2 Yes acetate inhibitory (34) Sulfolobales Sulfolobus solfataricus 38 80 (87) hetero cbh Yes aerobe (280) Sulfolobus tokodaii 32.8 80 (87) hetero cbh Yes aerobe (244) Thermococcus extreme gammatolerans 51.3 88 (95) hetero pep, aa No radiotolerance (121) Thermococcus anaerobic, So not Thermococcales kodakaraensis 52 85 (100) hetero pep, aa Yes required (20) Thermococcus barophilus 37.1 85 (95) hetero pep, aa No barophilic growth (156)

o Thermoproteales Thermoproteus tenax 56 88 (97) auto/hetero cbh, pep, CO2 Yes requires H2S and S (279)

Table 1.2. Thermotoga species and strains and their isolation sources Species Isolation Site Habitat Ref Thermotoga maritima Vulcano Island, Italy marine (108) Thermotoga neapolitana Volanic spring, Italy marine (29) Thermotoga sp str FjSS3-B.1 Hot spring on beach, Fiji marine (112) Thermotoga sp str SG1 Beach near active volcano, Indonesia marine * Thermotoga sp str KOL6 Submarine hydrothermal system, Iceland marine * Thermotoga sp str RQ7 Geothermal heated seafloor, Azores marine (108) Thermotoga sp str RQ2 Geothermal heated seafloor, Azores marine (108)

Thermotoga thermarum Solfataric spring, Djibouti continental (267) Thermotoga neapolitana LA4 Lake Abbe, Djibouti continental * Thermotoga neapolitana LA10 Lake Abbe, Djibouti continental * Thermotoga elfii Oil well, Sudan continental (201) Thermotoga lettingae Bioreactor continental (22) Thermotoga subterranea Oil well, France continental (116) Thermotoga hypogea Oil well, Cameroon continental (80) Thermotoga petrophilia Oil reservior, Japan continental (251) Thermotoga naphthophilia Oil reservior, Japan continental (251) * Stetter, K., unpublished data

Table 1.3. Biohydrogen yields of representative mesophiles and extreme thermophiles

MESOPHILES (Topt 30-40°C) H2 per unit glucose Ref

Mixed culture 2.1 (79) Ruminococcus albus 2.4 (113) Clostridium butyricum 2.2 (128) Enterobacter cloacale mutant 3.4 (144) Anaerobic sewage sludge 2.4 (149) Food waste culture 2.5 (176) Citrobacter sp. Y19 2.5 (178) Clostridium butyricum 2.0 (215) Clostridium sp. 2.4 (249)

EXTREME THERMOPHILES (Topt ≥ 70°C) H2 per unit glucose Ref Thermotoga sp. 3.6 (47) Pyrococcus furiosus 3.0 (136) Pyrococcus furiosus 3.0 (224) Thermotoga maritima 4.0 (247) Mixed bacterial culture 2.4 (198)

CHAPTER 2: Comparative genomic analysis of hyperthermophilic Thermotoga species

ABSTRACT

The genomes of several hyperthermophilic members of the bacterial genus Thermotoga

(T. maritima, T. neapolitana, T. sp. RQ2, T. petrophila, T. lettingae) were compared using bioinformatics tools in conjunction with cross-hybridization analysis of genomic

DNA to a cDNA microarray based on the T. maritima genome. Except for T. lettingae, the genomes of these Thermotoga species exhibited high degrees of homology when compared through either nucleotide or protein BLAST searches, with at least 80% of

ORFs being at least 70% identical at the nucleotide level using either comparison. The genomic differences between T. lettingae and the other Thermotoga species, combined with physiological aspects and the discovery of mesophiles with outer “toga” structures, suggest that a reevaluation of T. lettingae‟s taxonomy may be in order. The four closely related species share general genome organization traits, including locations for their origins of replication. Differences among these species include several genomic clusters containing unique operons with distinctly different GC content (nucleotide bias), and two large CRISPR-related inversions in T. petrophila and T. sp. RQ2. This high genome homology was confirmed experimentally through genomic DNA cross-hybridization to a

T. maritima cDNA microarray. Between 83-94% of the T. maritima probes showed a statistically significant level of cross-species hybridization for T. neapolitana, T. petrophila, and T. sp. RQ2. In addition, a novel but not yet sequenced strain, isolated from a North Sea oil field and designated Xyl.5.4, was also hybridized to the T. maritima array. Approximately 95% of the T. maritima probes successfully cross-hybridized to this

80 species, which was more than any of the sequenced Thermotoga species examined here.

This suggests the North Sea isolate may be a strain of T. maritima. Taken together the results reported here indicate that, with the exception of T. lettingae, the Thermotoga species examined share a core genome sequence of ~1470 ORFs, with 10 ORFs classified as one ORF in one species classified as two in others. These core genome ORFs presumably encode the essential genes defining Thermotoga physiology. Furthermore, genes unique to a particular species (or sub-group) likely reflect specific environmental or evolutionary factors.

INTRODUCTION

Among high temperatures microorganisms, the genus Thermotoga is one of the most widely studied (14). Thermotoga species have been isolated from a wide variety of locations, both continental (3, 61) and marine (4, 32), including petroleum reservoirs

(73), hot springs (79), and volcanically-heated sediments (31). Metagenomic studies have also found Thermotoga genus-specific DNA in oil fields (6). The biogeography of

Thermotoga is characterized by widely separated growth environments, a phenomenon referred to as “Island Biogeography” (22). This can be seen in Table 2.1, which lists all

Thermotoga species that have been identified along with their locales; Figure 2.1 provides a similar geographic representation. Note that Thermotoga neapolitana is thus far unique, strains having been isolated from both continental and marine environments.

It remains unknown why the very similar members of the Thermotoga genus are found at such widely dispersed locales, and precisely how the species have differentiated themselves in response to environmental and evolutionary pressures. One possibility is the “everything is everywhere, but the environment selects” theory that prokaryotes can disperse and survive nearly anywhere on earth, but require very specific conditions to thrive (2).

Also remaining unknown about Thermotoga is the genetic basis for the adaptability of the genus. This issue can now be addressed to a certain extent, given the recent surge in genome sequencing of members of the Thermotogales. As of July 2009, genome sequences of four Thermotoga species have been made publicly available in the

Genbank database (Table 2.2), the sequence of T. neapolitana has been done by the J.

Craig Ventner Institute (formerly, The Institute for Genome Research (TIGR)), and the sequence of Thermotoga naphthophila is in progress (K. Noll, unpublished data). Four of the sequenced strains share 99-100% identity of their 16S rRNA sequences (Thermotoga maritima, T. neapolitana, Thermotoga petrophila, Thermotoga species strain RQ2)

(hereafter, Tma, Nea, Pet, and RQ2, respectively), while Thermotoga lettingae shares about 90% identity with the other sequenced Thermotoga (3) (Table 2.3 ). T. lettingae has

98% 16S rRNA sequence identity with Thermotoga elfii, which also has ~90% 16S rRNA identity with the other four sequenced Thermotoga (61). Genome comparisons at the amino acid and nucleotide levels have recently provided evidence that classification of many Thermotoga ORFs as being from Archaea were inaccurate, and Thermotoga are actually more closely related to the Firmicutes (83). The various Thermotoga species can also be distinguished by traditional phenotypic and genomic classification; for example,

Pet can be differentiated from Tma in terms of growth on certain sugars, sulfur, and sensitivity to antibiotics (73).

These high levels of 16S rRNA homology for species within a genus are common

(41), and consequently, more detailed methods are needed to differentiate between species. 16S rRNA can be a useful taxonomic guide down to the genus level (71), corresponding to 80-90% identity among the sequences being compared (40). While 16S rRNA has been used as the basis for microarrays for analyzing environmental samples to look for both unique and known microorganisms (45), this analysis breaks down when

83 differentiating among individual species, such that classical, non-quantitative methods of differentiation of genotypes are used (8, 56). DNA-DNA hybridization levels of 70% typically correspond to ~85% DNA nucleotide identity between the two species (75, 76).

This standard has been used previously in DNA-DNA hybridization and Suppressive

Subtractive Hybridization work involving Thermotoga species (54). For differentiation on a species level, average nucleotide identity (ANI) has been found to be a simple and precise tool, when 16S rRNA is not specific enough. This technique compares homologous ORFs from two or more species and reports the average identity level (40,

41). In addition to direct ORF-to-ORF comparisons, genome alignment tools can add additional information about overall genome structure and arrangement (23, 80, 83).

Previous studies have shown that DNA microarrays can be used to resolve closely related microbial species, and in some cases more than one species at a time (64). They have also been used to examine the cDNA of an organism closely related to the species for which the array was created (7), a technique referred to as heterologous hybridization

(62). Heterologous hybridization has been useful for examining evolution of closely related species and strains of Drosophilia (63) and serotyping of pathogens (72). For example, a genomic DNA microarray for Shewanella oneidensis was able to distinguish between nine other Shewanella species, even though 16s rRNA identities were all above

93%, by determining the physical arrangement of the genes gained and lost among the species into several gene clusters (50). Similar work with several Brucella species also found distinct genomic islands gained and lost across closely related species, while

84 strains of the same species retained these islands (59). Species/strain detection and determination have also been done for yeast (78) and human respiratory pathogens (65).

Multi-species arrays have been used to measure total genome sequence differences between two closely related species by measuring effects of probe sequence difference on hybridization (24). These approaches can be problematic when the genome sequences of the closely related species do not exactly match the probes on the array. The differences in sequence can cause changes in hybridization that can give misleading transcriptomic information, and various methods have been created to account and correct for this factor.

One technique has involved re-normalization of the original input data to account for the

DNA mismatches (7), while another method eliminates all probes with less than 100% nucleotide identity from the analysis, leaving only perfect matches (39). Other techniques only examine a subset of the probes, but have other methods of choosing which probes to analyze. These include analyzing only those probes that give comparisons above or below a determined fold change cutoff (24, 64), and selecting only probes which have been previously seen to give successful heterologous hybridizations (60, 72). However, while there have been many techniques proposed, none of them have gained wide acceptance. Finally, it should be noted that experimental design for determining species in multi-microorganism samples must be done with great care, since any mismatch can be masked by other effects, including those arising from different dyes (60).

DNA microarrays have been used for transcript profiling in Thermotoga maritima, providing insights into phenomena as diverse as sugar utilization, heat shock,

85 biofilm formation, and hostile ecological interactions (15, 35, 36, 48, 55, 57, 58, 68). The utility of this cDNA microarray for studying other Thermotoga species was considered here, for both inter-species genomics analysis and as the basis for comparative transcriptomic analysis.

MATERIALS AND METHODS

Growth of microorganisms. Four genome sequenced Thermotoga species

(Thermotoga maritima, Thermotoga neapolitana, Thermotoga petrophila, and

Thermotoga species strain RQ2) and one new isolate (strain Xyl.5.4, provided by the

Doolittle lab at the Dalhousie University in Halifax, Nova Scotia) were used in this study.

Xyl.5.4 was isolated from an oil reservoir in the Norwegian sector of the North Sea. Each

Thermotoga species was grown in batch culture on artificial seawater media (ASW), supplemented with cellobiose (Sigma-Aldrich, St. Louis) to a final concentration of

0.25% (wt/vol), as reported previously (15). Cell densities were monitored by epifluorescence microscopy using acridine orange stain (69). Batch cultures were grown using 70 mL working volumes in 150 mL serum bottles sparged with N2. A 1% inoculum volume was used and the culture was allowed to grow for 12 hours, and then cooled to room temperature prior to being used to inoculate two sparged 500 ml bottles containing

330 mL ASW. Genomic DNA was then extracted from these 330mL cultures.

Genomic DNA extraction. Genomic DNA was extracted from early stationary phase cells, using procedures adapted from previous studies (11). Cells were grown to early stationary phase, and then harvested by centrifuging at 10,000 x g for 15 minutes.

The cells were re-suspended in 8 mL of TNE solution (0.1 M Tris (Sigma-Aldrich), 2.0

M NaCl (Sigma-Aldrich), 0.01 M EDTA pH 7.5 (Sigma-Aldrich), and added to 1 mL of

10% sarcosyl (Sigma-Aldrich) and 1 mL 10% SDS (Sigma-Aldrich). Then, 0.5 mL proteinase K solution (20 mg/mL) (Sigma-Aldrich) was added and the mixture was incubated at 50°C for 2-3 hours. The solution was transferred to 2 mL conical tubes

(USA Scientific, Ocala, FL). An equal volume of TE-saturated phenol (Ambion, Austin,

TX) was added, and then held on ice for 10 min. The tubes were then spun in a microcentrifuge at 15,000 RPM for 10 minutes, after which the aqueous layer was removed. The phenol extraction step was then repeated, followed by a final extraction with Phenol: Chloroform:IAA (25:24:1) (Ambion). Three volumes of 95% ethanol were then added to the solution, followed by incubation overnight at -20°C. Once removed from freezer, the solution was spun in a microcentrifuge at 15,000 RPM for 30 minutes.

The supernatant was discarded and the pellet re-suspended in 70% ethanol, before being spun again for 10 minutes at 10,000 RPM. The supernatant was discarded and the pellet air-dried for 3 min. The dry pellet was re-suspended in TE buffer (0.1 M Tris (Sigma-

Aldrich), 1 mM EDTA (Sigma-Aldrich)).

Comparative genomic analysis. Genomes were aligned using a full local alignment program: Mulan (http://mulan.dcode.org/) (43). The genomes were loaded into the program in FASTA format with the default settings. ORFs that produced statistically significant similarities by the BLASTZ program in Mulan were considered homologs and were visualized using Mulan in dot plot format.

Cross-species hybridization to the T. maritima cDNA microarray. Labeled cDNA from genomic preparations of the various Thermotoga species was used for cross- species hybridization. First, an aliquot of 4 g genomic DNA from a particular species was digested with 20 units HaeIII enzyme and the vendor-supplied buffer (New England

Biolabs, Ipswich, Massachusetts) for 30-60 minutes at 37oC. A „50X‟ solution of dNTP was prepared using dATP, dGTP, dCTP, dTTP, and amino allyl dUTP (Invitrogen) in the following ratios, 5:5:5:3:2, respectively. The „50X‟ dNTP solution was then diluted to

„3X‟ by adding 36.67 L of sterile water per 5 L of „50X‟ dNTP. The DNA samples were then removed from the 37oC incubator and purified using a PCR purification kit

(Qiagen, Inc), before being eluted with 30 L of EB buffer. Following elution, 1.6 L of random nonamers (Sigma-Aldrich) were added to the solution, which was placed in a

100°C bath for 5 min, and then immediately cooled on ice for 5 min. Once cooled, and while still on ice, 5 L 10X Klenow buffer (New England Biolabs), 4 L 3‟-5‟ exo-

Klenow enzyme (New England Biolabs), and 4 L of „3X‟ dNTP mixture were added.

The DNA and enzyme mixture was then incubated for 13-14 hours at 37°C. The samples

88 were removed and 5 L of 0.5 M EDTA, pH 8.0, (Ambion) was added to stop the reaction. The samples were again purified with a Qiagen PCR purification kit. From here, the genomic cDNA microarray experimental and analysis procedure followed exactly as the procedure using cDNA made from RNA. These procedures are described in previous work (10, 27, 28).

Mixed model analyses of microarray data. Replication of treatments, arrays, dyes, and cDNA spots allowed the use of analysis of variance (37, 38, 81) models for data analysis. For each experiment, a dye flip design was constructed and reciprocal Cy dye labeling utilized for all samples to estimate dye effects for each treatment. Slides were scanned using a Perkin-Elmer scanner with Scanarray software and spot intensities were imported into JMP Genomics (SAS Institute, Cary, NC). After local background subtraction and log transformation of spot intensities, a linear normalization analysis of variance model (81) was used to estimate global variation in the form of fixed effects

(dye [D], treatment [T]), random effects (array [A], spot A [S], block A [B]), and random error by using the model log2(yijklmno) = Ai + Dj + Tk + Ai(Sl) +Ai(Bm)+ εijklmn. A gene- specific analysis of variance model was used to partition the remaining variation into gene-specific effects using the model rijklmno = Ai + Dj + Tk + Ai(Sl) +Ai(Bm)+ εijklmn.

Gene annotations are based on published data including the Tma sequence (51), Tma microarray data (15, 35, 36), the COG database at the National Center for Biotechnology

Information (NCBI) (74) and the Conserved Domain Database at the NCBI (47), NCBI

BLAST searches (1, 34), and the Thermotoga sequences publicly available on

MEDLINE, but not yet officially published (Noll, K., unpublished data).

BLAST procedures. A web-based tool, GenomeBlast, was used for the full genome MegaBlast ORF-to-ORF nucleotide level comparisons (46). The web-based tool

Viroblast, (http://indra.mullins.microbiol.washington.edu/viroblast/viroblast.php) was used for complementary amino acid level comparisons (17). These results were supplemented with NCBI BLAST analysis of individual ORFs that produced errors or inaccuracies when used with one of the other programs (1, 34).

G+C skew analysis. The origins of replication of the various Thermotoga species were found by cumulative G+C skew analysis performed using the Munich Information

Center for protein sequences skew plot creator at http://mips.gsf.de/services/analysis/genskew (16, 26). The genomes were supplied in

FASTA format, and a skew plot minimum or maximum indicated the origin of replication.

RESULTS AND DISCUSSION

Bioinformatic comparative analysis of Thermotoga species genomes. A key motivation of this study was to design and create a genus-wide Thermotoga DNA

90 microarray that could be used for identifying Thermotoga species in environmental samples as well as to do transcriptional profiling of members of this genus. In order to create a Thermotoga genus level chip, the ORFs common to four genome sequenced species were used as a starting point: Tma, Nea, Pet, and RQ2; T. lettingae was not selected for this effort because of the lower homology of its genome sequence to the other four sequenced Thermotoga species. The core Thermotoga genome presumably represents the common characteristics of the genus. Conversely, ORFs that are unique to individual species can be used to differentiate it from members of the genus. ORFs in common with two or three species indicate shared genomic features within a subset of the genus. The genomes of the four sequenced Thermotoga were examined using

GenomeBlast, with a threshold of 70% nucleotide identity and coverage of 80% of a given ORF being the criteria for identity. ORFs that met this standard were considered to be “conserved”, while those that did not were considered “unique.” The 70% identity level was chosen for several reasons. In the genomic comparisons of Tma vs. either Pet or

RQ2, the genomes showed a high degree of conservation up to 90% identity, with a significant shift to “unique” status occurring between 90% to 100% identity (Figure 2.3 a,b). When Tma was compared to Nea, a high degree of conservation was observed up to

70% identity. Above 70% identity, the fraction of the ORFs in the genome that were not similar at the nucleotide level began to increase (see Figure 2.3c). The fractions of dissimilar ORFs in Pet and RQ2 did not begin increasing until above 90% identity (see

Figures 2.3a, b). Also, thresholds of 70-75% identity at the nucleotide level have been

91 shown to give reliable results in previous experimental work (12, 13, 50). Hybridization of Klebsiella pneumoniae genomic DNA to an E. coli array showed that 70% identity of a

DNA sequence to the array probe produced a signal the equivalent to only a 1.4- to 1.8- fold down-regulation in transcription levels (18). Another study used cluster analysis to determine the extent of cross-species hybridization for twelve well-characterized

Pseudomonas strains; this approach showed that 70% identity at the nucleotide level did resolve very closely related species due to significant cross hybridization (12).

The core Thermotoga genome. At 70% nucleotide identity, approximately 1470

ORFs were common to all four of the Thermotoga species (Table 2.4, Figure 2.3). The only uncertainty in this value relates to difficulty in differentiating certain ORFs because of annotation differences among the four species. For example, a single ORF in one genome annotation could be reported as two smaller separate ORFs in another. Based on the homology criteria established here, ORFs common to all four species comprised the

“core Thermotoga” genome. As might be expected, this set of ORFs includes central metabolic (glycolysis, pentose phosphate pathway) and key biosynthesis pathways

(amino acid, nucleotides, vitamins). Also included in the core genome are tRNAs, ribosomal proteins, and shock response proteins. Conserved across all four genomes were

ORFs in two operons recently shown to play a role in oxygen detoxification when Tma was exposed to microaerophilic conditions (42), and also play similar roles in other

Thermotoga species (77, 82). Microaerophilicity mechanisms are of interest due to the potential effect oxygen has on hydrogen production in Thermotoga species (20, 77).

ORFs common to three species. There are several sets of genes common to only three species. For example, 27 ORFs were absent in Tma, 42 were not in Pet, and 35 were not in RQ2. In contrast, 174 ORFs were not present in Nea, which is consistent with its larger evolutionary distance from the other Thermotoga (Table 2.4, Figure 2.3). Many of the ORFs in common to three species encoded sugar transporters. CRISPR segments were also among the ORFs in common to three species, indicating the possible influence of viruses on the evolution of the Thermotoga genus (16). Absent in Pet was a NADH- dehydrogenase-related respiratory chain (TM1209-17, CTN_1354-62, TRQ2_1609-01).

Absent in RQ2 were two sugar transporters, one that was associated with a LacI regulator

(TM1853-56, CTN0788-91, Tpet_0500-05), and the other with a D-tagatose 3-epimerase

(TM0416-22, CTN0249-53, Tpet_505-01). Thermotoga genomes characteristically encode a wide variety of glycoside hydrolases (51) and sugar ABC transporters (15).

ORFs common to two species. The 26 ORFs common to Pet and RQ2, but absent in Nea and Tma (Table 2.4), were widely scattered in the genome and produced only one significant contiguous region, from Tpet_0632-0642 and TRQ2_0657-0667.

This locus encoded proteins involved in sugar binding (Tpet_0636, TRQ2_0601) and transport (Tpet_0634-35, TRQ2_0659-660), although the specific sugar or sugars

93 involved are unknown. Twenty-two ORFs were in common between Nea and RQ2, but absent from the others (Table 2.4). These ORFs included an H+-transporting ATPase

(CTN_0913-21, TRQ2_1101-09) and six ORFs of a nucleotide sugars metabolism pathway used for DNA and RNA synthesis (CTN_0031-36, TRQ2_0307-03) (19). Only nineteen ORFs were in common between Nea and Pet only (Table 2.4), including a series of CRISPR proteins (CTN_0707-15, Tpet_1081-1088). Tma and RQ2 also had a CRISPR region among their 62 genes in common. The CRISPR regions that are not in common among all four species represent evolutionary divergence among the Thermotoga species.

CRISPR segments are involved in acquired immunity to viruses (9, 70). Given the widely disparate isolation sites for the two species, it suggests that the differences in the

CRISPR regions could be due to differences in viral exposure in their respective environmental niches. Tma and RQ2 had the most ORFs in common (Table 2.4). In addition to the CRISPRs, the ORFs in common with Tma and RQ2 included four sets of complete sugar transporters, binding proteins, and glycoside hydrolases. One of the four transporters was associated with a LacI regulator. Another sugar transporter with a LacI regulator (TM0946-59, CTN_1617-30) was included in the 42 ORFs in common between

Tma and Nea, but absent from the other species. Only 15 ORFs were in common only between Tma and Pet (Table 2.4), none of which were organized into operons, and most of which were annotated as hypothetical proteins.

ORFs unique to one species. Bioinformatic analysis revealed 54 ORFs unique to

Tma (Table 2.4), of which only 15 had definitive annotations. These ORFs included the locus TM0620-0631, which contains TM0624 and TM0627-8), thought to be part of a - linked exopolysaccharide biofilm production pathway (35, 36). Nea had 253 unique

ORFs, the most of the four species examined. Among these were two ABC transporters, likely to be involved in sugar transport and processing (CTN_1540-1555 and CTN_1168-

72). Differences in genome alignment between Tma and Nea, due to the presence and absence of CRISPR sequences, had been reported previously (16), specifically

CTN_0708-12 and CTN_0701. Differences in Thermotoga CRISPR segments were also detected in a recent wide-ranging comparison of newly sequenced Thermotogales genomes with genomes of organisms from multiple phyla (83). A unique locus encoding almost entirely hypothetical proteins was identified at GTN_0629-0637. The remaining unique ORFs in Nea were scattered around the genome and did not conform to any functional groups. Pet had 53 unique ORFs, including Tpet_1790-1794 which contains a unique sugar ABC transporter annotated as similar to rbsD, a ribose transporter in E. coli

(5). Also of note were two entirely unique regions, Tpet_1750-1754 and Tpet_1765-

1779, which contained mostly hypothetical proteins and had no clear functional organization.

Disposition of T. lettingae. The large number of ORFs in common among these four sequenced Thermotoga species suggests that a genus level DNA microarray could be

95 created, which took advantage of the common ORFs. While four of the sequenced

Thermotoga proved to be very similar, BLAST comparisons with T. lettingae showed it to be different enough at the nucleotide level that it was not a good candidate for this array. This was true even though T. lettingae has 90% identity in 16S rRNA to the other four Thermotoga species (3). While the other four Thermotoga shared over 75% of their

ORFs, based on the 70% nucleotide sequence identity criterion, T. lettingae had only

12% of its ORFs in common with Tma by this measure (Figure 2.5). As can be seen in

Figures 2.2 and 2.5, the percentages of similar ORFs are lower than in comparison to the other Thermotoga at all nucleotide percent identity levels. Genome alignment further confirmed the differences between Tma and T. lettingae (Figure 2.6). Alignments for the other four Thermotoga species revealed significant genome organization features, while the alignment with T. lettingae did not. This was true using either nucleotide sequence or overall genome alignment comparisons. However, given differences in codon usage, these species could be more closely related at the amino acid sequence level. However, further examination using pair-wise ORF to ORF comparisons at the amino acid level indicated that this was not the case (Figure 2.5). It should also be noted that there are significant phenotypic differences between T. lettingae and the other four Thermotoga species, including optimal growth temperature - which for T. lettingae is 65°C (3) compared to 78-80°C for the others (31, 54, 73). T. lettingae differs from the four sequenced Thermotoga in other aspects of growth physiology, as well. For example Pet grows more slowly on some sugars and grows in the presence of rifampicin (73), while T.

96 lettingae does not (3). T. lettingae does have the outer “toga” sheath common to the

Thermotoga (3), but recent discoveries have found mesophiles, now dubbed

“mesotogas,” also have the characteristic toga-like envelope (52). Finally, it should be noted that T. lettingae was isolated from a waste treatment bioreactor (3) and has not yet been isolated from environmental samples. Taken together, the genome sequence differences and physiological attributes suggest that T. lettingae does not closely resemble the other four Thermotoga species examined here.

Genome alignment and origin of replication. Evolutionary and biogeographic effects on the genomes of the various Thermotogas were also investigated on the basis of genome alignments. A full genome alignment study has previously been done for Tma and Nea (16). That study found that, while the overall chromosome structure was very similar, there were numerous CRISPR-associated DNA rearrangements located on the second half of the chromosome – corresponding to TM1000-1805 and CTN_0700-1620.

CRISPR segments have been proposed to function as phage resistance genes (70), suggesting that bacteriophage attack may be implicated. It should also be noted that while no Thermotoga-specific phages have yet been identified, thermophilic bacteriophages and archaeaphages have been detected (67, 84). An alignment with Nea was also done as a control, which confirmed the results of the previous study (16) (Figure 2.7). Alignments of Tma with Pet and RQ2 provided similar insights (Figures 2.8 and 2.9).

Genome alignments indicated that RQ2 and Pet group together, as do Tma and

Nea. Alignment of the Tma and RQ2 genomes revealed three significant inversions. The regions perpendicular to the main line of the genome in the dot plots of the genome alignments indicate additional reversals when Tma is compared to Pet and RQ2 (Figures

2.8 and 2.9). Further examination of the genome neighborhoods around these inversions in each species showed that the ORFs surrounding the ends of all three inversions are

CRISPR-associated segments (Table 2.5). The numeric differences of the loci annotations are due to the reversed and shifted genome labeling in Pet and RQ2, compared to Tma and Nea. The large inversions in Pet and RQ2 contained similar genetic material, only oriented in the opposite direction. All of the major inversions were located on the same half of the chromosome, as determined by the direction of DNA replication, defined in the Nea comparison study as a replichore (16). This region is also where the major rearrangements in the Nea genome occurred. (16) Like the Nea rearrangements, the inversions in Pet and T. sp. RQ2 are associated with CRISPR segments unique to that organism (Table 2.5). Genome alignment results of Tma and Pet (Figures 2.8 and 2.9) indicated that Pet shares one of the CRISPR-related inversions (homologous to TM0377-

0390) with RQ2 (Table 2.5). The inversion is common to both RQ2 and Pet, suggesting that it is evolutionarily older than the subsequent inversions on the other end of their respective genomes. The other inversion in Pet is located in a region that is also inverted in RQ2, but the start and end points are clearly different for the Pet inversion, indicating an alternate event as a cause. This is the first large inversion in the sequenced

Thermotoga without any CRISPR segments located at the ends, previous large genome inversions had all been CRISPR related (16). This suggests an alternate method of genome disruption than was used to create the other inversions. In addition, genome rearrangements associated with CRISPRs have repeatedly been linked with viral infection

(70). The existence of these CRISPR-related inversions in Thermotoga species suggests not only the existence of phages that affect Thermotoga, but also that the method of genome modification can involve viruses. It should also be noted that while the

CRISPRs provide some evidence for the existence of viruses that infect Thermotoga, no examples of Thermotoga-specific viruses have been characterized.

In addition to the whole genomes being in excellent general alignment across all four Thermotoga species, the origin of replication is located in the same place on all four genomes. Initial analyses of Tma failed to find the origin of replication (51, 66), but a later cumulative G+C skew analysis (26) succeeded. The G+C content of a genome varies depending on the direction of genome replication and the origin of replication is associated with minimum or maximum values (44). A similar cumulative G+C skew analysis of the other sequenced Thermotoga species indicates that the origin is conserved across all four species (Figures 2.10 a, b, c). The cumulative G+C skew analysis produced minimum/maximum values at base pair 938233 between ORFs CTN_0535-6 for Nea (Figure 2.10 a), at base pair 785714 in Pet (Figure 2.10 b), between ORFs

Tpet_0774-3, and at base pair 822127 between ORFs TRQ2_0797-6 in RQ2 (Figure 2.10 c). The location of the origin found by this technique in Tma matches the previously

99 identified location of the origin. The location is conserved across all four species with similar DNA sequences and the same two hypothetical proteins on either side of the origin.

Other comments on comparative bioinformatics analysis. Overall, the analysis here was consistent with previous reports focusing on differences between selected

Thermotoga species that pre-dated the availability of genome sequence information.

Nesbo and Doolittle created a lambda library of RQ2 genes not in Tma for subtractive hybridization analysis (54). Genes found to be absent by this technique included an archaeal type ATPase (TRQ2_1101-07), rhamnose biosynthetic genes (TRQ2_0299-

0302), ORFs with similarity to an arabinosidase (TRQ2_0657-61), and a novel archaeal

Mut-S homolog (TRQ2_1785-6), all of which were found to be unique by the 70% identity criterion. Based on the RQ2 lambda library, additional ORFs, not originally screened for, were found in clusters or “islands” of ORFs. These clusters displayed evidence of lateral gene transfer, such as different G+C content and best BLAST matches to organisms outside the Thermotoga genus (53).

The genome sequence information on the various Thermotoga species is also useful for examining metabolic potential in relation to environmental conditions. For example, while Pet was isolated from an oil field, there has been no evidence that it metabolizes petroleum or other long-chain hydrocarbons, either as an energy source or for any other purpose. The only petroleum-metabolizing organism with a full genome

100 sequence and identified pathway for petroleum metabolism is Alcanivorax borkumensis

(25). A BLAST comparison of the A. borkumensis genome to the full Thermotoga genomes produced no hits to any of the genes of the petroleum-metabolizing pathway.

The potential influence of petroleum and other oils on the growth physiology any

Thermotoga species remains unclear.

Inter-species cross hybridization in the genus Thermotoga. To complement the bioinformatics-based inter-genomic analysis for the four Thermotoga species, genomic

DNA hybridizations were performed for T. sp. RQ2, Nea and Pet against an existing Tma cDNA microarray containing 1,926 probes, which had previously been used to study Tma physiology and ecology experiments (15, 35, 36, 48, 49, 57). The heterologous hybridization of genomic DNA from RQ2, Pet, and Nea to the Tma microarray was done to assess the feasibility of developing a genus level array, to see if the genomic islands detected with the bioinformatics 70% identity criterion could be found, and to examine the relationships among species on the basis of their entire genomes and not just 16S rRNA sequences (Table 2.6). RQ2 had an overall genomic nucleotide identity of 86% with Tma, the highest of the three newly sequenced species. Using a two-fold difference in probe intensities as a criterion, as had been used for transcriptomic studies (15, 35, 36,

48), RQ2 also had the highest number of successful hybridizations with 1813, or 94% of the ORFs on the array. Nea was least similar to Tma, based on full genome sequence comparisons, and also gave the lowest number of successful cross-hybridizations, with

101 only 1606 ORFs showing significant cross hybridization. (Table 2.6) Pet‟s similarity to

Tma was, again, in between the other two sequenced species, with 1754 ORFs giving significant cross hybridization and an overall genomic similarity of 79.4%. (Table 2.6)

The ORFs that failed to cross-hybridize were frequently consecutively arranged in the Tma genome, confirming the 70% identity analysis that indicated the presence of distinct genomic islands in each species. As expected, ORFs unique to species other than

Tma could not be detected because the array was based on Tma ORFs. The Nea cross- hybridization results (Table 2.7) revealed seven segments of at least four or more consecutive Tma ORFs that failed to hybridize by the two-fold intensity criterion used.

Five of these (TM0089-92, TM0161-166, TM0707-15, TM1406-10, TM1786-1801) corresponded to genome segments that with less than 70% nucleotide sequence identity between Tma and Nea. The Pet genomic DNA cross-hybridization (Table 2.7) produced the largest number of “islands,” with nine regions of four or more ORFs failing to hybridize by the “two-fold” standard. Only one of these groupings (TM0972-77) was not an island. Cross-hybridization with RQ2 (Table 2.7) produced seven “islands” of four consecutive weakly hybridizing ORFs. Four of these regions (TM0411-22, TM0620-31,

TM0945-62, TM1143-47) matched regions with less than 70% identity. None of these genomic islands included either a tRNA or integrase ORF typically associated with horizontal transfer. However, less than 50% of horizontal transfer events include a tRNA gene, although 85% of them exhibit distinct nucleotide bias compared to the rest of the genome (30). Nucleotide bias analysis reveals several such genomic clusters found in

102

Tma. However, none of the other Thermotoga species exhibited such a bias, including

TM410-420, TM620-30, TM641-53, TM934-52, TM987-94, andTM1282-85, suggesting that they might be true genomic islands. It is interesting that none of these genomic islands are within the CRISPR-related sections found inverted in one or more of the other

Thermotoga species.

Thermotoga Xyl.5.4. Cross-species hybridization was used to examine the relatedness of a new Thermotoga isolate from a petroleum reservoir in the North Sea for which genome sequence information is not available. Thermotoga strain Xyl.5.4 showed strong homology to Tma, with 93% of the probes having significant cross hybridization.

This was the highest hybridization percentage of any other species or strain tested against the Tma array. The Xyl.5.4 cross-hybridization also produced no more than two consecutive ORFs with unsuccessful hybridization (Table 2.7). All of the other species tested had multiple examples of at least four consecutive ORFs absent in the other species but present in Tma. This lack of consecutive absent ORFs suggests that there were no significant “genomic islands” in Tma that are not the Xyl.5.4 strain. For example, the

Xyl.5.4 strain matched the Tma probes for several ORFs in the TM0620-30 region that are not present in any of the other Thermotoga species examined (Table 2.7). These results indicate that strain Xyl.5.4 is more closely related to Tma than are the other species. In fact, the presence of these ORFs suggests that Xyl.5.4 might be a strain of

Tma. In this case, Xyl.5.4 could be adapted for oilfield environments, as opposed to the

103 volcanically-heated sediments from which Tma was isolated. However, no further direct nucleotide or protein sequence comparisons are possible to confirm this because the genome for this strain has not been sequenced. If Thermotoga strain Xyl.5.4 is an oilfield ecotype of Tma, then it is further evidence that the “core” Thermotoga genes provide for adaptability to many environments.

The unique genes of each species provide an opportunity to see how each species has adapted to specific environmental conditions. However, due to the fact that Tma and

Xyl.5.4 appear to have few ORFs that are present in one species, but not the other, they do not provide much insight into any major adaptive changes. This suggests that the genomic changes needed to adapt from one the environment of heated volcanic sediment isolation site of Tma to the oilfield of Xyl.5.4 are either quite minimal or can be handled through gene regulation. The ORFs found only in Nea give no clear indication of the basis for its ability to inhabit both maritime and continental environments. There are many sugar transport and processing related ORFs, and hypothetical proteins, but no identified ORFs that code for osmotic regulation proteins. The absence of unique adaptive genes suggests that the ability to survive in both types of environments is apparently encoded in the ~1470 core Thermotoga genes. It further suggests that the other

Thermotoga species have this ability, but simply may not yet have been detected in an alternate environment. Unique CRISPR segments in hyperthermophiles may be related to unique biogeographic features in the local environment (29). Thus, the unique CRISPR segments in each Thermotoga species may reflect local biogeography of bacteriophages.

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Finally, the unique sugar processing and transport ORFs in each species suggest some sort of adaptation to localized nutrient environments. Some of the transporters have been examined in Tma (15). However, for the ORFs unique to the other Thermotoga species, the role of either specific sugars transported and/or modified by the corresponding proteins or the availability of specific sugars in their isolation sites is not clear.

CONCLUSIONS

Bioinformatic analysis of several Thermotoga genomes have shown that, despite differences in environmental niche, there remains a core, defining set of genes that give the Thermotoga genus its essential properties. However, this core set of genes is not as evident in T. lettingae. This bioinformatics analysis was confirmed experimentally by heterologous hybridization of genomic DNA from the sequenced Thermotoga to a Tma cDNA microarray. The microarray also could be used to access the similarity of Tma to a new Thermotoga isolate, Xyl.5.4. Also, the successful heterologous hybridization of multiple Thermotoga species indicates that this microarray could be used as a basis to examine other Thermotoga species with significant genomic homology.

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FIGURES

Figure 2.1 Map of Thermotoga species and strain isolation sites. Different color rings indicate different species and strains.

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C Figure 2.2 Fractions of unique and similar ORFs as a function of percent identity for the sequenced Thermotoga

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Figure 2.3 Venn diagram of the shared ORFs of the sequenced Thermotoga as defined by having 70% nucleotide identity.

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Fraction of unique and common ORFs in T. maritima vs T. lettingae (DNA and amino acid)

0.8 common ORFs (A.A.) 0.6 unique ORFs (A.A.) 0.4 common ORFs (DNA) unique ORFs (DNA)

0.2 fraction of genome of fraction 0 0% 20% 40% 60% 80% 100% percent identity

Figure 2.4 Fraction of ORFs unique and in common between T. maritima and T. lettingae above a percent identity theshold using nucleotide (dashed lines) and amino acid comparisons (solid lines)

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Figure 2.5 Genome alignment dot plot of T. maritima vs. T. lettingae. Each dot indicates an ORF with significant similarity by the alignment program standards. Highly aligned genomes produce a diagonal line across the area of the plot.

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Figure 2.6 Dot plot genome alignment results of T. maritima vs. T. neapolitana. Each dot indicates an ORF with similarity above the BLAST search threshold. The solid diagonal line indicates high genome alignment. The several CRISPR related inversions can easily be seen in the latter quarter of the genomes.

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Figure 2.7 Dot plot genome alignment results for T. maritima vs. T. petrophila. Each dot indicates an ORF with similarity above the BLAST search threshold. The solid diagonal line indicates high genome alignment.

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Figure 2.8 Dot plot genome alignment results: T. maritima vs. T. sp. RQ2. Each dot indicates an ORF with similarity above the BLAST search threshold. The solid diagonal line indicates high genome alignment.

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Figure 2.9 Cumulative G+C Skew plots of 10a T. neapolitana 10b T. petrophila and 10c T. sp. RQ2. The origin of replication is indicated by a maximum or minimum.

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Tables Table 2.1. Biogeography of Thermotoga isolation sites. Thermotoga species have been isolated from marine and continental environments as well as locations with and without petroleum. Only T. neapolitana has strains isolated from both marine and continental environments.

Isolation sites and Biogeography of Thermotoga species Species Isolation Location Habitat Reference Thermotoga maritima Vulcano Island, Italy marine (31) Volanic spring near Naples, Thermotoga neapolitana Italy marine (4) Thermotoga sp FjSS3-B.1 Hot spring on beach, Fiji marine (32) Beach near active volcano, Stetter, K. Unpublished Thermotoga sp SG1 Indonesia marine Data Submarine hydrothermal Stetter, K. Unpublished Thermotoga sp KOL6 system, Iceland marine Data Geothermal heated seafloor, Thermotoga sp str RQ7 Azores marine (31) Geothermal heated seafloor, Thermotoga sp RQ2 Azores marine (31)

Species Isolation Location Habitat Reference

continental, no Thermotoga thermarum solfataric spring, Djibouti petroleum (79)

continental, no Stetter, K. Unpublished Thermotoga neapolitana LA4 Lake Abbe, Djibouti petroleum Data

continental, no Stetter, K. Unpublished Thermotoga neapolitana LA10 Lake Abbe, Djibouti petroleum Data continental, no Thermotoga lettingae Bioreactor petroleum (3)

Species Isolation Location Habitat Reference

continental, Thermotoga elfii Oil well, Sudan petroleum (61) continental, Thermotoga subterranea Oil well, France petroleum (33) continental, Thermotoga hypogea Oil well, Cameroon petroleum (21) continental, Thermotoga petrophilia Oil reservior, Japan petroleum (73) continental, Thermotoga naphthophilia Oil reservior, Japan petroleum (73)

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Table 2.2. Genome properties of the four sequenced Thermotoga species examined in detail in this chapter.

sequencing G+C protein Name organization content ORFs coding total bp publication Craig T. maritima Ventner Inst. 46% 1928 1858 1,860,725 (51) Craig T. neapolitana Ventner Inst. 47% 1954 1905 1,884,540 unpublished Noll lab, U. of T. petrophila Conn. 46% 1864 1785 1,823,511 (83) unpublished, Noll lab, U. of available on T. sp RQ2 Conn. 46% 1905 1819 1,877,693 NCBI

Table 2.3. 16S rRNA % identity in the of the sequenced Thermotoga.

T. maritima T. neapolitana T. petrophila T. sp RQ2 T. lettingae T. maritima 100.0 T. neapolitana 99.4 100.0 T. petrophila 99.1 99.6 100.0 T. sp RQ2 99.7 99.4 99.2 100.0 T. lettingae 90.8 90.6 91.0 90.9 100.0

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Table 2.4. The number of ORFs shared among the Thermotoga based on a 70% nucleotide identity threshold. The precise number of ORFs conserved across all four species is uncertain due to differences in genome annotation. M = T. maritima. N =T. neapolitana. P = T. petrophila. R = T. sp RQ2.

Genes found in these species Number of genes M+N+P+R ~1470 M+N+P 35 M+N+R 42 M+P+R 174 N+P+R 27 M+N 42 M+P 15 M+R 59 N+P 18 N+R 22 P+R 26 M 54 N 253 P 53 R 50

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Table 2.5. CRISPR related genomic inversions in T. petrophila and T. sp RQ2 compared to T. maritima. The two species share an inversion between TM0377-390, but contain different inversions and CRISPR regions in other sections of their genomes.

Tma-RQ2 inversions inversion site annotation TM0377 TM0388 Both ORFs CRISPR in both species TM0389 TM0390 Both ORFs CRISPR in both species TM1404 TM1414 Both ORFs CRISPR in both species TM1523 TM1524 Both ORFs CRISPR in both species TM1523 TM1524 Both ORFs CRISPR in both species TM1642 TM1643 Both ORFs CRISPR in both species

Tma-Pet inversions inversion site annotation TM0377 TM0388 Both ORFs CRISPR in both species TM0389 TM0390 Both ORFs CRISPR in both species TM1677 TM1678 Both ORFs hypothetical protein in both species riboflavin-specific deaminase, hypothetical TM1828 TM1829 respectively in both species

Table 2.6. Genomic DNA cross hybridization success rates for the sequenced Thermotoga species and the novel Thermotoga strain.

Sequenced strain failed hybridized success rate RQ2 116 1813 94.00% T. petrophila 175 1754 90.90% T. neapolitana 323 1606 83.30% Novel strain Xyl.5.4 91 1838 95.30%

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Table 2.7. ORFs that failed to hybridize to the T. maritima array during homologous genomic DNA hybridization for T. neapolitana, T. petrophila, and T. sp RQ2. A hybridization was considered a failure if the difference of the log-squared mean (LSmeans) values for T. maritima and the other species for that ORF were greater than 1 – equivalent of a two-fold change in transcription levels in an RNA/cDNA array. T. neapolitana on T. T. petrophila on T. T. sp RQ2 on T. maritima maritima maritima LSmeans LSmeans LSmeans ID difference ID difference ID difference TM0022 1.1 TM0022 1.1 TM0318 2.4 TM0036 1.5 TM0087 1.4 TM0411 2.3 TM0037 1.3 TM0121 1.6 TM0412 2.0 TM0038 1.0 TM0303 2.4 TM0413 2.9 TM0039 1.1 TM0305 1.8 TM0414 1.9 TM0044 1.2 TM0318 3.0 TM0415 2.9 TM0048 1.8 TM0320 1.1 TM0416 3.3 TM0050 1.8 TM0411 1.5 TM0417 3.6 TM0087 1.1 TM0412 1.3 TM0418 3.1 TM0089 1.3 TM0413 2.0 TM0419 3.3 TM0090 1.8 TM0414 1.9 TM0420 3.1 TM0091 1.3 TM0415 1.9 TM0421 2.1 TM0092 1.7 TM0416 2.5 TM0422 2.7 TM0096 1.3 TM0417 2.9 TM0431 3.0 TM0097 1.6 TM0418 1.4 TM0432 1.1 TM0107 1.4 TM0419 1.3 TM0435 1.6 TM0108 1.4 TM0420 1.3 TM0448 1.4 TM0120 1.5 TM0431 2.6 TM0520 2.2 TM0124 1.3 TM0432 1.3 TM0525 1.4 TM0130 1.5 TM0433 2.3 TM0526 1.1 TM0132 1.7 TM0435 1.5 TM0601 1.6 TM0133 1.1 TM0448 1.8 TM0616 1.9 TM0134 2.0 TM0533 1.5 TM0618 2.6 TM0139 1.2 TM0591 1.3 TM0620 3.8 TM0155 1.8 TM0616 2.1 TM0622 1.1 TM0158 1.7 TM0618 2.0 TM0623 2.2 TM0161 1.3 TM0620 3.1 TM0624 2.7 TM0162 1.1 TM0623 2.1 TM0625 2.1 TM0163 1.6 TM0625 1.5 TM0626 2.5

128

Table 2.7 continued TM0164 1.7 TM0626 2.1 TM0628 3.0 TM0165 1.3 TM0628 2.4 TM0629 3.3 TM0177 1.7 TM0631 1.6 TM0634 1.9 TM0178 1.9 TM0634 1.2 TM0639 1.4 TM0180 1.1 TM0636 1.2 TM0641 1.9 TM0183 1.6 TM0639 1.3 TM0642 1.4 TM0188 1.8 TM0641 2.3 TM0644 1.4 TM0189 1.8 TM0642 1.2 TM0645 1.5 TM0190 1.5 TM0644 1.9 TM0646 1.8 TM0191 1.4 TM0645 1.7 TM0648 1.3 TM0193 1.8 TM0646 2.5 TM0649 1.9 TM0202 1.1 TM0649 1.3 TM0650 1.4 TM0203 1.2 TM0650 1.7 TM0651 1.0 TM0204 1.2 TM0652 1.3 TM0653 1.5 TM0212 1.1 TM0653 1.8 TM0654 1.3 TM0215 1.2 TM0654 1.6 TM0663 2.6 TM0218 1.4 TM0663 2.8 TM0774 2.5 TM0225 1.3 TM0728 1.0 TM0934 1.8 TM0233 1.4 TM0825 1.1 TM0935 1.2 TM0234 1.6 TM0946 2.7 TM0936 1.2 TM0237 1.3 TM0947 2.9 TM0937 1.6 TM0238 1.5 TM0948 2.7 TM0938 1.4 TM0245 1.5 TM0949 2.2 TM0939 1.4 TM0246 1.1 TM0950 2.8 TM0940 1.6 TM0255 1.1 TM0951 4.0 TM0941 2.5 TM0258 1.2 TM0952 3.1 TM0942 3.0 TM0261 1.5 TM0953 1.8 TM0945 1.3 TM0262 1.0 TM0954 3.4 TM0946 2.2 TM0296 1.4 TM0955 3.8 TM0947 3.0 TM0354 1.1 TM0956 2.8 TM0948 2.4 TM0357 1.1 TM0957 3.9 TM0949 2.8 TM0367 1.5 TM0958 2.4 TM0950 3.0 TM0388 1.1 TM0960 1.5 TM0951 3.5 TM0406 1.3 TM0961 1.4 TM0952 2.4 TM0409 1.4 TM0962 1.3 TM0953 1.9 TM0411 1.1 TM0963 1.3 TM0954 3.0 TM0416 1.1 TM0964 1.4 TM0955 3.4 TM0417 1.8 TM0965 1.7 TM0956 3.0 TM0422 1.7 TM0966 2.3 TM0957 3.5

129

Table 2.7 continued TM0424 1.1 TM0967 2.4 TM0958 3.0 TM0425 1.5 TM0968 3.4 TM0959 1.1 TM0426 2.4 TM0969 1.8 TM0960 1.5 TM0431 1.6 TM0970 1.7 TM0961 2.2 TM0433 2.1 TM0972 3.0 TM0962 1.0 TM0435 2.0 TM0974 2.2 TM0974 2.0 TM0449 1.1 TM0975 3.8 TM0975 3.6 TM0461 1.7 TM0976 1.3 TM0976 1.7 TM0472 1.1 TM0977 1.8 TM0977 1.4 TM0493 1.2 TM0987 2.4 TM0992 2.8 TM0498 1.1 TM0988 2.7 TM0994 1.2 TM0499 1.6 TM0989 1.6 TM1000 1.3 TM0517 2.4 TM0990 3.5 TM1001 1.5 TM0519 1.1 TM0991 2.8 TM1024 1.7 TM0520 1.4 TM0992 2.8 TM1065 1.1 TM0523 1.1 TM0993 1.3 TM1067 2.0 TM0536 1.4 TM0994 1.3 TM1070 2.7 TM0537 1.8 TM1000 1.3 TM1071 1.8 TM0567 1.3 TM1001 1.4 TM1078 1.6 TM0573 1.3 TM1007 1.1 TM1114 1.1 TM0577 1.3 TM1009 1.1 TM1116 1.1 TM0589 1.1 TM1016 2.3 TM1117 1.4 TM0590 1.4 TM1018 1.9 TM1125 1.6 TM0618 2.5 TM1019 1.3 TM1127 1.6 TM0620 2.2 TM1020 1.3 TM1143 1.6 TM0623 1.5 TM1024 2.2 TM1144 2.2 TM0624 1.1 TM1063 1.1 TM1145 1.8 TM0625 1.1 TM1066 1.1 TM1146 1.6 TM0626 2.0 TM1067 1.7 TM1147 1.9 TM0628 2.9 TM1078 1.9 TM1152 1.3 TM0629 3.3 TM1111 1.3 TM1167 1.2 TM0630 2.0 TM1120 3.3 TM1168 1.2 TM0661 1.1 TM1121 2.1 TM1169 1.2 TM0663 2.1 TM1122 3.1 TM1170 1.1 TM0670 1.3 TM1125 2.4 TM1173 2.1 TM0672 1.8 TM1127 1.7 TM1174 1.7 TM0691 1.1 TM1143 2.1 TM1189 1.5 TM0707 1.2 TM1144 2.2 TM1263 1.5 TM0708 1.4 TM1145 2.3 TM1265 1.7

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Table 2.7 continued TM0709 1.1 TM1146 1.8 TM1310 1.9 TM0711 1.1 TM1147 1.7 TM1429 1.8 TM0712 1.1 TM1173 2.3 TM1670 1.6 TM0713 1.1 TM1174 2.0 TM1795 1.1 TM0714 1.3 TM1189 1.4 TM1847 1.7 TM0715 1.1 TM1197 2.0 TM1854 3.1 TM0724 1.7 TM1198 2.5 TM1855 2.9 TM0725 1.6 TM1199 1.6 TM0731 1.1 TM1205 1.3 TM0732 1.7 TM1206 2.0 TM0737 1.3 TM1207 1.6 TM0740 1.2 TM1209 3.1 TM0745 1.4 TM1210 1.5 TM0746 1.2 TM1211 1.8 TM0749 1.5 TM1212 2.5 TM0759 1.7 TM1213 1.6 TM0764 1.4 TM1214 2.0 TM0774 1.4 TM1215 2.8 TM0776 1.8 TM1216 2.6 TM0777 2.1 TM1217 1.5 TM0784 1.4 TM1271 1.1 TM0790 1.4 TM1282 2.0 TM0794 1.3 TM1283 2.8 TM0795 1.6 TM1284 2.1 TM0796 1.1 TM1285 1.4 TM0797 1.8 TM1287 1.4 TM0817 1.0 TM1288 2.0 TM0829 1.4 TM1289 1.6 TM0830 1.1 TM1290 1.8 TM0834 1.4 TM1291 3.3 TM0835 1.4 TM1292 2.6 TM0838 1.1 TM1293 1.3 TM0840 1.7 TM1294 1.8 TM0848 1.0 TM1295 1.0 TM0850 1.4 TM1296 1.7 TM0852 1.4 TM1310 2.0 TM0854 1.0 TM1406 3.0 TM0870 1.6 TM1407 2.1 TM0871 1.4 TM1408 2.7

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Table 2.7 continued TM0872 1.0 TM1409 2.4 TM0875 2.0 TM1410 2.3 TM0876 1.1 TM1429 1.8 TM0878 1.3 TM1589 3.0 TM0879 1.5 TM1625 1.4 TM0880 1.1 TM1646 1.1 TM0884 2.0 TM1716 1.1 TM0885 1.5 TM1725 2.0 TM0889 1.3 TM1746 4.3 TM0893 1.9 TM1748 1.3 TM0900 1.7 TM1749 1.6 TM0901 1.3 TM1750 1.1 TM0909 1.2 TM1751 3.0 TM0910 1.3 TM1752 1.5 TM0912 1.4 TM1790 1.6 TM0915 1.6 TM1791 1.8 TM0923 1.4 TM1792 2.8 TM0924 1.4 TM1793 2.4 TM0925 1.3 TM1794 2.0 TM0928 1.7 TM1795 2.9 TM0930 1.5 TM1796 2.6 TM0931 1.6 TM1797 2.8 TM0937 1.1 TM1798 2.7 TM1023 1.4 TM1799 1.3 TM1024 2.2 TM1800 2.4 TM1029 1.1 TM1801 1.8 TM1032 1.1 TM1802 1.6 TM1042 1.2 TM1044 1.6 TM1045 1.5 TM1047 1.3 TM1051 1.1 TM1061 1.1 TM1063 1.1 TM1065 1.1 TM1079 1.3 TM1089 1.1 TM1091 1.2 TM1111 2.2

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Table 2.7 continued TM1120 2.2 TM1121 1.1 TM1122 2.1 TM1141 1.4 TM1142 1.3 TM1143 1.5 TM1144 2.0 TM1145 1.8 TM1146 1.1 TM1147 1.3 TM1173 1.6 TM1182 1.1 TM1188 1.3 TM1189 1.3 TM1202 1.1 TM1230 1.3 TM1251 1.1 TM1252 1.2 TM1256 1.3 TM1279 1.0 TM1282 1.2 TM1283 2.2 TM1284 1.0 TM1285 1.4 TM1288 1.3 TM1289 1.4 TM1290 1.4 TM1291 2.2 TM1292 1.6 TM1382 1.1 TM1383 1.1 TM1387 1.5 TM1388 1.6 TM1389 2.5 TM1406 1.8 TM1407 1.3 TM1408 1.4 TM1409 1.3 TM1410 1.5

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Table 2.7 continued TM1418 1.3 TM1439 1.5 TM1440 1.3 TM1447 1.4 TM1449 1.4 TM1450 1.1 TM1484 1.0 TM1504 1.0 TM1505 1.1 TM1506 1.3 TM1508 1.2 TM1513 1.1 TM1514 2.4 TM1528 1.1 TM1529 1.3 TM1541 1.1 TM1548 1.6 TM1549 1.2 TM1551 1.2 TM1554 1.3 TM1555 1.1 TM1556 1.5 TM1559 1.1 TM1562 2.0 TM1564 1.1 TM1576 1.5 TM1577 1.1 TM1586 1.1 TM1587 1.1 TM1588 1.5 TM1607 1.0 TM1608 1.1 TM1621 1.7 TM1622 1.3 TM1625 1.9 TM1626 1.6 TM1634 1.7 TM1635 1.9 TM1636 1.1

134

Table 2.7 continued TM1645 1.2 TM1656 1.3 TM1670 2.6 TM1673 1.4 TM1674 1.8 TM1676 1.0 TM1677 1.4 TM1679 1.3 TM1689 1.4 TM1691 1.0 TM1695 1.4 TM1702 1.4 TM1708 1.3 TM1710 1.3 TM1711 1.3 TM1712 1.3 TM1716 1.5 TM1725 1.2 TM1727 1.3 TM1739 1.2 TM1740 1.1 TM1746 3.4 TM1751 3.1 TM1755 1.5 TM1757 1.2 TM1767 1.8 TM1768 1.1 TM1769 1.3 TM1786 1.2 TM1787 1.7 TM1788 2.1 TM1789 2.5 TM1790 3.0 TM1791 2.8 TM1792 3.7 TM1794 1.1 TM1795 2.0 TM1796 2.1 TM1797 1.1

135

Table 2.7 continued TM1798 2.4 TM1800 1.8 TM1801 1.0 TM1808 2.7 TM1809 2.4 TM1810 1.5 TM1813 2.1 TM1830 1.5 TM1831 1.5 TM1832 2.2 TM1833 2.7 TM1836 1.7 TM1838 1.0 TM1864 1.1 TM1865 1.2 TM1872 1.0 TM1877 1.3

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Chapter 3: Glucose and polysaccharide transcriptomes reveal physiological differences in hyperthermophilic Thermotoga species grown in pure and mixed cultures

Steven R. Gray, Andrew D. Frock, Sara Blumer-Schuette, Clemente I. Montero and Robert M. Kelly*

Department of Chemical and Biomolecular Engineering,

North Carolina State University, Raleigh, NC 27695-7905

Portions of this chapter to be submitted to: Applied and Environmental Microbiology

*Address correspondence to: Robert M. Kelly Department of Chemical and Biomolecular Engineering North Carolina State University EB-1, 911 Partners Way Raleigh, NC 27695-7905 Phone: (919) 515-6396 Fax: (919) 515-3465 Email: [email protected]

137

ABSTRACT

Growth of four Thermotoga species (T. maritima, T. neapolitana, T. petrophila, and T.

RQ2) on glucose and a polysaccharide mixture (glucomannan, galactomannan, xylan, carboxymethylcellulose, pectin and lichenan) was examined to assess physiological similarities and differences among the closely related bacteria (based on 16S rRNA phylogeny). The fact that these Thermotoga share a “core” genome of 1470 out of approximately 1900 ORFs formed the basis of a multi-species, cDNA microarray, which was used to evaluate pure and multi-species transcriptomes. Growth of the four

Thermotoga species on either glucose or the polysaccharide mixture was such that all cultures had similar lag phases (5 hours), grew at approximately the same rate (td of ~ 70 minutes), reached the same final cell density (1.5-2.2 x 108 cells/ml), and entered into stationary phase at around 10 hours. Furthermore, H2 production was comparable for all pure and mixed cultures. Nonetheless, significant differences between the four species were observed with respect to their pure culture transcriptomes. Transcriptional changes were more pronounced in glucose culture, with T. maritima and T. petrophila each having over 600 core ORFs change at least two-fold compared to the mixed culture. In the polysaccharide culture, T. neapolitana had the most core ORFs change with 464, while T. petrophila had the least with 121. Also for polysaccharide growth, the pure and mixed cultures had core genome ORFs encoding ABC transporters and glycoside hydrolases for β-mannans, and xylan up-regulated significantly. The mixed culture, but not the pure cultures, exhibited up-regulation of pectin utilization ORFs. Non-core ORFs

138 up-regulated on glucose included an exopolysaccharide synthesis operon unique to T. maritima and an uncharacterized ABC transporter found in T. neapolitana and T. sp.

RQ2. Based on ORFs unique to each species, population composition (T. sp RQ2: T. maritima: T. petrophila: T. neapolitana) for glucose-batch (6.3:1.5:1.1:1), glucose- chemostat (2.8:1.9:1.6:1) and polysaccharide-batch (1.4:1.3:1:1.1) mixed cultures differed. Furthermore, the mixed culture transcriptome indicated that ORFs encoding genes with ecological significance responded. In particular, components of a conserved genome locus containing putative proteins and peptides with sequence and organizational homology to a Bacillus subtilis bacteriocin (subtilosin A) and several hic-family toxin- antitoxin pairs were significantly up-regulated in T. neapolitana growing in the mixed culture. Consistent with previous studies of the HicAB system, lon protease, the ζE factor and its regulatory system, and ribosomal protein ORFs were also up-regulated. This study demonstrated that transcriptional response analysis for related bacteria could be accomplished using a cDNA microarray based on a core genome, which also facilitated estimates of population composition.

139

INTRODUCTION

The Thermotogales comprise a geographically diverse Order of bacteria that have optimal growth temperatures ranging from hyperthermophilic (Topt ≥ 80 °C) (23) to mesophilic (Topt ≤ 50°C) (45). Characteristic of many members of the Thermotogales is a sheath-like envelope resembling a toga that drapes around the cell. The functional role of the toga has not been definitively established, although it has been shown to contain at least one porin (49), and may play a role in compartmentalizing enzymes for glycoside hydrolysis and acquisition (53). Within the Thermotogales is the genus Thermotoga, that contains extremely thermophilic species (3, 50, 58), some of which are also hyperthermophilic (23). Thermotoga species have been isolated from thermal environments world-wide, including petroleum reservoirs (57), hot springs (63), volcanically-heated sediments (23), deep sea vents (38) and geothermally-heated pools

(21). Metagenomic studies also indicate that Thermotogales are present in oilfields (47), ranging from the North Sea (13), to the South China Sea (31), to western Siberia in

Russia (4).

Given the wide-range of locales inhabited by Thermotoga species, it is interesting to consider the underlying microbial ecology that makes them so adaptive. There have been some efforts along these lines. Thermotoga species are oligotrophic, capable of growing on a wide variety of carbohydrates, ranging from simple sugars, such as glucose, to complex carbohydrates, such as xylan (12), forming hydrogen, acetate and CO2 as primary metabolic products. The Thermotoga maritima transcriptome was found to vary

140 considerably with carbohydrate growth substrate (11), implicating a large number of

ABC transporters in sugar uptake (11, 12, 14, 43). Certain ecological behaviors have been noted in members of the genus Thermotoga. T. neapolitana contains a number of

CRISPR sequences that may be related to phage attack resistance (14). In laboratory pure cultures and co-cultures with another hyperthermophile, Methanococcus jannaschii,

Thermotoga maritima forms biofilms (48, 51). Syntrophic interactions with this methanogen have been noted (42) and peptide-based quorum sensing has also been observed (25, 26).

Recently, a number of genomes from the Thermotogales have been sequenced, facilitating comparative genomics analyses that have provided new insights and raised questions about the appropriate phylogeny within the Thermotogales (65). To date, genome sequence analysis has been reported for several Thermotoga species, including T. maritima (44), Thermotoga petrophila (65) and Thermotoga lettingae (65). Thermotoga neapolitana and Thermotoga species strain RQ2 have sequences available on NCBI, but these have yet to appear in the literature. With the exception of T. lettingae, these

Thermotoga species have 16S rRNA sequence identities of 98-100%, and share a “core” genome, representing approximately 75% of their ORFs (Figure 3.1). It is interesting to consider the physiological characteristics encoded in the “core” genome, as well as the differentiating features that are associated with the “non-core” ORFs for members of this genus.

141

Here, a multi-species cDNA microarray was developed for four Thermotoga species to examine the relationship between genotype and phenotype for growth on glucose and a polysaccharide mixture in pure culture and a “zoo” of the four species.

Transcriptional response analysis for pure and mixed cultures was used to explore the similarities and differences within this group. The findings here reveal that even among closely related microorganisms from a phylogenetic standpoint differentiating physiological characteristics can exist that presumably play a role in their ability to survive and inhabit specific environmental niches.

MATERIALS AND METHODS

Growth of individual Thermotoga species in batch culture. Four Thermotoga species (Thermotoga maritima MSB8, Thermotoga neapolitana, Thermotoga petrophila, and Thermotoga species strain RQ2) were each grown in pure culture on artificial sea water media (ASW), supplemented with either glucose (0.25% w/v) (Sigma-Aldrich, St.

Louis) or a polysaccharide mix (0.083% w/v) consisting of equal parts lichenan (>85% purity) (Megazyme, Wicklow, Ireland), konjac glucomannan (90% purity) (Jarrow

Formulas, Los Angeles), pectin (>85% purity) (Sigma-Aldrich), galactomannan (95% purity) (Sigma-Aldrich), caboxymethylcellulose (~99% purity) (Sigma-Aldrich), xylan from oat spelts (90% purity) (Sigma-Aldrich), and xylan from birchwood (90% purity)

(Sigma-Aldrich). Prior to determining growth rates and maximum cell yields, cultures

142 were passaged at least six times on either glucose or the polysaccharide mixture, using a

1% (v/v) inoculum. Cell densities were determined by epifluorescence microscopy using acridine orange stain, as described previously (56).

Thermotoga species (pure and mixed cultures) were grown anaerobically at 80°C in a batch 2 L 5-neck round-bottom flask with a 1 L working volume, sparged with high purity (99.998 %) N2 (Airgas/National Welders, Charlotte, NC). Growth temperature was controlled at 79 ± 10C, using a type K thermocouple and a Digi-Sense controller (Cole-

Palmer, Vernon Hills, Illinois) connected to a heating mantle (Fisher Scientific, Waltham,

Massachusetts). Culture pH was monitored using an autoclavable pH probe connected to a Chemcadet pH controller (Cole-Palmer). The flask was positioned on a stir plate, such that it could be mixed at 400 rpm with a Teflon stir bar. The inert gas sparging rate was controlled at 11 mL/min by a rotameter (Cole-Palmer). To achieve anaerobic conditions prior to inoculation, the flask was sparged with N2 and reduced with 6 mL/L of 10%

(w/v) Na2S. For all experiments, cultures were passaged six times from the same stock as

70 mL batch cultures (1% inoculum) in 150 mL serum bottles sparged with N2. The sixth passage was grown for 12 hours, and cooled to room temperature, prior to inoculation at an initial cell density of ~6 x 106 cells/ml. Cell counts were done on samples taken hourly until early/mid log phase, when biomass samples for RNA extraction were taken.

Batch mixed cultures of the Thermotoga “zoo”. Growth of the Thermotoga

“zoo” for RNA extraction was performed in the same 2 L (1 L working volume) 5-neck

143 round-bottom flask used for batch culture of the single species cultures. To establish the mixed culture, T. maritima, T. neapolitana, T. petrophila, and T. sp RQ2 were passaged six times in N2-sparged, batch pure cultures (1% inoculum) in ASW, with glucose or polysaccharide mix supplemented, as described above. The sixth passages were grown for 12 hours then cooled to room temperature, and used to inoculate a mixed culture with equal amounts of each species, such that an initial overall cell density of ~6x106 cells/ml was established. The mixed „zoo‟ culture containing the four Thermotoga species was again grown for six passages anaerobically at 800C on ASW, supplemeneted with glucose or polysaccharide mix. Following the sixth pass, the mixed culture was grown for 12 hours, cooled to room temperature, and then used to inoculate the sparged, batch 2 L flask, as was done for the pure cultures.

DNA extraction from batch and continuous mixed cultures of Thermotoga species to determine population distribution. Mixed cultures were established by inoculating the first batch culture pass or the initial chemostat startup with equal amounts of T. maritima, T. neapolitana, T. petrophila, and T. sp RQ2, to obtain an initial cell density of approximately 6x106 cells/ml. For continuous culture, Thermotoga “zoo” experiments were performed in the same 2 L/5-neck round-bottom flask used for batch culture (1 L working volume), based on methods described previously (48). The reactor was operated in batch mode until a cell density of approximately 108 cells was achieved

(~7-8 hours). At that point 250 mL of culture was removed for subsequent DNA

144 extraction. The reactor was refilled to 1 L volume and continuous operation was initiated at a dilution rate of 0.25 h-1. Biomass samples for genomic DNA were harvested at 12,

24, 36, 48 and 60 hours after mechanical steady state was achieved. Cell densities were monitored by epifluorescence microscopy using acridine orange stain, as described previously (56). For batch growth, the cultures were run in 500 mL bottles containing 330 mL of glucose-supplemented ASW, as described above. Each pass was allowed to grow for 12 hours, after which a 1% inoculm was taken and used for the next culture passage.

The remainder of the culture was removed and used for DNA extraction.

Design of the multi-species cDNA microarray. The Thermotoga multi-species cDNA microarray used a combination of previously constructed cDNA probes from T. maritima, along with new probes representing ORFs specific to the other three species.

Vector NTI Advance 10 (Invitrogen, Carlsbad, CA) was used for probe design. Probes were produced by PCR, using primers obtained from Integrated DNA Technnologies

(Coralville, Iowa). The primers were used with genomic DNA and spotted to slides, according to the established procedures (6). For choosing probes to add to create the multi-species array, two approaches were used. For T. neapolitana and T. petrophila, in silico analysis was used in conjunction with heterologous cross-hybridization results

(data not shown). GenomeBlast (http://bioinfo-srv1.awh.unomaha.edu/genomeblast/) provided an ORF-to-ORF pair-wise comparison of the whole genomes (32). NCBI

BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (2, 24) was done for individual ORFs

145 that produced errors or inaccuracies in GenomeBlast. For example, ORFs that were annotated in T. maritima as a “Frame Shift” would not produce matches on GenomeBlast under any conditions. T. neapolitana and T. petrophila ORFs added to the array were chosen based on a combination of BLAST homology of less than 70% nucleotide identity, taken together with DNA cross-species hybridization results. When the BLAST results and the experimental data conflicted, experimental data were given greater weight.

For T. sp RQ2, the full sequence was not yet available at the time the array was created, so instead experimental results from suppressive subtractive hybridization experiments reported previously (46) were used to design probes for the ORFs and genome fragments.

Note that the T. sp RQ2 genome sequence has recently been made available on NCBI (K.

Noll, unpublished data). As such only 58 ORFs identified in the T. sp RQ2 genome are not included in the version of the multi-species array used here.

RNA and genomic DNA extraction procedures. Unless otherwise specified, all chemicals used for RNA and DNA preparations were supplied by Sigma-Aldrich, St.

Louis. Biomass samples for total RNA isolation were taken from cells growing on glucose or polysaccharides in early- to mid-exponential phase, using previously described protocols (17). Genomic DNA was extracted from early stationary phase cells, using procedures adapted from those described previously (7). For genomic DNA, cells were grown to early stationary phase, and then harvested by centrifuging at 10,000 x g for 15 minutes. These cells were re-suspended in 8 mL of TNE solution (0.1 M Tris, 2.0 M

146

NaCl, 0.01 M EDTA, pH 7.5) and mixed with 1 mL of 10% sarcosyl and 1 mL of 10%

SDS. Then, 0.5 mL of proteinase K solution (20 mg/mL) were added, and the mixture was allowed to incubate at 50°C for 2-3 hours. The solution was transferred, 1 mL at a time, to 2 mL conical tubes (USA Scientific, Ocala, FL). An equal volume of TE- saturated phenol (Ambion, Austin, TX) was added, after which the tube was placed on ice for ten minutes. The tubes were then spun in a microcentrifuge at 15,000 RPM for 10 minutes, after which the aqueous layer was removed. The phenol extraction step was then repeated, followed by a final extraction with 25:24:1 Phenol:Chloroform:IAA (Ambion).

Three volumes of 95% ethanol were then added to the solution, followed by incubation overnight at -20°C. Once removed from the freezer, the solution was spun at 15,000 RPM for 30 minutes. The supernatant was discarded and the pellet re-suspended in 70% ethanol, before being spun again for 10 minutes at 10,000 RPM. The supernatant was discarded, and the pellet air-dried for three minutes. The dry pellet was re-suspended in

TE buffer (0.1 M Tris, 1 mM EDTA).

cDNA microarray protocols. The creation of cDNA from the extracted RNA, and subsequent RNA-based microarray analysis, was done using methodologies discussed previously (6, 26). Hybridizations were carried out for 18-20 hours following modified TIGR protocols, described elsewhere (6, 19, 20). Hybridized slides were scanned on a Perkin-Elmer ExpressLite Scanner (Perkin-Elmer, Waltham,

Massachusetts) and quantitated by using ScanArray 2.1 (Perkin-Elmer). The arrays were

147 probed with genomic DNA from the various species. An aliquot of 4 µg genomic DNA was digested with 20 units HaeIII enzyme and supplied buffer (New England Biolabs,

Ipswich, Massachusetts) for 30-60 minutes at 37oC. A „50X‟ solution of dNTP was prepared using dATP, dGTP, dCTP, dTTP, and amino allyl dUTP (Invitrogen) in the following ratios, respectively, 5:5:5:3:2. The „50X‟ dNTP solution was then diluted to

„3X‟ by adding 36.67 µL of sterile water per 5 mL of „50X‟ dNTP. The DNA samples were removed from the 37oC incubator and purified with a PCR purification kit (Qiagen,

Valencia, California), finally being eluted with 30 µL of EB buffer. Following elution,

1.6 µL random nonamers (Sigma-Aldrich) were added to the solution and the mixture placed in a 100oC bath for five minutes and then immediately cooled on ice for five minutes. Once cooled, and while still on ice 5 µL 10X Klenow buffer (New England

Biolabs), 4 µL 3‟-5‟ exo- Klenow enzyme (New England Biolabs), and 4 µL of „3X‟ dNTP mixture were added. The DNA and enzyme mixture was then incubated 13-14 hours at 37oC. The samples were removed and 5 µL of 0.5 M EDTA, pH 8.0, (Ambion) was added to stop the reaction. The samples were again purified with a Qiagen PCR purification kit. From here, the genomic cDNA microarray experimental and analysis procedure followed exactly as the procedure using cDNA made from RNA.

Mixed model analyses of microarray data. Replication of treatments, arrays, dyes, and cDNA spots allowed for the use of analysis of variance models (29, 30, 64) for data analysis. For each experiment, a loop design was constructed and reciprocal Cy dye- labeling was utilized for all samples to estimate dye effects for each treatment. For the

148 glucose and polysaccharide experiments, these loops had five slides each, (Figure 3.1).

For the Thermotoga zoo time course experiments, each loop had three slides (Figure 3.2).

For the experiments comparing each species on glucose and polysaccharide culture the loop had two slides (Figure 3.3). Slides were scanned using PerkinElmer Scanarray software and spot intensities were imported into JMP Genomics (SAS Institute, Cary,

NC). After local background subtraction and log transformation of spot intensities, a linear normalization analysis of variance model (64) was used to estimate global variation in the form of fixed effects (dye [D], treatment [T]), random effects (array [A], spot A

[S], block A [B]), and random error by using the model log2(yijklmno) = Ai + Dj + Tk +

Ai(Sl) +Ai(Bm)+ εijklmn. A gene-specific analysis of variance model was used to partition the remaining variation into gene-specific effects using the model rijklmno = Ai + Dj + Tk +

Ai(Sl) +Ai(Bm)+ εijklmn. Gene annotations are based on published data including the T. maritima sequence (44), T. maritima transcriptomics (12, 25, 26), the COG database at the National Center for Biotechnology Information (NCBI) (60), the Conserved Domain

Database at the NCBI (36), NCBI BLAST searches (2, 24), and the Thermotoga sequences available on MEDLINE.

BLAST procedures. For comparisons of individual ORFs, both nucleotide and protein BLAST searches were performed using the NCBI BLAST tool at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). (2, 24)

149

Identification of Toxin-Antitoxin loci. Toxin-Antitoxin systems were located with the web-based finder RASTA (http://genoweb.univ-rennes1.fr/duals/RASTA-

Bacteria) (54). Genomes were loaded in to the program in FASTA format and the default parameters were used.

Estimating population composition in the Thermotoga zoo. In order to estimate the individual species composition in the Thermotoga zoo for glucose and polysaccharide growth, the LSM (Least Squares Mean) differences of the genes unique to each organism were averaged for the ORFs found by BLAST search to be 100% unique to that organism

(Table 3.1). The LSM differences were used to compute a “fold change” that provided a basis for enumerating the amount of the particular ORF in the population. The average of the “fold changes” for the unique ORFs was used to determine the amount that particular species had increased or decreased between the two conditions being compared. The initial composition of the Thermotoga zoo contained equal amounts of cells from the four species, such that the fold changes could be used to estimate new the compositions from that initial point.

RESULTS AND DISCUSSION

Design of the multi-Thermotoga species cDNA microarray. Previously, a T. maritima cDNA microarray had been established to investigate a range of physiological and ecological issues pertaining to the growth of this bacterium (6, 9, 10, 22-24, 38, 39,

150

46, 47, 55). With the recent availability of genome sequence information for other

Thermotoga species, the prospect for modifying the T. maritima array to function for multiple members of this genus was considered. The use of DNA microarray technology has been considered for probing the genetic content of microbial systems (8, 9). Here, the development of the multispecies Thermotoga array was pursued for several specific reasons. First, if in fact there was sufficient homology among the genomes of interest, common probes could be used, facilitating the expansion of transcriptomics analysis to several Thermotoga species at minimal expense. Second, the multispecies array could also be used for investigating mixed culture transcriptional dynamics to explore interspecies interactions. Third, in mixed cultures, probes for ORFs unique to certain species could be used for enumeration, thereby providing estimates of population composition for very closely related microorganisms for which 16S rRNA probes would not provide meaningful information. And, finally, the multi-species array could be used to interrogate environmental samples for Thermotoga genotypes.

The Thermotoga species for which genomes are available are listed in Table 3.2.

Note that by 16S rRNA phylogeny, T. neapolitana (99.1%), T. petrophila (99.1%), T. sp

RQ2 (99.7%) are much more closely related to T. maritima than T. lettingae (90.8%)

(Table 3.3). Previous reports showed that 70-75% nucleotide sequence identity correlated to a 2-fold ratio when comparing 83 homologous genes between Escherichia coli K-12 and Klebsiella pneumoniae 342 (15). Based on this, a 70% nucleotide identity threshold was used here to define homology among genes represented in the five Thermotoga

151 genomes. T. sp RQ2 (1772), T. neapolitana (1616) and T. petrophila (1721) had significantly more ORFs meeting this threshold than T. lettingae (215). This is not surprising since T. lettingae has a much lower growth Topt and different 16S rRNA sequence than the other genome sequenced Thermotoga species. Because addition of the

T. lettingae genome to a multi-species array would have necessitated more than 1800 new probes (see Table 3.4), it was not included in this study.

When genomic DNA from the three Thermotoga species was hybridized to the T. maritima array, 94.0% (T. sp RQ2), 90.9% (T. petrophila), and 83.3% (T. neapolitana) of the probes responded 2-fold or more (Table 3.4). These hybridization results likely reflect a certain amount of cross-hybridization for genes containing regions with sufficient homology to the probes represented on the microarray; for example, note that although

146 ORFs in T. sp RQ2 were not expected to hybridize based on the 70% nt identity criterion, only 116 T. sp RQ2 ORFs actually were not recognized by the 2-fold criterion.

However, the results in Table 3.4 suggest that a multi-species microarray created through expansion of the existing T. maritima chip was plausible.

Core and non-core Thermotoga genomes. Based on 70% nt sequence homology, a core genome of approximately 1470 ORFs could be defined for the four Thermotoga species (Table 3.5 and Figure 3.1); The value is approximate due to 10 ORFs reported as a single ORF in at least one species and multiple ORFs in at least one other species. Until further experimental evidence is available, the correct annotation of these ORFs cannot

152 be determined. These ORFs of the core genome include ones that define the essential characteristics of the Thermotoga genus. They include central metabolism pathways such as Embden-Myerhof and pentose phosphate, as well as the majority of the ABC transporters found in the genus. The core also includes many RNA ORFs, such as those coding for rRNA and tRNA. The non-core ORFs are, therefore, the ORFs that define each species within the Thermotoga genus. Some examples of non-core genome features are listed in Table 3.6. While most ABC transporters are common to all four species, each

Thermotoga has some ABC transporter ORFs not found in the other species. Notable features found in only one species include an exopolysaccharide synthesis operon (26) in

T. maritima, and a PTS in T. sp RQ2. The CRISPR-related proteins of T. maritima and T. neapolitana have been shown to be partially different from one another (14), and additional differences were found when the CRISPR-related proteins of T. petrophila and

T. sp RQ2 were compared with the other species (Table 3.6).

Determining population composition of the Thermotoga zoo. In each of the four Thermotoga species, ORFs that are less than 70% identical to the other individual species could be identified: 54 in T. maritima, 253 in T. neapolitana, 53 in T. petrophila, and 50 in T. sp RQ2. However, some ORFs were completely unique to each species, based on 100% identity by megablast comparison using the GenomeBLAST program: 34 in T. maritima, 88 in T. neapolitana, 18 in T. petrophila, and 76 in T. sp RQ2 (Table 3.1).

These ORFs were used to estimate population composition for the Thermotoga zoo

153 growing in batch or continuous culture on glucose and in batch culture on the polysaccharide mixture (glucomannan, galactomannan, xylan-birchwood, xylan-oat spelt, carboxymethylcellulose, pectin, and lichenan) (see below).

Growth and H2 production for Thermotoga species in pure and mixed culture. Growth of the four Thermotoga species on either glucose or the polysaccharide mixture was such that all cultures had similar lag phases (5 hours), grew at approximately

8 the same rate (td of ~70 minutes), reached the same final cell density (1.5-2.2 x 10 cells/ml), and entered into stationary phase at around 10 hours. Furthermore, when H2 production was adjusted for cell density at time of harvest, production rates were

-1 -1 -1 comparable for all pure and mixed cultures at ~2.5x10-11 mol H2 l hr cell (see chapter 4).

Figure 3.4 and Table 3.7 report the estimates for population composition for the

Thermotoga zoo. These estimates are based on 2-fold differences between the starting composition (equal number of cells from each of the four species), inoculated from exponentially growing cultures on the same medium. Note that after the first pass in the batch culture, the population composition was approximately 6.3:1.5:1.1:1 for T. sp RQ2:

T. maritima : T. petrophila: T. neapolitana, respectively, which changed slightly to

4.2:1.5:1:1 after the 6th pass. In chemostat culture, the population composition initially was approximately 2.8:1.9:1.6:1 for T. sp RQ2: T. maritima: T. petrophila: T. neapolitana, respectively, which changed to 3.6:2.4:1.3:1 after 60 hours. For

154 polysaccharide culture, the zoo population composition was 1.4:1.3:1.1:1 for T. sp RQ2:

T. maritima : T. neapolitana : T. petrophila, respectively,after the 1st pass. This composition changed to 2.5:2.4:1.1:1 for T. sp RQ2: T. maritima : T. neapolitana : T. petrophila, respectively, after the 6th pass. Chemostat cultures were not examined for the polysaccharide mixture due to problems with feeding and removing the viscous solution from the bioreactor. While this approach for estimating population composition can be further refined, it does provide information on the relative amounts of phylogenetically- and physiologically-similar microorganisms growing in mixed culture. For the glucose- grown cultures, population composition was time-invariant in both batch and continuous culture. The relative amounts of each species established in the first pass on the batch culture or during start-up of the chemostat were maintained; neither five additional batch passes, nor 15 reactor volumes (60 hours) of growth in continuous culture significantly changed the population species distribution (Figure 3.5). Consequently, whatever underlying factors defining the composition of the mixed culture took hold in the initial growth stages. The differences in cell population composition between the batch and continuous culture could be related to the fact that extracellular products do not accumulate in the chemostat (Table 3.5).

The composition of the Thermotoga zoo in the polysaccharide-based media was estimated in a similar manner. Only batch culture was examined for the polysaccharide mix. The composition analysis of the polysaccharide mix batch culture again showed T. sp RQ2 as the most prevalent species at both pass 1 and pass 6 (Table 3.7, Figure 3.5)

155 comprising 28-35% of the culture. T. maritima was similar to T. sp RQ2 in increasing its‟ prevalence to 27-33% of the culture. T. neapolitana and T. petrophila each comprised less than 25% of the culture at both pass 1 and pass 6. Unlike the glucose cultures, T. neapolitana was the not the least prevalent species, but was slightly more prevalent than

T. petrophila. The polysaccharide batch culture was also the only growth condition where all of the species compositions at the earlier and later time point were not statistically similar. The amounts of T. maritima and T. petrophila increase and decrease, respectively, from the 1st to the 6th pass. T. sp. RQ2 and T. neapolitana show only statistically insignificant changes. These results may indicate a slow change in the composition of the Thermotoga zoo during culture on polysaccharides.

Glucose and polysaccharide transcriptomes for Thermotoga species grown in pure culture. RNA was collected from mid-exponential phase of the four Thermotoga species pure cultures grown on glucose and a polysaccharide mixture (glucomannan, galactomannan, xylan (birchwood and oat spelt), carboxymethylcellulose, pectin and lichinen) so that the impact of growth substrate could be evaluated by transcriptional response analysis. Table 3.8 reports the number of ORFs in the core and non-core genomes responding 2-fold or more to the polysaccharide mixture compared to glucose for each Thermotoga species (dye-flip). Of the 1470 ORFs (excluding tRNAs) in the core genome, 281 were differentially transcribed for at least one species for the polysaccharide-glucose contrast. Only two of these ORFs responded in the same way for

156 all four species and the mixed culture: TM0056 and TM0071, both binding proteins for xylan ABC transporters. T. maritima (178 ORFs) and T. neapolitana (197 ORFs) exhibited more differential transcription than T. petrophila (48 ORFs) or T. sp RQ2 (84

ORFs).

There were distinctive features of the four species with respect to the transcriptional response to polysaccharides. Table 3.9 summarizes the response of ORFs encoding glycoside hydrolases (GH) and ABC sugar transporters identified in the genomes of these Thermotoga species. Out of 42 GH-encoding ORFs identified in the genomes of these Thermotoga species, 23 responded 2-fold or more in at least one species for the polysaccharide:glucose contrast. Only 5 GH ORFs responded in T. petrophila and 2 of these were down-regulated on polysaccharides. While significant up- regulation of core genome xylanases (TM0061, TM0070 and homologs) and mannanases

(TM1227, TM1524 and homologs) was evident in the other Thermotoga species on polysaccharides, these were not induced in T. petrophila; in fact, the T. petrophila homolog to TM1524 was down-regulated. For all individual Thermotoga species and the

Thermotoga zoo, ORFs from previously identified xylan transporters (TM0056-61, and

TM0070-77) (12) were up-regulated on the polysaccharide mix (Table 3.9). Also up- regulated in all individual species and the mixed culture were ORFs from the TM1218-23

ABC transporter, previously identified as β-mannan-related (12) (Table 3.9). In the

Thermotoga zoo, ORFs encoding a pectin ABC transporter (TM1195-1200) and pectin- active glycoside hydrolases were up-regulated in polysaccharide culture (Table 3.9).

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None of the individual species had pectin transport ORFs up-regulated. The unique response of the Thermotoga zoo suggests that, in the mixed culture, competition increases the range of sugar utilization. Non-core ORFs also played a role in the response of the individual Thermotoga species. In T. maritima, several ORFs of an operon thought to be involved in exopolysaccharide synthesis (TM0622-30) were significantly up-regulated on glucose (Table 3.10). This change suggests that T. maritima may utilize available β- glucans to form exopolysaccharide, but has to synthesize them when none are available in the growth medium. An unclassified ABC transporter found only in T. neapolitana and T. sp. RQ2 (TRQ2_970-76) had significantly higher transcription levels on glucose (Table

3.9), suggesting that it could be a transporter for the monosaccharide. T. petrophila was the only organism not to have any unique sugar utilization ORFs respond in pure culture to either glucose or polysaccharides, and it also had the fewest ORFs with significant transcription changes between the two conditions. This suggests that T. petrophila may be in a state such that it is prepared for polysaccharide utilization at all times.

Pure and mixed culture transcriptomes. While genomic DNA hybridization and tracking the relative amounts of constitutively transcribed genes can be done to estimate population composition, this information does not shed any light on the underlying changes in regulation and transcription that define the mixed culture. As a result, the transcriptomes of the pure and mixed cultures were examined for clues to the factors that were responsible. All transcriptional response experiments were performed in

158 batch culture. Transcriptional variation was reflected in the number of significantly up- or down-regulated ORFs. During glucose batch culture, over 600 ORFs of the core

Thermotoga genome in T. maritima and T. petrophila were differentially transcribed 2- fold or more compared to the mixed culture, while less than 300 showed differential regulation in T. neapolitana and T. sp RQ2 (Table 3.11). Using a three-fold change in transcription as the cut-off, the differences in the number of ORFs showing significant changes in transcription became more pronounced (Table 3.11). Finally, use of a four- fold cut-off to examine the data resulted in large differences in number of ORFs exhibiting differential transcription among the various species. At the four-fold cut-off level, T. sp RQ2 has only 2 ORFs differentially transcribed, compared to T. petrophila which had 200 (Table 3.11). In polysaccharide culture, the Thermotoga zoo had fewer core ORFs differentially transcribed compared to the pure cultures (Table 3.12). T. neapolitana was the exception with over 450 ORFs differentially transcribed compared to the Thermotoga zoo, an increase of over 50% compared to the glucose culture. T. petrophila had the greatest decrease in number of ORFs differentially transcribed, with only 121 ORFs changing significantly in polysaccharide culture, as opposed to the more than 600 that changed in glucose culture. T. maritima and T. sp RQ2 also had decreases in differential transcription with 241 and 141 ORFs showing significant changes, respectively (Table 3.12).

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Interspecies interactions in the Thermotoga zoo. A key issue to be considered here is whether the population composition relates in any way to interspecies interactions in the mixed culture. To address this, the mixed culture transcriptomes were compared to the pure cultures growing on either glucose or polysaccharides. While differential transcription of ORFs implicated in these interactions was minimal for the polysaccharide-grown cultures, there were several ORFs that responded in the mixed culture compared to the pure cultures.

TM1300 Locus. Previous work examining the transcriptome of T. maritima growing on the spent media of another hyperthermophile, Pyrococcus furiosus (16), identified a putative small peptide of 31 amino acids (TM1316) up-regulated 18–fold

(39). This ORF was subsequently found to be an ortholog to a bacteriocin from Bacillus subtilis, subtilosin A, with anti-listerial activity (28, 61). The similarities extend to the loci and operons encoding TM1316 and subtilosin A in T. maritima and B. subtilis, respectively. Both operons encode a small peptide (or two in the case of T. maritima) which contains a signal peptide region, followed by an SAM radical family protein, and a putative ABC transporter set (66, 67). Several similar operons were found in the surrounding genome locus in T. maritima (TM1300-1336) (Figure 3.6). These operons were, for the most part, conserved among the four Thermotoga species, with some minor modifications (Figure 3.6). Neither TM1316, nor any of the ORFs in this operon or genomic locus operon, exhibited any significant response in the mixed culture on

160 glucose. However, TM1300 was significantly up-regulated in the mixed culture compared to each of the pure batch cultures on glucose, ranging from 2.6-fold against T. sp RQ2 and T. neapolitana to 8-fold against T. maritima (Table 3.8). Unlike the case for

TM1316 up-regulation for T. maritima growing on spent P. furious media, the rest of the

ORFs in the TM1300-04 operon were not affected. Furthermore, TM1300 did not respond in the mixed culture growing on polysaccharides, perhaps suggesting that competitive behaviors are minimized in richer growth media.

Also located within the 1300s locus are putative components of HicAB toxin- antitoxin pairs, some either previously not identified as ORFs or annotated as hypothetical proteins in the Thermotoga genomes (Table 3.13 and Figure 3.6). One toxin

(TM1312) and three antitoxins (TM1311, TM1313, TM1321) were identified in T. maritima by homology to recently discovered HicAB genes in other bacteria (35). Two putative toxins (TM1310a, TM1320) were found by a combination of the RASTA TA locus-finding program (54) and BLAST searches. TM1320 is apparently the missing partner of an antitoxin (TM1321) previously identified elsewhere (35), and this ORF had been annotated as a frame shift in the T. maritima genome. Furthermore, TM1311 had previously been identified as a HicB antitoxin without a cognate toxin. RASTA analysis showed that a toxin was encoded between base pairs 1332392 to 1332540, placing the previously undetected ORF between the TM1310 and TM1311. This new toxin was designated here as TM1310a. Because it was not yet identified when the array was constructed, no probe was available to track the transcription of the associated gene.

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Homologs to TM1310a were identified in T. neapolitana (CTN_1274a) and T. petrophila

(Tpet_1472a). It is interesting that this ORF, like its cognate antitoxin partner TM1311, is absent in the T. sp RQ2 genome (Table 3.13, Figure 3.5). BLAST searches also indicate that the toxin encoded in TM1320 is absent in T. neapolitana. Otherwise, all of the other toxins and antitoxins were conserved across the four Thermotoga species studied here, as well as in T. lettingae. TM1311, TM1312, TM1313, and TM1321 are also conserved in

Marinotoga piezophila, but no homology was noted with any more distantly related organism.

Growth on glucose triggered transcription of the toxin-antitoxin loci in the

Thermotoga zoo mixed culture (Table 3.13). Both toxins on the array (TM1312, TM1320 and orthologs) were up-regulated in the Thermotoga zoo against the pure cultures, with

TM1312 81-fold higher in the mixed culture compared to T. petrophila. In addition to

TM1312, ORFs of the toxin-antitoxin pair TM1320 and TM1321 were up-regulated 16.6 and 15.8 fold, respectively, in the mixed culture compared to T. petrophila. In contrast,

TM1313 and homologs were down-regulated in mixed culture compared to T. maritima and T. petrophila, which is interesting in view of the TM1312 response.

Previous work with E. coli under various stresses indicated that having additional toxin-antitoxin loci either has no effect (62) or gives a competitive advantage (52). Here,

T. sp RQ2 dominated the mixed culture, despite missing one of the toxin-antitoxin pairs found in the other Thermotoga species (Table 3.13). Little is known about the effect of the absence of a HicA toxin from a HicAB toxin-antitoxin pair, as seen in T. neapolitana.

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It was seen that HicB could reverse the bacteriostatic effects of HicA, but the effect of a

HicA knockout or HicB overexpresssion was not reported (27).

The T. maritima 1300s locus and its homologs in the other Thermotoga species were not the only genes responding in the mixed culture. A phage tail-like bacteriocin with proteolytic activity and structural similarity to known bacteriocins was previously discovered in T. maritima (22), which has subsequently been found to be TM0785 (44).

Although not responding to T. maritima growth on spent P. furiosus (39), TM0785 and its homologs were up-regulated in the Thermotoga zoo by up to 4.8-fold, in comparison to the T. neapolitana pure culture (Table 3.13).

Stress response in glucose culture. In addition to the transcriptional responses of the 1300s locus, the Thermotoga zoo transcriptome showed signs of stress response. The sigma-E (Sigma 24) factor plays a role in stress response in microorganisms, including acid stress in Salmonella (41), and extracytoplasmic accumulation of misfolded or unfolded protein in E. coli. (1). The sigma-E (or ζE) factor in the Thermotoga genomes

(TM1598, CTN0860, Tpet_1194, and TRQ2_1261) was up-regulated in the glucose- grown mixed culture by 2- to 6.8-fold against the pure cultures (Table 3.13). BLAST searches indicate that the E. coli sigma-E, RseP, and DegS (a sigma-E activator) (1) have homologs in T. maritima as TM0890 and TM0571, respectively. For growth on glucose, the DegS homologs were up-regulated in the Thermotoga zoo by 2.0- to 6.0-fold against the individual species (Table 3.13), suggesting a similar role for the ζE regulatory system

163 in Thermotoga. ζE and degS transcription was also observed to increase under chloramphenical challenge in T. maritima (40). The ζE ORF was up-regulated 5 and 30 minutes after addition of chloramphenicol to wild-type T. maritima by 2.3- and 3.8-fold, respectively. The ORF encoding degS (TM0571) was also up-regulated two-fold at 30 minutes after addition of chlorampheincol. In contrast, a chloramphenicol resistant T. maritima mutant showed no significant transcriptional response (40). No similar transcriptional changes of ζE and associated ORFs were observed in the polysaccharide mix.

Also up-regulated in the Thermotoga zoo during glucose growth were ORFs encoding two lon proteases (Table 3.13). The Thermotoga are unusual in that they contain both the bacterial (LonA: TM1633, CTN_0825, Tpet_1158, TRQ2_1299) and membrane-bound archaeal (LonB: TM1869, CTN_0805, Tpet_0928, TRQ2_0950) versions of the Lon protease (44). The up-regulation of LonB in the glucose mixed culture ranged from 2.2-fold versus T. maritima to 5.3-fold against T. petrophila. LonA was up-regulated 2.5-fold in the mixed culture against T. sp RQ2 and 6.8-fold for the same contrast with T. petrophila. (Table 3.8) Lon proteases are induced by a variety of stresses including glucose starvation (33) and heat in E. coli (18). Up-regulation of LonA or B may correlate to the HicAB toxin-antitoxin response observed in the TM 1300 locus

(27). While the E. coli Lon protease was up-regulated in heat shock (18), the genes encoding T. maritima LonA and LonB proteases were down-regulated for this kind of stress (48). Outside of this stress response, the LonA and LonB ORFs have not exhibited

164 any significant up- or down-regulation in any other T. maritima microarray study (10, 25,

26, 40, 55). However, the Lon protease of Pseudomonas aeruginosa has been shown to be involved in quorum sensing (59) and biofilm formation (37).

ORFs encoding 21 ribosomal proteins were up-regulated in the mixed culture compared to the pure cultures (Table 3.14). This contrast was most evident for T. petrophila, which has three ribosomal protein ORFs down-regulated over 15-fold compared to the mixed culture. Increased ribosome synthesis is often associated with faster growth, but the observed growth rate for the mixed culture (td of ~ 65-70 min) was similar to the monocultures. Although no clear correlation could be found, the relationship between Lon protease, TA loci, and ribosomal proteins in the mixed culture merits further consideration.

Conclusions. The four Thermotoga species studied here had similar growth rates and cell densities when grown in pure culture on either glucose or polysaccharides. The mixed culture also had similar growth properties, indicating that neither competition nor substrate affected overall growth. However, species composition of the mixed culture was different on glucose as compared to polysaccharides. When the four Thermotoga were grown in mixed culture, differences in numbers of expressed ORFs compared to the pure cultures suggest that the comprehensive transcriptome of the mixed culture resembles that of T. sp. RQ2 and T. neapolitana, not T. petrophila nor T. maritima. ORFs encoding putative bacteriocins, stress proteins (ζE and Lon protease), and toxin-antitoxin

165 pairs, common to all four species, were up-regulated in the glucose-grown mixed cultures, but not for growth on polysaccharides, perhaps indicating competition among species when growing on the monosaccharide.

166

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Figures

Figure 3.1. Venn diagram of the shared ORFs of the selected Thermotoga species, based on 70% identity at the nucleotide level. Numbers indicate the ORFs shared by subsets of species. Legend: T. maritima – black; T. neapolitana– green;T. petrophila– blue;T.sp RQ2 - red.

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T. sp RQ2

T. Thermotoga petrophila Zoo

T. T. maritima neapolitana

Figure 3.2. Loop design for growth on glucose and polysaccharides, incorporating T. maritima, T. neapolitana, T. petrophila, and T. species strain RQ2, as well as RNA from the Thermotoga zoo. The head of the arrow represents Cy5 dye labeling and the tail represents Cy3 dye labeling.

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4 species mix

T. zoo T. zoo Pass 1 Pass 6

Figure 3.3. The loop experimental design for the Thermotoga zoo batch culture experiment. The head of the arrow represents Cy5 dye labeling and the tail represents Cy3 dye labeling.

4 species mix

T. zoo T. zoo 0 hours 60 hours

Figure 3.4. The loop experimental design for the Thermotoga zoo continuous culture experiment. The head of the arrow represents Cy5 dye labeling and the tail represents Cy3 dye labeling.

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Figure 3.5. Population composition for Thermotoga zoo growing on glucose-based medium for batch culture (A) and chemostat culture (B) and the polysaccharide mixture for the zoo (C). Estimates based on hybridization of genomic DNA from cultures to Thermotoga multi-species cDNA microarray. For batch culture, estimates after 1st and 6th passage are shown. For chemostat culture, estimates at start-up and after 60 hours (15 generations) of operation are shown.

177

Tm1305 Tm1308 Tm1311 Tm1299 Tm1306 Tm1309 Tm1312 Tm1300 Tm1307 Tm1313 Tm1301 Tm1302 Tm1303 Tm1304 Tm1310 Tm1314

Tpet

Tnea

TRQ2

Tm1315 Tm1320 Tm1316 Tm1321 Tm1317 Tm1319

Tpet

Tnea

TRQ2

Tm1329 Tm1326 Tm1330 Tm1323 Tm1327 Tm1331 Tm1333 Tm1324 Tm1325 Tm1328 Tm1332 Tm1334 Tm1335

Tpet

Tnea TRQ2 T. maritima T. neapolitana T. petrophila T. sp RQ2 TM1299 CTN_1283 Tpet_1482 TRQ2_1521 TM1300 CTN_1281a Tpet_1481 TRQ2_1520a TM1301 CTN_1281 Tpet_1480 TRQ2_1520 TM1302 CTN_1280 Tpet_1479 TRQ2_1519 TM1303 CTN_1279 Tpet_1478 TRQ2_1518 TM1304 CTN_1278 Tpet_1477 TRQ2_1517 TM1305 CTN_1277a Tpet_1476 TRQ2_1516 TM1306 CTN_1277 Tpet_1475 TRQ2_1515 TM1307 TM1308 CTN_1276 Tpet_1474 TRQ2_1514 TM1309 TM1310 CTN_1275 Tpet_1473 TRQ2_1513 TM1310a CTN_1274a Tpet_1472a

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Figure 3.6 continued TM1311 CTN_1274 Tpet_1472 TM1312 CTN_1273a Tpet_1471 TRQ2_1512 TM1313 CTN_1273 Tpet_1470 TRQ2_1511 TM1314 CTN_1272 Tpet_1469 TRQ2_1510 TM1315 CTN_1271 Tpet_1468 TRQ2_1509 TM1316 CTN_1270a Tpet_1467a TRQ2_1508a TM1317 CTN_1270 Tpet_1467 TRQ2_1508 TM1318 CTN_1269 Tpet_1466 TRQ2_1507 TM1319 CTN_1268 Tpet_1465 TRQ2_1506 TM1320 Tpet_1464 TRQ2_1505 TM1321 CTN_1267 Tpet_1463 TRQ2_1504 TM1322 CTN_1266 Tpet_1462 TRQ2_1503 CTN_1265 Tpet_1461 CTN_1264 Tpet_1460 CTN_1263 Tpet_1459 CTN_1262 Tpet_1458 TM1323 Tpet_1457a TRQ2_1502a TM1324 CTN_1261 Tpet_1457 TRQ2_1502 TM1325 CTN_1260 Tpet_1456 TRQ2_1501 TM1326 CTN_1259 Tpet_1455 TRQ2_1500 TM1327 CTN_1257 Tpet_1454 TRQ2_1499 TM1328 CTN_1256 Tpet_1453 TRQ2_1498 TM1329 CTN_1255 Tpet_1452 TRQ2_1497 TM1330 CTN_1254 Tpet_1451 TRQ2_1496 TM1331 CTN_1252 Tpet_1450 TRQ2_1495 TM1332 Tpet_1449 TRQ2_1494 TM1333 Tpet_1448 TM1334 CTN_1251 Tpet_1447 TRQ2_1493 TM1335 CTN_1250 Tpet_1446 TRQ2_1492 TM1336 CTN_1249 Tpet_1445 TRQ2_1491

Figure 3.6. Comparison of the TM_1299 through TM_1336 gene neighborhood across multiple Thermotoga spp. T. maritima locus numbers are located within the polygon representing each gene when possible. Grey shaded polygons indicate the presence of nucleotide homology to annotated genes from other Thermotoga species. Black shaded polygons indicate annotated pseudo-genes. Polygons that are placed above carrots indicate the addition of loci to the specific gene neighborhood, not present in other Thermotoga species. Dashed lines indicate where no nucleotide sequence is present, and serve to space homologs evenly in the figure. Polygons with a double-lined border within the gene neighborhood of T. neapolitana signify a non-continuous block of loci that are homologous to the corresponding area in T. maritima.

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Tables

Table 3.1. Unique ORFs/probes used to determine the composition of the Thermotoga zoo. T. maritima T. neapolitana T. petrophila T. sp. RQ2 TM0001 CTN_0679 Tpet_0257 2A5 TM0002 CTN_0674 Tpet_0298 2A8 TM0003 CTN_0673 Tpet_0943 2C2 TM0004 CTN_0667a Tpet_0944 2C9 TM0154* CTN_0647 Tpet_1500 2D12 TM0377 CTN_0617 Tpet_1621* A11 TM0390 CTN_0545 Tpet_1682 A12 TM0391 CTN_0531 Tpet_1723 A8 TM0411 CTN_0527 Tpet_1729 B2 TM0479 CTN_0508a Tpet_1735 C1 TM0504 CTN_0429 Tpet_1737* C12 TM0507 CTN_0383 Tpet_1741 C2 TM0589 CTN_0373 Tpet_1751 C9 TM0623 CTN_0372 Tpet_1753 D5 TM0625 CTN_0359 Tpet_1770 E2 TM0626 CTN_0345 Tpet_1771 E4 TM0995 CTN_0329a Tpet_1772 E6 TM0999 CTN_0316 Tpet_1773 E7 TM1024 CTN_0288 KJ3B1 TM1025 CTN_0245 KJ3D1 TM1144 CTN_0242 NTPC TM1145 CTN_0236 ORF1 TM1173 CTN_0201 ORF11 TM1236 CTN_0192 ORF12 TM1242 CTN_0147 ORF13 TM1299 CTN_0109 ORF14 TM1338 CTN_0102a ORF15 TM1411 CTN_0094 ORF16 TM1412 CTN_0074 ORF17 TM1429 CTN_0049a ORF5 TM1671 CTN_0048 ORF6 TM1779 CTN_0047 ORF7 TM1829 CTN_0046a RMLC TM1838 CTN_0046 TAA19 CTN_0045a TAA67

180

Table 3.1 continued CTN_0045 TAA68 CTN_0039a TAA96 CTN_0034 TAB07 CTN_0032 TAB30 CTN_0030 TAC01 CTN_0029 TAC02 CTN_0027 TAC08 CTN_0026 TAC14 CTN_1935 TAC19 CTN_1934 TAC39 CTN_1933 TAC43 CTN_1932 TAC44 CTN_1931 TAC62 CTN_1915a TAC83 CTN_1868 TAC87 CTN_1794 TAD30 CTN_1650a TAD42 CTN_1599a TAD43 CTN_1574 TAD55 CTN_1547 TAD64 CTN_1546 TAE11 CTN_1540 TAE28 CTN_1504 TAE30 CTN_1415a TAE43 CTN_1407 TAE44 CTN_1387 TAE53 CTN_1285 TAE64 CTN_1281a TAE65 CTN_1263 TAF05 CTN_1262 TAF09 CTN_1259a TAF14 CTN_1248 TAF15 CTN_1175a TAF46 CTN_1175 TAF50 CTN_1174 TAF61 CTN_1173 TAF65 CTN_1105a TAF85

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Table 3.1 continued CTN_1102a TAF91 CTN_1074 TAGB CTN_1055 TXX4 CTN_1024a WBAQ CTN_0918 CTN_0917 CTN_0916 CTN_0915 CTN_0913 CTN_0782 CTN_0715 CTN_0710 CTN_0709 CTN_0708 CTN_0697 CTN_0696 * ORFs TM0154, Tpet_1621, and Tpet_1737 were not used in calculations for the polysaccharide mix because the analysis software did not produce usable data.

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Table 3.2. Thermotoga species with full genome sequences

16S rRNA Protein Isolation site Growth homology to G+C Total coding T (°C) T. maritima Name opt Total bp (%) ORFs ORFs

Sulfate- reducing bioreactor, T. lettingae Belgium 65 90.8 2,135,342 38 2110 2040

Vulcano T. maritima Island, Italy 80 100.0 1,860,725 46 1928 1858

Volcanic spring near T. neapolitana Naples, Italy 77 99.4 1,884,540 47 1954 1905

Oil reservoir, T. petrophila Japan 80 99.1 1,823,511 46 1864 1785

Geothermally heated seafloor, T. sp RQ2 Azores 80 99.7 1,877,693 46 1905 1819

Table 3.3. 16S rRNA phylogeny for genome-sequenced Thermotoga species

T. maritima T. neapolitana T. petrophila T. sp RQ2 T. lettingae

T. maritima 100.0

T. neapolitana 99.4 100.0

T. petrophila 99.1 99.6 100.0

T. sp RQ2 99.7 99.4 99.2 100.0

T. lettingae 90.8 90.6 91.0 90.9 100.0

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Table 3.4. Hybridization of Thermotoga species genomic DNA to T. maritima cDNA microarray

Species # ORFs with ORFs not ORFs hybridizing % ORFs T. maritima 70% expected to T. maritima recognized by probes not homology (nt) to array based on 2- T. maritima hybridizing to T. maritima hybridize fold criterion array genome (out of 1929)

T.sp RQ2 1772 146 1813 94.0 116

T. petrophila 1721 197 1754 90.9 175

T. neapolitana 1616 302 1606 83.3 323

T. lettingae 215 1825

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Table 3.5. Common ORFs in selected Thermotoga genomes based on 70% homology at the nucleotide level. Thermotoga species# Number of ORFs M  N  P  R 1470* M  N  P only 35 M  N  R only 42 M  P  R only 174 N  P  R only 27 M  N only 42 M  P only 15 M  R only 59 N  P only 18 N  R only 22 P  R only 26 M only 54 N only 253 P only 53 R only 50 # M = maritima; N = neapolitana; P = petrophila; R = sp RQ2 *note that 10 ORFs not in the core genome are reported as two separate ORFs in other species

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Table 3.6. Non-core genome features in Thermotoga species

T. maritima T. neapolitana T. petrophila T.sp RQ2 exopolysaccharide synthesis TM0620-29 Tpet_1790-95 GTN_0328-31 Uncharacterized ABC GTN_0438-47 transporters GTN_1829-34 TRQ2_0970-75 TM0403-06 TRQ2_0627-30 mannans ABC transporter TM1746-50 TRQ2_1075-79 myo-inositol transporter TM0418-21 GTN_0432-35 Tpet_0500-03 ribose ABC transporter TM0955-59 GTN_0997-1001 TRQ2_0313-16 Tpet_1084-85 GTN_1892-93 CRISPR segments GTN_1889-91,94,96 Tpet_1081-83,86,88 TM1806-10 Tpet_1092-96 TRQ2_1010-14 TM1791-94, 1796-1802 TRQ2_1018-24,26-30 phosphotransferase system (PTS) TRQ2_0637-40 oxidoreductase TM0424-28 Tpet_0492-96 TRQ2_0516-20 electron transport system TM1207-17 GTN_1252-62 TRQ2_1601-11

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Table 3.7. Estimation of Thermotoga zoo population composition

Batch Chemostat Unique Species ORFs Glucose (%) Polysaccharide (%) Glucose (%)

1st pass 6th pass 1st pass 6th pass Start 60 h @ μ= 0.25 h-1 T. maritima 32 15.1  10.7 20.4  11.5 27.3  2.6 33.6 5.8 25.7  9.7 28.7  7.1

T. neapolitana 87 10.3 9.7 13.2  12.5 22.4 4.1 16.3 7.6 13.5  6.6 12.4  7.4

T. petrophila 15 11.5  7.0 11.7 10.9 21.7 3.1 14.6 5.2 21.7  6.3 15.8  7.8

T.sp RQ2 75 63.2  14.9 54.6  12.7 28.6 3.3 35.5 8.1 39.0  11.3 43.0  10.4

Estimates based onthe average of least squares mean estimates of unique ORFs in each species computed from ANOVA mixed effects model

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Table 3.8. Differential transcription of Thermotoga spp. for growth on the polysaccharide mixture compared to glucose.

Up-regulated on Down-regulated on Species polysaccharides polysaccharides total core non-core total core non-core T. maritima 64 57 7 114 87 27 T. neapolitana 31 30 1 166 125 41 T. petrophila 34 31 3 14 14 0 T. sp RQ2 40 29 11 44 30 14 Thermotoga zoo 93 42 50 75 40 35

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Table 3.9. ORFs encoding sugar utilization proteins in Thermotoga genomes. Positive values indicate up-regulation in polysaccharides vs. glucose, shaded squares indicate not present in the genome.

ID Substrate Annotation Tma Nea Pet RQ2 Zoo TM0025 β-glucosidase NC NC 3.5 NC -2.0 TM0027 ATP-binding protein NC NC 7.2 -2.9 -2.2 laminarin TM0029 permease protein 2.9 NC 4.4 -2.7 -3.0 TM0030 permease protein NC NC 2.2 NC NC TM0031 periplasmic binding protein NC NC NC -2.8 -3.0 TM0056 periplasmic binding protein 6.9 2.2 NC 7.7 6.6 TM0057 ATP-binding protein 4.2 2.8 3.7 6.0 5.6 TM0058 ATP-binding protein 2.4 2.2 4.7 NC NC TM0060 permease protein NC 2.1 NC NC NC TM0061 endo-1,4- β -xylanase A 12.1 5.9 NC 14.3 13.0 TM0070 xylan endo-1,4- β -xylanase B 4.1 2.3 NC 3.2 2,1 TM0071 periplasmic binding protein 10.7 2.2 3.6 13.7 10.0 TM0072 permease binding protein NC NC NC NC 2.1 TM0073 permease protein 2.3 NC NC 2.5 2.9 TM0074 ATP-binding protein NC 2.2 NC NC 2.9 TM0075 ATP-binding protein NC NC NC NC NC TM0077 acetylxylan esterase NC 2.4 NC 2.4 2.9

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Table 3.9 continued TM0102 unknown basic membrane protein NC -2.3 3.7 -2.2 NC TM0105 sugar permease protein -2.2 NC NC NC NC TM0310 β-D-galactosidase NC NC NC 3.3 3.2 TM0431 permease protein NC 3.8 TM0433 pectin pectatelyase 2.2 2.3 4.3 TM0437 exo-poly- α-D-galacturonosidase 2.9 NC NC NC TM0752 α-glucosidase NC NC NC 2.6 2.1 TM1062 β-glucuronidase -2.0 NC NC NC TM1068 α-glucosidase -2.4 NC NC NC NC TM1195 β-galactosidase NC NC NC NC 2.1 TM1198 pectin permease protein NC NC NC 2.3 TM1199 periplasmic binding protein NC NC NC 2.9 TM1219 ATP-binding protein 3.2 2.5 -2.7 2.5 3.1 TM1220 ATP-binding protein NC 2.1 -2.4 NC NC TM1221 permease protein NC NC -2.9 NC NC TM1222 permease protein 3.2 NC NC NC NC mannan TM1223 periplasmic binding protein 38.3 13.6 -5.4 15.5 10.9 TM1224 transcriptional regulator, XylR NC NC NC NC 7.4 TM1226 periplasmic binding protein 24.5 NC NC 16.9 11.3 TM1227 endo-1,4-β-mannosidase 27.8 12.4 -4.2 16.0 10.5 TM1281 6-phospho-β-glucosidase 2.1 NC NC NC 2.1 TM1524 endoglucanase/mannanase 2.3 2.2 NC 4.7 NC TM1525 endoglucanase/mannanase NC 2.8 NC NC NC TM1624 β-mannosidase 4.5 NC NC NC NC

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Table 3.9 continued TM1746 periplasmicoligopeptide protein 5.5 4.2 2.8 TM1747 permease protein 12.6 8.9 5.1 mannan TM1749 ATP-binding protein 10.5 4.6 3.6 TM1750 ATP-binding protein 10 6.3 5.2 TM1751 endoglucanase 3.5 2.9 2.7 TM1834 α-glucosidase 3.1 NC NC -3.9 -4.4 TM1836 permease protein -2.6 NC NC -2.8 -2.9 TM1837 maltose permease protein -2.3 NC NC NC NC TM1839 periplasmic maltose-binding protein -8.2 -7.8 NC -8.8 -3.0 TM1844 GH13 NC NC 2.8 -5.3 -4.1 TM1845 pullulanase NC NC 3.2 NC NC TM1848 cellobiosephosphorylase 13.6 4.7 -3.1 9.3 5.0 TRQ2_0632 GH43 -2.0 NC TRQ2_0658 α-N-arabinofuranosidase NC 2.3 TRQ2_0661 extracellular sugar-binding protein NC 4.7 unknown TRQ2_0662 GH43 NC 2.5 sugar TRQ2_0664 GH43 NC 2.4 TRQ2_0667 GH43 NC 3.4 TRQ2_0970 extracellular solute-binding protein -3.0 NC -6.7 -22.2 TRQ2_0972 unknown sugar binding protein NC NC -14.4 -5.2 TRQ2_0973 sugar periplasmic binding protein -4.2 -2.5 -18.7 TRQ2_0974 (glucose?) monosaccharide-transporting ATPase -2.1 -3.1 -4.7 TRQ2_0975 monosaccharide-transporting ATPase NC -2.2 -3.8 TRQ2_0976 ABC transporter related, ROK family -2.0 NC -3.4 TRQ2_1647 α-glucanphosphorylase NC -3.3

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Table 3.9 continued unknown CTN_0244 sugar permease protein NC -6.1 CTN_0632 endo-1,4-β-xylanase A 4.7 NC CTN_0781 α-amylase NC 5.2 CTN_1407 α -glucanphosphorylase NC -3.0 Tpet_0636 sugar binding protein NC 3.5 Tpet_0642 GH43 NC 2.1 Tpet_1794 monosaccharide-transporting ATPase NC 2.4

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Table 3.10. Exopolysaccharides synthesis operon regulatation on glucose and polysacahrides. ( positive values indicate up- regulation in polysaccharides vs. glucose, shaded squares indicate not present in the genome) ID Annotation T. maritima T. neapolitana T. petrophila T. sp RQ2 TM0622 lipopolysaccharide biosynthesis protein, putative -3.38 TM0625 hypothetical protein -2.08 TM0627 lipopolysaccharide biosynthesis protein -3.21 TM0628 hypothetical protein -2.72 TM0630 nucleotide sugar epimerase, putative -3.07 TM0631 lipopolysaccharide biosynthesis protein -2.25 TM0632 extracellular polysaccharide biosynthesis-related protein -2.48 NC

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Table 3.11. ORFs differentially transcribed in the mixed culture compared to the pure cultures of each Thermotoga species growing on glucose.

2-fold 2-fold 3-fold 3-fold 4-fold 4-fold  mixed  pure  mixed  pure  mixed  pure Species culture culture culture culture culture culture

T. maritima 379 243 231 109 152 48

T. neapolitana 233 49 71 19 26 8

T. petrophila 472 257 328 127 242 51

T. sp RQ2 168 105 2 16 1 1

Table 3.12. ORFs up- or down-regulated ≥2-fold in the Thermotoga zoo compared to pure cultures up-regulated down-regulated in the T. zoo in the T. zoo T. maritima 134 107 T. neapolitana 353 111 T. petrophila 81 40 T. sp. RQ2 107 40

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Table 3.13. Response of selected ORFs in Thermotoga species in mixed culture vs. pure culture.

GENE ID Fold up-regulation in Thermotogazoo vs. – Annotation T. maritima T. neapolitana T. petrophila T. sp. RQ2 T. maritima T. neapolitana T. petrophila T. sp. RQ2 TM0785 GTN_0824 Tpet_0144 TRQ2_0142 Bacteriocin 3.0 2.2 4.8 1.7 TM1300 GTN_1334 Tpet_1481 TRQ2_1520a Bacteriocin 8.0 2.6 6.9 2.6 TM1310a GTN_1342a Tpet_1472a HicA toxin (RASTA) not on chip not on chip not on chip TM1311 GTN_1343 Tpet_1472 HicB antitoxin 2.6 NC 2.1 TM1312 GTN_1343a Tpet_1471 TRQ2_1512 HicA toxin 1.8 9 81.1 2 TM1313 GTN_1344 Tpet_1470 TRQ2_1511 HicB antitoxin -2.1 NC -2.7 NC TM1320 Tpet_1464 TRQ2_1505 HicA toxin (Frame Shift) 8.9 16.6 2.8 TM1321 GTN_1350 Tpet_1463 TRQ2_1504 HicB antitoxin 11.5 4 15.8 3.5 TM0571 GTN_0588 Tpet_0347 TRQ2_0365 DegS-type ζE regulator 6.2 2.1 4.5 4.0 RNA polymerase ζE TM1598 GTN_1751 Tpet_1194 TRQ2_1261 factor 2.0 2.0 4.1 2.7 TM1633 GTN_1786 Tpet_1158 TRQ2_1299 Lon protease 4.8 2.6 6.8 2.5 TM1869 GTN_1805 Tpet_0928 TRQ2_0950 Lon protease 4.0 2.3 5.3 2.0

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Table 3.14. Differentially transcribed ribosomal protein ORFs in mixed culture during growth on glucose.

GENE ID fold up-regulation in Thermotoga zoo vs. - T. sp. T. maritima T. neapolitana T. petrophila T. sp. RQ2 annotation (T. maritima) T. maritima T. neapolitana T. petrophila RQ2 TM0451 GTN_0463 Tpet_0469 TRQ2_0484 ribosomal protein L33 5.9 5.3 27.9 3.1 TM0454 GTN_0466 Tpet_0466 TRQ2_0481 ribosomal protein L11 3.6 3.4 11.3 2.1 TM0457 GTN_0469 Tpet_0463 TRQ2_0478 ribosomal protein L7/L12 17.9 4.6 21.2 3.8 TM1399 GTN_1425 Tpet_1384 TRQ2_1430 ribosome recycling factor 8.1 2.5 6.4 3.0 TM1453 GTN_1573 Tpet_1341 TRQ2_1345 ribosomal protein S9 4.3 2.3 5.3 2.2 TM1454 GTN_1574 Tpet_1340 TRQ2_1346 50S ribosomal protein L13 3.3 2.3 5.2 2.0 TM1473 GTN_1595 Tpet_1319 TRQ2_1367 30S ribosomal protein S4 6.5 2.8 7.8 2.7 TM1474 GTN_1596 Tpet_1318 TRQ2_1368 30S ribosomal protein S11 5.0 2.9 8.4 2.9 TM1475 GTN_1597 Tpet_1317 TRQ2_1369 30S ribosomal protein S13 6.1 3.0 8.8 2.9 TM1477 GTN_1599 Tpet_1315 TRQ2_1371 translation initiation factor IF-1 3.5 2.6 7.0 2.5 TM1484 GTN_1606 Tpet_1308 TRQ2_1378 ribosomal protein L18 4.2 2.3 5.0 2.1 TM1488 GTN_1610 Tpet_1304 TRQ2_1382 50S ribosomal protein L5 8.2 2.5 6.3 2.3 TM1492 GTN_1614 Tpet_1300 TRQ2_1386 ribosomal protein L29 2.2 3.4 11.2 2.2 TM1495 GTN_1617 Tpet_1297 TRQ2_1389 ribosomal protein L22 3.5 2.4 5.8 2.2 TM1496 GTN_1618 Tpet_1296 TRQ2_1390 ribosomal protein S19 3.6 2.2 5.0 2.1 TM1501 GTN_1623 Tpet_1291 TRQ2_1395 ribosomal protein S10 5.0 2.3 5.5 2.6 TM1504 GTN_1626 Tpet_1288 TRQ2_1398 30S ribosomal protein S7 4.9 2.7 7.3 2.2 TM1505 GTN_1627 Tpet_1287 TRQ2_1399 30S ribosomal protein S12 3.0 2.5 6.2 2.7

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CHAPTER 4: Biohydrogen production by Thermotoga species growing on simple and complex α- and β- linked carbohydrates

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ABSTRACT

The growth physiology of the hyperthermophilic bacterium Thermotoga maritima on α- vs. β-linked sugars and presence vs. absence of elemental sulfur was examined using batch and continuous cultures. Unlike the case for Pyrococcus furiosus, a fermentative hyperthermophilic archaeon, the T. maritima transcriptome varied little, whether grown on cellobiose or maltose or in the presence or absence of elemental sulfur.

However, similar to P. furiosus, growth was better on cellobiose than maltose, perhaps related to a cellobiose phosphorylase (TM1848) that mediated superior bioenergetics on that disaccharide. Biohydrogen production by Thermotoga maritima and three other

Thermotoga species (T. maritima, T. neapolitana, T. petrophila, and T. sp RQ2) was examined for pure and mixed culture growth on glucose and a polysaccharide mixture containing carboxymethylcellulose, galactomannan, glucomannan, lichenan, pectin, xylan (birchwood), and xylan (oat spelts). Under an inert gas (N2) sparge rate of 10

-4 ml/min, H2 production differed among the four species, ranging from 7.4 x 10 mol H2

-1 -1 -3 -1 -1 L h for T. neapolitana on glucose to 1.6 x 10 mol H2L h for the mixed culture on polysaccharides. The mixed culture exhibited the highest volumetric hydrogen production on both substrates, but when adjusted for cell density at time of cell harvest, production rates for all four species in pure or mixed culture were statistically similar. The effect of

N2 sparging rates was examined for T. maritima. Increasing the sparge rate from 10 ml/min to 50 ml/min reduced the doubling time from 62 to 47 minutes, concomitant with a maximum headspace hydrogen reduction from 5.3% to 1.6%. Final cell densities were

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~3.0x108 cells/ml for all cases. The maximum hydrogen production rate increased from

-1 -1 -1 -1 6.0 mmol H2 L h for the 10 ml/min sparged culture to 7.0 mmol H2 L h for the 50 ml/min sparged culture, but in both cases occurred at the transition from log to stationary phase. While increased sparging resulted in an increase in maximum production rate, it also caused a reduction in the total amount of H2 produced over the life of the culture; the

-1 50 ml/min sparged culture produced 45.1 mmol H2 L and the 10 ml/min sparged culture

-1 produced 53.5 mmol H2 L . These results indicate that for Thermotoga fermentation, operating conditions must be chosen to maximize either H2 production rate or total amount of H2 produced.

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INTRODUCTION

Biofuels could provide a way to reduce U.S. dependence on fossil fuels, specifically imported petroleum (12). Liquid biofuels, especially ethanol, are currently receiving the most attention (4). However, corn-based ethanol usage for fuels conflicts with food requirements, and even cellulosic ethanol does not completely avoid problems with elevated greenhouse gas emissions (21). Consequently, interest in hydrogen as a way to replace fossil fuels has increased (12, 18). As such, microbial approaches for biohydrogen production are being carefully examined. Extremely thermophilic microorganisms (Topt  70°C) are of particular interest, given their higher yields of H2 per mole of sugar consumed compared to mesophilic bacteria (17, 50). Production levels of

H2 near the “theoretical” (Thauer) limit of four H2 molecules per glucose substrate have been reported (29, 64). The highest H2 production levels have been achieved with species in the genus Thermotoga, which in some cases approach the Thauer limit (12, 50, 55, 60); in these studies, glucose or xylose were used as substrates to elucidate hydrogen production pathways and yields (9). Industrial scale production of hydrogen, however, will be based on complex carbohydrates (7, 42), such as paper sludge hydrolysate (28) and cow manure (65), so that microorganisms with versatile, oligotrophic growth physiologies will be required. Evaluation of biohydrogen production for these types of substrates has been limited to date.

Biohydrogen production through dark fermentation must address the issue of self- inhibition due to high partial pressures of hydrogen (58). In Thermotoga species, high H2

200 partial pressures inhibit growth, resulting in lower cell densities than facultatively hydrogenotrophic mesophiles, such as E. coli (50). High levels of hydrogen also cause a shift in metabolic byproducts from H2 to alanine (30) or lactate (6). The most commonly used solution to reduce the partial pressure of H2 is sparging with N2 (36). Sparging stationary phase batch cultures of Thermotoga neapolitana has been shown to increase total H2 production over the total growth cycle of the batch cultures (41). T. neapolitana can also maintain H2 production rates (15) in the face of microaerophilic conditions (37).

Another method of alleviating hydrogen inhibition is to co-culture Thermotoga species with H2-metabolizing methanogens; T. maritima grown with Methanococcus janaschii achieved increased cell densities, although methane, and not H2, was the major gaseous energy product (25).

The metabolic routes to H2 generation from carbohydrates for Thermotoga species have yet to be fully characterized (10, 23), but progress has been made with the characterization of the primary hydrogenase used by T. maritima (51). One known difference between hydrogenesis in Thermotoga species and P. furiosus is the direct conversion of acetyl-CoA to acetate by means of an acetyl-CoA synthetase (49). In the archaeal system, the reducing equivalents are generated by glycolysis and then transferred to ferredoxin (5), which acts as electron donor to a membrane-bound hydrogenase (5, 48). During growth on sulfur, this archaeal hydrogenase system is replaced in Thermotoga species by an oxidoreductase system that moves the electrons from ferredoxin to a sulfur reductase (52). In contrast, T. maritima metabolizes sugars to

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CO2, acetate, lactate and H2 by a conventional Embden-Myerhof pathway (1). Hydrogen sulfide replaces H2 as a product in the presence of polysulfide, and alanine is also produced from sugar fermentation (46). Thermotoga species characteristically have an exceptionally broad range of glycoside hydrolases and a complete Entner-Doudoroff pathway (38, 61); these are absent in the only extremely thermophilic bacterial genus with similar high production level of H2 per mole of glucose (14), namely

Caldicellulosiruptor (57).

Recently, a number of Thermotoga genomes have been sequenced: T. maritima

(38), Thermotoga petrophila (66), and Thermotoga lettingae (66). The genome sequence of another Thermotoga species, strain RQ2, was recently released (Noll, K, unpublished data). Finally, the sequence of Thermotoga neapolitana was completed by the Craig

Ventner Institute (formerly The Institute for Genome Research) (K. Nelson, personal communication). With the exception of T. lettingae, the genome-sequenced Thermotoga species appear to have approximately 75% of their ORFs in common with T. maritima, as well as 16S rRNA identities of 98-100%. Table 4.1 summarizes the similarities and differences of the genomes. The genome similarities were confirmed by heterologous hybridization to a T. maritima whole genome microarray (see Chapter 2). This array was then used as a basis for creating a multi-species array for use with any of the four closely related Thermotoga (see Chapters 2 and 3).

Here, the effect of sulfur on the transcriptome of T. maritima growing on either maltose or cellobiose was examined. Then, hydrogen production by the four Thermotoga

202 species in pure and mixed culture was examined for growth on glucose and a mix of seven polysaccharides (carboxymethylcellulose, galactomannan, glucomannan, lichenan, pectin, xylan (birchwood), and xylan (oat spelts)). Finally, the effect of varying the N2 sparge rate was examined using batch culture of T. maritima.

MATERIALS AND METHODS

Louis) or a polysaccharide mix (0.083% w/v) consisting of equal parts lichenan (> 85% purity) (Megazyme, Wicklow, Ireland), konjac glucomannan (90% purity) (Jarrow

(Sigma-Aldrich). Prior to determining growth characteristics, cells were grown for at least six passes on either glucose or the polysaccharide mixture, using a 1% (v/v) inoculum. Cell densities were monitored by epifluorescence microscopy using acridine orange stain, as described previously (54).

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Palmer, Vernon Hills, Illinois) connected to a heating mantle (Fisher Scientific, Waltham,

Massachusetts). Culture pH was monitored using an autoclavable pH probe connected to a Chemcadet pH controller (Cole-Palmer). The flask was positioned on a stir plate, such that it could be mixed at 400 rpm with a Teflon stir bar. For comparisons of H2 production by the various Thermotoga species and the mixed culture, the inert gas sparging rate was controlled at 11 ml/min by a rotameter (Cole-Palmer). For experiments comparing H2 production over the full T. maritima growth cycle, the inert gas sparging rate was controlled by a rotameter for the 10 ml/min condition, and a digital mass flowmeter for the 50 ml/min condition. To achieve anaerobic conditions prior to inoculation, the flask was sparged with N2 and reduced with 6 mL/L of 10% (w/v) Na2S.

For all experiments, cultures were passed six times from the same stock as 70 mL batch cultures (1% inoculum) in 150 mL serum bottles sparged with N2. The sixth pass was grown for 12 hours, and cooled to room temperature prior to inoculation at an initial cell density of ~6 x 106 cells/ml. Cell counts were done hourly until early/mid log phase, when biomass samples for RNA extraction were taken.

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Batch, mixed cultures of the Thermotoga “zoo”. Thermotoga “zoo” growth experiments for RNA extraction were performed in the same 2 L (1 L working volume)

5-neck round-bottom flask used for batch culture of the single species cultures. To establish the mixed culture, T. maritima, T. neapolitana, T. petrophila, and T. sp RQ2 were passed six times in N2 sparged, batch pure cultures (1% inoculum) of ASW, with glucose or polysaccharide mix supplemented, as described above. The sixth passes were grown for 12 hours, cooled to room temperature, and then used to inoculate a mixed culture in equal proportions, such that an initial cell density of ~6x106 cells/ml was established. These mixed „zoo‟ culture containing the four Thermotoga species was again grown for six passes anaerobically at 800C on ASW with glucose or polysaccharide mix supplemented. Following the sixth pass, the mixed culture was grown for 12 hours, cooled to room temperature, and then used to inoculate the 2L sparged batch flask, again as done for the single cultures.

Batch culture of T. maritima in presence and absence of sulfur. To examine the effect of elemental sulfur, T. maritima was established on ASW media, supplemented with either cellobiose or maltose, as described above. As appropriate, elemental sulfur was added to a level of 10 g/L (w/v). Once established, the cultures were used to inoculate two 500 mL bottles containing 330 mL of ASW supplemented with the appropriate sugar and, if needed, sulfur. Cell densities were monitored by epifluorescence microscopy using acridine orange stain, as described previously (54).

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Continuous culture of T. maritima The continuous culture experiments were performed in a continuous culture system with a 2 L 5-neck round-bottom flask with a 1

L working volume, sparged with high purity (99.998 %) N2 (Airgas/National Welders,

Charlotte, NC), and used methods described previously (44). Cultures of T. maritima were grown anaerobically for six passes at 800C on ASW supplemented with either cellobiose or maltose (0.25% w/v). Growth temperature was controlled at 79 ± 10C, using a type K thermocouple and a Digi-Sense controller (Cole-Palmer, Vernon Hills, Illinois), connected to a heating mantle (Fisher Scientific, Waltham, Massachusetts). Culture pH was monitored using an autoclavable pH probe, connected to a Chemcadet pH controller

(Cole-Palmer). The flask was positioned on a stir plate, such that it could be mixed at 400 rpm with a Teflon stir bar. The inert gas sparging rate was controlled at 11 ml/min by a rotameter (Cole-Palmer). To achieve anaerobic conditions prior to inoculation, the flask was sparged with N2 and reduced with 6 mL/L of 10% (w/v) Na2S. For all experiments, cultures were passed six times from the same stock as 70 mL batch cultures (1% inoculum) in 150 mL serum bottles sparged with N2. The sixth pass was grown for 12 hours, and cooled to room temperature prior to inoculation at an initial cell density of ~6 x 106 cells/ml.

After inoculation, the reactor was operated in batch mode until a cell density of

~1.0x108 cells was achieved, at which point continuous operation at a dilution rate of

0.25 h-1 was initiated. Samples for cell counts were taken from the reactor every 4 hours.

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Cell density, pH, and gas concentrations usually reached steady state after three culture volume changes, such that samples harvested after ~6 volume changes (typically about

15 hours after initiating continuous operation) were at biological steady state. In order to examine gene regulation during growth on maltose or cellobiose, the continuous culture was initially begun with media containing either cellobiose or maltose (0.25% w/v). Once steady state was achieved, biomass samples were taken for RNA extraction, following which ASW with an equal amount of cellobiose and maltose (0.125% w/v each) was added to the chemostat and allowed to return to steady state. Once steady state was achieved for the mixed sugar culture, biomass samples were once again removed for

RNA extraction. The media was then replaced with ASW supplemented by the original sugar (cellobiose or maltose) at 0.25% w/v. This culture was allowed to reach steady state and then biomass was again removed for RNA extraction. Cell densities were monitored by epifluorescence microscopy using acridine orange stain, as described previously (54).

Measurement of product gas composition. Product gas from the sparged batch reactor was measured every 15 minutes with a Gow-Mac G400C on-line gas chromatograph (GC) with a thermoconductivity detector, using Chromperfect 5.0 software (Justice Laboratory Software, Palo Alto) to program auto-sampling frequency.

The product gas from the sparged batch reactor was passed through a manifold to a cooling tower to minimize evaporative water loss. Prior to reaching the GC, the product gas was passed through an adsorber filled with drierite (Fisher Scientific, Pittsburgh) to

207 remove residual traces of H2O. Peaks were detected by the gas chromatograph and recorded by the Chromperfect 5.0 software. The curves were later integrated and the gas composition was calculated by comparing the peak area to calibration curves generated from standard gas mixtures (Airgas/National Welders, Charlotte).

Design of the genus level cDNA microarray. The Thermotoga genus cDNA microarray used a combination of previously constructed cDNA probes from T. maritima, along with new probes representing ORFs specific to the other three species.

Vector NTI Advance 10 (Invitrogen, Carlsbad, CA) was used for probe design. Probes were made by PCR using primers designed with Vector NTI Advance 10 and ordered from Integrated DNA Technologies (Coralville, Iowa). The primers were used with genomic DNA and spotted to slides, according to the established procedures (8). For deciding what probes to add to create the multi-species array, two approaches were used.

For T. neapolitana and T. petrophila, in silico analysis was used in conjunction with heterologous cross-hybridization results (see Chapter 2). GenomeBlast (http://bioinfo- srv1.awh.unomaha.edu/genomeblast/) provided an ORF-to-ORF pair-wise comparison of the whole genomes (34). NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (3, 24) was done for individual ORFs that produced errors or inaccuracies in GenomeBlast. For example, ORFs that were annotated in T. maritima as a “Frame Shift” would not produce matches on GenomeBlast under any conditions. T. neapolitana and T. petrophila ORFs added to the array were chosen based on a combination of BLAST homology of less than

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70% nucleotide identity, taken together with DNA cross-species hybridization results.

When the BLAST results and the experimental data conflicted, experimental data were given greater weight. For T. sp RQ2, the full sequence was not available, so instead experimental results from suppressive subtractive hybridization experiments reported previously (39) were used to design probes for the ORFs and genome fragments. Note that the T. sp RQ2 genome sequence has since been made available on NCBI (K. Noll, unpublished data).

RNA extraction procedure. Unless otherwise specified, all chemicals used for

RNA preparations were supplied by Sigma-Aldrich, St. Louis. Biomass samples for total

RNA isolation were taken from cells growing on glucose or polysaccharides in early- to mid-exponential phase, using previously described protocols (16).

cDNA microarray protocols. The creation of cDNA from the extracted RNA, and subsequent RNA-based microarray analysis, was done using methodologies discussed previously (8, 27). Hybridizations were carried out for 18-20 hours following modified TIGR protocols, described elsewhere (8, 19, 20). Hybridized slides were scanned on a Perkin-Elmer ExpressLite Scanner (Perkin-Elmer, Waltham,

Massachusetts) and quantitated by using ScanArray 2.1 (Perkin-Elmer).

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Mixed model analyses of microarray data. Replication of treatments, arrays, dyes, and cDNA spots allowed the use of analysis of variance (31, 32, 63) models for data analysis. For each experiment, a loop design was constructed and reciprocal Cy dye- labeling was utilized for all samples to estimate dye effects for each treatment. For the glucose and polysaccharide experiments, these loops had five and six slides each, respectively (Figures 4.1 and 4.2). For the Thermotoga maritima sulfur experiment, a four slide loop was used. The loop contained two slides each for the two sugars, one with sulfur and one without sulfur (Figure 4.3). The T. maritima continuous culture experiment examined single and two-sugar growth transcriptomes based on a six slide loop (Figure 4.4). Slides were scanned using PerkinElmer ScanArray software and spot intensities were imported into JMP Genomics (SAS Institute, Cary, NC). After local background subtraction and log transformation of spot intensities, a linear normalization analysis of variance model (63) was used to estimate global variation in the form of fixed effects (dye [D], treatment [T]), random effects (array [A], spot A [S], block A [B]), and random error by using the model log2(yijklmno) = Ai + Dj + Tk + Ai(Sl) +Ai(Bm)+ εijklmn. A gene-specific analysis of variance model was used to partition the remaining variation into gene-specific effects using the model rijklmno = Ai + Dj + Tk + Ai(Sl) +Ai(Bm)+ εijklmn.

Gene annotations are based on published data including the T. maritima sequence (38), T. maritima transcriptomics (13, 26, 27), the COG database at the National Center for

Biotechnology Information (NCBI) (56), the Conserved Domain Database at the NCBI

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(35), NCBI BLAST searches (3, 24), and the Thermotoga sequences available on

MEDLINE.

RESULTS

Effect of S0 and glucan disaccharide linkage on the growth physiology of T. maritima. There were few differences in the T. maritima transcriptome for batch growth on cellobiose compared to maltose in either the presence or absence of elemental sulfur

(S0) (Table 4.1). Previous work with P. furiosus looking at the effect of glucoside linkage and sulfur found that, in the absence of S0, cellobiose-grown cultures generated twice as much protein and 50% more hydrogen than maltose-grown cells. The addition of S0 changed overall metabolism differently for each species, with maltose-grown cultures showing larger increases in hydrogen and protein production. S0 also induced up- regulation of 135 ORFs and down-regulation of 137 ORFs (11). The response in T. maritima had many fewer changes, with only three ORFs down-regulated on sulfur for

211 both sugars, and eight up-regulated (Table 4.1). All of the ORFs exhibiting differential regulation The three up-regulated ORFs were a putative iron-sulfur binding protein

(TM1292), a dihydrofolate reductase (TM1641), and a hypothetical protein (TM1681)

(Table 4.1). Eight ORFs were up-regulated in the culture with S0 for both sugar cultures

(Table 4.1). These ORFs included hypothetical proteins organized in an operon

(TM0980-0982). Other ORFs that were up-regulated included phosphoglycerate kinase

(TM0689) and the transcriptional regulator TetR (TM0823).

Chemostat culture was used to examine the effect of glucoside linkage (maltose vs. cellobiose) on the T. maritima transcriptome in the absence of S0. Three ORFs were up-regulated at least two-fold on maltose compared to cellobiose: a maltose-binding protein (TM1839), an α-glucsidase (TM1834), and an α-glucan phosphorylase (TM1168)

(Table 4.2). The maltose binding protein and –glucosidase are part of a maltose processing operon. Eleven ORFs were up-regulated in cellobiose compared to maltose, including several involved in cellobiose metabolism such as cellobiose phosphorylase

(TM1848), and a cellobiose transporter operon (TM1219-1223) (Table 4.2). Also up- regulated on cellobiose were a Lac family transcriptional regulator, CelR (TM1218), and an NADH polysulfide reductase (TM0379). The CelR transcriptional regulator has been shown to be involved in the breakdown of cellulose and cellobiose in other species (62).

Also examined in chemostat culture was the impact of the simultaneous presence of maltose and cellobiose in the media on the T. maritima transcriptome. This was investigated by changing the sugar in the culture media from either cellobiose or maltose

212 alone to an equal mixture of the two glucan dimers. Later, the media was again replaced with one containing only the original single sugar, cellobiose or maltose. Twelve ORFs responded significantly to addition of maltose to the cellobiose culture, while only five

ORFs responded when maltose was removed and cellobiose became the only substrate

(Table 4.3). Twenty-six ORFs were up-regulated upon the addition of cellobiose to the maltose culture, while 25 ORFs were up-regulated upon return to maltose culture. With the addition of cellobiose to maltose ORFs involved in cellobiose transport (TM1219-23) and metabolism (TM1848) were significantly up-regulated (Table 4.4). The transcriptomes of the single sugar cultures (M1 and M2, C1, and C2 in Figure 4.2) showed no significant differences, indicating that there was no hysteresis effect for the glucan dimer cultures.

Biohydrogen production by Thermotoga species. As described in Chapter 3, all of the Thermotoga species in pure and mixed culture exhibited similar doubling times, final cell densities, transitions from lag phase to log phase, and transition to stationary phase on both glucose and polysaccharides. Such similar growth properties suggest that

H2 production levels might also be similar across the Thermotoga genus. The generation

-4 of H2 through the fermentation of glucose in batch culture ranged from 7.4 x 10 mol H2

-1 -1 -3 -1 -1 L h for T. neapolitana on glucose to 1.5 x 10 mol H2 L h for the Thermotoga zoo.

-4 The fermentation of polysaccharides produced H2 at rates ranging from 9.8 x 10 mol H2

-1 -1 -3 -1 -1 L h for T. petrophila to 1.6 x 10 mol H2 L h for the mixed culture (Figure 4.3). In

213 order to account for the different cell densities of each culture, the H2 production levels were determined on a per cell basis, using the final cell counts and the H2 production level before RNA sampling. Production of H2 on a per cell basis was found to be within the margin of error of the cell counts for all four species as well as the Thermotoga zoo during growth on either glucose or polysaccharides (Figure 4.4). The values for

-11 -1 -1 -1 polysaccharide culture ranged from 2.4 x 10 mol H2 L h cell for T. petrophila to

-11 -1 -1 -1 2.7 x 10 mol H2 L h cell for the Thermotoga zoo. The glucose cultures produced

-11 -1 -1 -1 -11 values that ranged form 2.4 x 10 mol H2 L h cell for T. neapolitana to 3.0 x 10

-1 -1 -1 mol H2 L h cell for T. sp. RQ2.

Effect of sparge rate on T. maritima growth and biohydrogen production properties. Increasing the sparge rate from 10 ml/min to 50 ml/min changed some growth properties of T. maritima, while leaving others unaffected (Table 4.5, Figure 4.5).

The increase reduced the doubling time of the T. maritima culture from 62 minutes for the 10 ml/min culture to 47 minutes for the 50 mL/min culture (Table 4.5). Pressure was maintainted at 1 atm while ambient and culture chamber temperatures were maintained at

18-21oC and 80oC, respectively. The final cell density was unaffected, with both cultures having final cell densities near 3.1x108 (Table 4.5, Figure 4.6). The change in the sparge rate also affected H2 production by the cultures. The specific volumetric hydrogen

-1 production rate in the 50 mL/min culture reached a maximum value of 7.0 mmol H2 L

-1 -1 -1 h , and the 10 ml/min culture had a maximum of 6.0 mmol H2 L h (Table, 4.5,

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Figure 4.6). Both of these maximum values occurred at the transition from log to stationary phase (Figures 4.5 and 4.6). The hydrogen content of the headspace also peaked at the transition to stationary phase, with the headspace of the 10 ml/min sparged culture containing 5.3% hydrogen and the 50 ml/min sparged culture containing 1.6% hydrogen at their maximum values (Table 4.5).

DISCUSSION

T. maritima: effects of S0 and glucan dimer linkage. Among the hyperthermophiles, the organism with the best studied system for glucan dimer uptake and metabolism is Pyrococcus furiosus (11, 33). Previous work with P. furiosus had shown significant and varied response to the presence of S0, with ORFs encoding many hydrogenase subunits showing significant changes (2). A similar study using P. furiosus in continuous culture showed S0 and glucan dimer linkage each had effects on both biohydrogen and biomass production. P. furiosus produced twice the biomass and 50%

0 more H2 on cellobiose than maltose. Also, addition of S to the P. furiosus cultures caused an increase in protein production and shift to H2S production for both sugars, but the observed changes in metabolism were larger in maltose culture (11). Compared to P. furiosus, the response of T. maritima is different and more specific. One factor in this difference is that T. maritima is inhibited to a much more significant extent by H2 than P. furiosus and uses S0 to alleviate the inhibition. The presence or absence of S0 produced

215 similar statistically significant transcriptional changes in the same ORFs for both cellobiose and maltose (Table 4.1). The similarity of these changes suggests that sulfur acts independently of glucan dimer linkage in T. maritima.

The most interesting transcriptional change induced by S0 was the differential transcription of the TM0980-82 ORFs. A BLAST search of these ORFs indicated that the previously uncharacterized TM0981 was similar to a DsrE family sulfur reductase (43).

Additionally, the homologs of the hypothetical protein encoded in TM0980 ORF in T. petrophila and T. sp. RQ2 are annotated as “Uncharacterized protein involved in the oxidation of intracellular sulfur-like protein.” The up-regulation of these ORFs on sulfur further suggests that they may code for a sulfur reducing protein. This operon is wholly conserved in T. neapolitana and T. petrophila, but only partially conserved in T. sp. RQ2.

In T. sp. RQ2, TM0892 is absent, which suggests that the sulfur response of T. sp. RQ2 might be different from the other sequenced Thermotoga.

T. maritima also clearly indicated a sugar preferences with cellobiose preferred to maltose. There are two major reasons T. maritima may prefer cellobiose to maltose. The first has to do with cellular energetics. T. maritima contains a cellobiose phosphorylase

(TM1848) which simultaneously cleaves and phosphorylates cellobiose (45). Unlike P. furiosus, which contains an α-glucan metabolism pathway based on a maltodextran phosphorylase which both cleaves and phosphorylates α-glucans (33), T. maritima does not have a similar enzyme for maltose metabolism. Thus, the breakdown of maltose produces two glucose molecules, while the cleavage of cellobiose produces one glucose

216 molecule and one glucose phosphate. Some of the energy used by breaking the cellobiose glucan dimer bond is stored in the glucose phosphate and it can directly enter the

Embden-Myerhof pathway. In contrast, maltose is metabolized to two glucose molecules, both of which require phosphorylation before entering metabolic pathways.

Consequently, cellobiose metabolism requires less energy input than maltose metabolism.

A second explanation for the preference for cellobiose to maltose comes from the nature of T. maritima biofilms. T. maritima biofilms consist primarily of β-linked glucans (27,

44, 47), and as, T. maritima produces, destroys, or recycles biofilm, it handles β-glucans like cellobiose. As a result, the metabolism of cellobiose and similar molecules are more closely integrated into the lifestyle of T. maritima. A pathway for β-linked biofilm synthesis in T. maritima has been proposed (27) and biofilm production has been noted in both pure culture (44) and co-culture with Methanococcus jannaschii (26, 27). These

ORFs were also up-regulated in T. maritima during glucose culture compared to culture on the polysaccharide mix (see Chapter 3). It should also be noted that T. maritima is the only Thermotoga thus far known to form biofilms. Comparisons of the T. maritima genome to the genomes of the other Thermotoga indicate that three of the ORFs

(TM0624, TM0627-8) are absent in T. sp. RQ2 and T. petrophila, and all four of the

ORFs in the proposed pathway (TM0624, TM0627-8, TM0767) are absent in T. neapolitana. These same genomic comparisons also indicate that TM1848 cellobiose phosphorylase ORF is conserved across all four organisms. These genomic differences suggest that β-glucan metabolism might be less closely integrated in the overall

217 metabolism of these other Thermotoga, but that the energetics of cellobiose metabolism would be similar to T. maritima. A similar glucan dimer preference experiment performed with T. neapolitana, which completely lacks the β-linked biofilm synthesis pathway, could help determine whether energetics or biofilm formation is the key factor in glucan dimer linkage preference.

Influence of sugars on hydrogen production by Thermotoga species. The

Thermotoga species all produced H2 at statistically similar rates on a cell density basis as

-1 -1 -1 measured by mmol H2 L hr cell and had similar transcriptional profiles in pure batch culture sparged with N2 (see Chapter 3). Previous work with T. maritima found a pentose- hexose effect such that H2 production per sugar glucose metabolized was twice the H2 production during growth on an equal mixture of glucose and xylose. However, no detailed examination of this issue on complex sugar mixtures was considered (9). The results here suggest that polysaccharide-grown cultures produce H2 at rates similar to those determined for growth on glucose. Also, the comparable hydrogen production rates for pure and mixed cultures suggest that there was no impact from any interactions or competition among the Thermotoga species. The similarity of the H2 production levels among the four species and the conservation of the known, characterized portions of the

H2 production pathway (10, 51) also suggests that any as yet unidentified or uncharacterized portions of the H2 production pathway are also likely to be conserved across the Thermotoga genus.

218

Volumetric H2 production was slightly higher than those seen previously in log phase Thermotoga batch culture. A log phase culture of T. neapolitana was reported to

4 -1 -1 have a volumetric production rate of 5.97x 10 mol H2 L h for growth on glucose, but different media and culture conditions were used here, as such the results may not be directly comparable (59).

Previous work with sparged Thermotoga cultures examined only either intermittent sparging (41) or sparge rates of 10 ml/min or less (27, 53). Comparing the results of the 10 ml/min sparged culture to the 50 ml/min sparged culture shows that the increased sparging reduced both the H2 content of the culture headspace and the doubling time of T. maritima. This is consistent with previous work that suggested H2 inhibition of

Thermotoga cultures works through bottlenecking of metabolism (22, 50). The final cell density remaining unchanged is also consistent with previous work which showed no difference in final cell density for sparged and quiescent cultures of T. maritima (27). The only condition which H2 removal resulted in significant increases in cell density was co- culture with Methanococcus jannaschii. Co-culture increased the cell density of T. maritima approximately ten-fold to densities in excess of 109 cells per ml (27). The key difference between removal of hydrogen using co-culture with M. jannaschii and sparging the culture vessel is that the co-culture forms a biofilm containing both organisms allowing M. jannaschii to grow directly adjacent to T. maritima and remove

H2 from the immediate area around the cells. For sparging, the H2 must be transported away from the cells before being removed in the headspace by the sparge gas. This

219 difference in location of H2 removal is a possible cause for the difference in final cell density observed under the two conditions.

Both the 10 ml/min sparged culture and the 50 ml/min sparged culture achieved their peak H2 headspace content and H2 production rates at the transition from log to stationary phase, but the production rates and headspace content differed between the two cultures. The hydrogen production rates of the 50 ml/min sparged batch culture had a higher peak production rate, but H2 production in that culture decreased in stationary phase more rapidly than in the 10 ml/min culture. This faster decrease resulted in the 50 ml/min sparged culture producing less H2 over a full growth cycle than the 10 ml/min sparged culture. This result indicates that conditions that maximize H2 production rate for

Thermotoga are not the same as those that maximize overall H2 production and that conditions must be chosen to optimize one of the desired properties: production rate or total production. The reasons for the difference in optimal conditions are unknown, but could be due to depletion of essential nutrients or accumulation of waste products that ultimately reduce the ability of the stationary phase culture to produce H2. Further experiments, possibly involving additional microarrays and analysis of media samples, would be required to determine the exact physiological and biochemical natures of this phenomenon.

The full culture growth cycle H2 production values are consistent with previously published data from T. neapolitana in unsparged batch culture with pretreated cellulose

-1 as substrate. The T. neapolitana cellulose culture produced 44.2 mmol H2 L over its

220

-1 lifetime (40), almost identical to the 45.1 mmol H2 L produced by the 50 ml/min sparged culture of T. maritima. The results presented here, along with results from other studies of various Thermotoga grown on simple and complex sugar substrates (13, 40) are further confirmation that the metabolic pathways and responses are conserved across the Thermotoga genus. The ORFs involved in H2 production, and sugar metabolism are highly conserved and form part of the core genome of the Thermotoga genus (see

Chapter 2), and while each species contains unique sugar ABC transporters, the vast majority of the sugar transport ORFs are also part of the core genome. Consequently, the growth properties, transcriptional response (see Chapter 3), and production of metabolic products such as hydrogen are all similar for the Thermotoga species studied here. The non-core sugar ABC transporters and glycoside hydrolases likely help each individual

Thermotoga species adapt to a niche in their specific natural habitats, but they do not appear affect growth and response in the general laboratory cultures examined here.

These ORFs would likely cause significantly different behavior only under specific conditions not yet examined, where those non-core ORFs are required for adaptation or survival.

CONCLUSIONS.

There are several conclusions that can be drawn from this study. First, T. maritima exhibits a preference for β-linked saccharides, which is likely due to a combination of energetic and biofilm metabolism effects. No hysteresis effect for α-

221 linked sugars was detected. Second, unlike the strong, genome-wide response of P. furiosus, T. maritima has only a few genes respond to the presence of sulfur. Third, H2 production on a per cell basis is similar for the four Thermotoga species studied for culture on either glucose or a polysaccharide mix. Adding additional competing

Thermotoga species does not alter the production levels, at least through mid-log phase.

Examining longer term studies may provide additional useful information about hydrogen production in stationary phase or continuous competitive culture. Finally, full growth cycle studies of T. maritima show that increasing sparge rate increases cell growth and H2 production rate, as but decreases the total amount of H2 produced. Consequently, for

Thermotoga, the sparge rate must be chosen to maximize either H2 production rate or total production.

222

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229

Figures

Figure 4.1. Experimental loop design for cellobiose-maltose batch experiment. Cy3 is at the base of the arrow and Cy5 at the tip of the arrow.

Figure 4.2. Experimental loop design for cellobiose-maltose chemostat experiment. Cy3 is at the base of the arrow and Cy5 at the tip of the arrow.

230

H2 production for Thermotoga cultures (volumetric basis)

1.8E-03

1.6E-03

1.4E-03

1.2E-03

1.0E-03 glucose /liter*hour

2 8.0E-04 polysaccharides

6.0E-04 mol H 4.0E-04

2.0E-04

0.0E+00 Tma Nea Pet RQ2 Zoo

Figure 4.3. H2 production on a volumetric basis for both glucose and polysaccharide batch culture.

Figure 4.4. Volumetric H2 production after accounting for differences in cell densities at time of measurement and RNA sampling.

231

1.00E+09

1.00E+08

10 ml/min

1.00E+07 50 ml/min cell density cell

1.00E+06 0 2 4 6 8 10 12 time (hr)

Figure 4.5. Full T. maritima growth curves for glucose culture with 10 and 50 ml/min sparge rates.

Volumetric hydrogen production (mmol/(l*hr))

8 7 6 5 10 ml/min 4 50 ml/min 3 2

Hydrogen production rate production Hydrogen 1 0 0 5 10 15 20 25 time (hr)

Figure 4.6 H2 production rates for the full growth cycle of 10 and 50 ml/min sparged batch cultures of T. maritima on glucose.

232

Tables

Table 4.1. ORFs differentially transcribed in T. maritima during culture with and without sulfur.

Cellobiose Maltose GENE fold fold ID Annotation change change ORFs up-regulated without sulfur TM1641 dihydrofolate reductase 2.4 2.5 TM1292 iron-sulfur cluster-binding protein, puta 2.2 2.2 TM1681 hypothetical protein 2.0 2.0 ORFs up-regulated with sulfur TM0689 phosphoglycerate kinase/triose-phosphate 2.0 2.1 TM0266 DNA-binding protein, HU 2.0 2.0 TM0477 outer membrane protein alpha 2.1 2.1 TM0823 transcriptional regulator, TetR family 2.1 2.1 TM1485 ribosomal protein L6 2.1 2.2 TM0982 conserved hypothetical protein 2.3 2.3 TM0980 Uncharacterized protein involved in the oxidation 2.4 2.4 TM0981 Uncharacterized ACR involved in intracellular sulfur 2.5 2.5

233

Table 4.2. ORFs differentially transcribed between cellobiose and maltose during chemostat growth. GENE Cellobiose 1 - Cellobiose 1 - Cellobiose 2 - Cellobiose 2 ID Annotation Maltose 1 Maltose 2 Maltose 1 - Maltose 2 ORFS up-regulated on cellobiose 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2- TM0066 oxoglutarate aldolase 2.6 2.3 2.2 NC TM0067 2-keto-3-deoxygluconate kinase 2.4 2.4 2.6 2.7 TM0068 D-mannonate oxidoreductase, putative 2.8 3.0 2.5 2.7 TM0379 NADH oxidase 4.2 6.9 4.4 7.1 TM1218 transcriptional regulator, LacI family 6.2 5.5 7.3 6.5 TM1219 oligopeptide ABC transporter, ATP-binding protein 7.3 6.7 6.4 5.9 TM1220 oligopeptide ABC transporter, ATP-binding protein 6.2 5.5 5.9 5.3 TM1221 oligopeptide ABC transporter, permease protein 5.3 6.0 5.6 6.3 TM1222 oligopeptide ABC transporter, permease protein 3.3 3.5 3.0 3.2 TM1223 oligopeptide ABC transporter, periplasmic binding protein 29.3 38.4 26.4 34.7 TM1848 cellobiose-phosphorylase 7.2 9.4 10.2 13.4

ORFs up-regulated on maltose TM1168 α-glucan phosphorylase -2.6 NC -3.0 -2.1 TM1834 α-glucosidase -2.0 NC -2.1 NC TM1839 maltose ABC transporter, periplasmic maltose-binding protein -3.0 -2.1 -5.0 -3.5 NC = no significant change Cellobiose 1: cellobiose culture before cellobiose and maltose dual substrate culture. Cellobiose 2: cellobiose culture after cellobiose and maltose dual substrate culture. Maltose 1: maltose culture before cellobiose and maltose dual substrate culture. Maltose 2: maltose culture after cellobiose and maltose dual substrate culture

234

Table 4.3. ORFs differentially transcribed upon transition from cellobiose culture to cellobiose and maltose culture and then back to cellobiose.

Transcription changes upon switch from cellobiose to mixed substrates GENE ID Annotation fold change TM0044 hypothetical protein 2.1 TM0373 dnaK protein -2.4 TM0374 heat shock protein, class I -2.1 TM0394 hypothetical protein -2.2 TM0395 NADH oxidase, putative -2.2 TM0758 flagellin 5.9 TM0850 grpE protein, putative -2.4 TM0979 hypothetical protein 2.1 TM1168 maltodextran phosphorylase -2.7 TM1400 aspartate aminotransferase, putative -2.2

Transcription changes upon switch from mixed substrates to cellobiose GENE ID Annotation fold change TM0373 dnaK protein -3.4 TM0505 groES protein -2.6 TM0506 groEL protein -2.8 TM0560 hypothetical protein 2.0 TM0606 hypothetical protein 2.3 TM0767 maltodextrin glycosyltransferase -2.4 TM0787 putative thiazole biosynthetic enzyme -2.1 TM0850 grpE protein, putative -2.1 TM0872 hypothetical protein 2.0 TM1168 α-glucan phosphorylase -3.1 5-methyltetrahydropteroyltriglutamate-- TM1286 homocysteine methyltransferase -2.3 TM1850 hypothetical protein -2.0

235

Table 4.4. ORFs differentially transcribed upon transition from maltose culture to maltose and cellobiose culture and then back to maltose culture. Changes upon switch from maltose to mixed substrates fold GENEID Annotation change TM0308 α-xylosidase YicI 3.0 TM0312 hypothetical protein 2.8 TM0313 K+ channel, β-subunit 2.4 TM0560 hypothetical protein -2.6 TM0767 maltodextrin glycosyltransferase -2.6 TM0943 glutamine synthetase 2.0 TM1168 α-glucan phosphorylase -2.3 TM1218 transcriptional regulator, LacI family 9.0 TM1219 oligopeptide ABC transporter, ATP-binding protein 8.0 TM1220 oligopeptide ABC transporter, ATP-binding protein 7.4 TM1221 oligopeptide ABC transporter, permease protein 6.8 TM1222 oligopeptide ABC transporter, permease protein 4.7 TM1223 oligopeptide ABC transporter, periplasmic binding 42.9 TM1245 phosphoribosylformylglycinamidine synthase I 2.1 TM1246 phosphoribosylformylglycinamidine synthase II 2.0 TM1249 phosphoribosylaminoimidazolecarboxamide formyltransferase 2.2 TM1286 methyltransferase 2.1 TM1439 hypothetical protein -2.3 TM1524 endoglucanase 7.5 TM1525 endoglucanase 14.6 TM1742 nagD protein, putative 3.0 TM1837 maltose ABC transporter, permease protein -2.1 TM1839 maltose ABC transporter, periplasmic maltose-binding protein -2.6 TM1841 hypothetical protein -2.7 TM1842 hypothetical protein -2.0 TM1848 cellobiose-phosphorylase 10.1

236

Table 4.4 continued Changes upon switch from mixed substrates to maltose fold GENEID Annotation change TM0064 glucuronate isomerase -2.1 TM0070 endo-1,4-β-xylanase B -2.2 TM0211 aminomethyltransferase -2.3 TM0269 hypothetical protein -2.1 TM0302 oligopeptide ABC transporter, permease protein -2.4 TM0308 α-xylosidase YicI -3.5 TM0312 hypothetical protein -3.0 TM0313 K+ channel, β-subunit -3.3 TM0324 hypothetical protein -2.1 TM0326 transcriptional regulator, RpiR family -2.1 TM0758 flagellin -2.1 TM1218 transcriptional regulator, LacI family -8.0 TM1219 oligopeptide ABC transporter, ATP-binding protein -7.4 TM1220 oligopeptide ABC transporter, ATP-binding protein -6.6 TM1221 oligopeptide ABC transporter, permease protein -7.7 TM1222 oligopeptide ABC transporter, permease protein -5.0 TM1223 oligopeptide ABC transporter, periplasmic binding -56.3 TM1437 dimethyladenosine transferase 2.2 TM1439 hypothetical protein 2.6 TM1524 endoglucanase -8.3 TM1525 endoglucanase -21.1 TM1748 binding- dependent transport inner membrane component -2.0 TM1792 CRISPR-associated RAMP protein, Cmr4 family 2.0 TM1848 cellobiose-phosphorylase -13.2 TM1856 transcriptional regulator, LacI family -2.1

237

Table 4.5. Growth and H2 production properties of 10 ml/min and 50 ml/min sparged batch cultures of T. maritima.

doubling time final cell density peak H2 production rate Peak headspace total H2 produced 1 -1 -1 -1 (min) (cells ml- ) (mmol L hr ) H2 ( mol %) (mmol L ) sparge rate 10 ml/min 61 3.09 x 108 6.0 5.3 53.4 50 ml/min 47 3.06 x 108 7.0 1.6 45.1

238

APPENDICES

239

APPENDIX A: ORFs of the examined Thermotoga that are unique by 70% nucleotide identity.

240

Table A1. T. maritima unique ORFs by 70% nt identity ID Annotation TM0001 hypothetical protein TM0002 hypothetical protein TM0003 hypothetical protein TM0004 hypothetical protein TM0145 secreted metalloendopeptidase Gcp, putative TM0147 hypothetical protein TM0149 fatty acid/phospholipid synthesis protein TM0154 hypothetical protein TM0377 hypothetical protein TM0390 hypothetical protein TM0391 hypothetical protein TM0411 transcriptional regulator, XylR-related TM0479 hypothetical protein TM0504 hypothetical protein TM0507 hypothetical protein TM0589 hypothetical protein TM0618 hypothetical protein TM0620 lipopolysaccharide biosynthesis protein TM0622 lipopolysaccharide biosynthesis protein, putative TM0623 hypothetical protein TM0624 N-acetylglucosaminyl-phosphatidylinositol biosynthesis-related protein TM0625 hypothetical protein TM0626 hypothetical protein TM0627 lipopolysaccharide biosynthesis protein TM0628 hypothetical protein TM0629 hypothetical protein TM0630 nucleotide sugar epimerase, putative TM0631 lipopolysaccharide biosynthesis protein TM0995 hypothetical protein TM0999 hypothetical protein TM1024 hypothetical protein TM1025 hypothetical protein TM1067 oligopeptide ABC transporter, periplasmic oligopeptide-binding protein TM1143 methyl-accepting chemotaxis protein TM1144 hypothetical protein TM1145 hypothetical protein

241

Table A1 continued TM1146 methyl-accepting chemotaxis protein TM1147 hypothetical protein TM1173 hypothetical protein TM1189 hypothetical protein TM1236 hypothetical protein TM1242 hypothetical protein TM1271 type IV pilin-related protein TM1299 hypothetical protein TM1338 hypothetical protein TM1411 helicase-related protein TM1412 hypothetical protein TM1429 glycerol uptake facilitator protein TM1671 hypothetical protein TM1779 hypothetical protein TM1790 hypothetical protein TM1795 hypothetical protein TM1829 hypothetical protein TM1838 hypothetical protein

242

Table A2. T. neapolitana unique ORFs by 70% nt identity

ID Annotation CTN_0026 hypothetical protein CTN_0027 hypothetical protein CTN_0028 glycosyl transferase, group 1 family protein CTN_0029 hypothetical protein CTN_0030 glycosyl transferase, group 2 family protein CTN_0034 conserved hypothetical protein CTN_0039 lipopolysaccharide biosynthesis protein CTN_0039a hypothetical protein CTN_0045 hypothetical protein CTN_0045a hypothetical protein CTN_0046 hypothetical protein CTN_0046a hypothetical protein CTN_0047 putative ATP/GTP-binding protein CTN_0048 hypothetical protein CTN_0049 hypothetical protein CTN_0049a lipopolysaccharide biosynthesis protein CTN_0050 HD domain protein CTN_0073 hypothetical protein CTN_0087 pyrroline-5-carboxylate reductase CTN_0094a hypothetical protein CTN_0103 putative membrane protein CTN_0109 hypothetical protein CTN_0130 transposase, IS605 OrfB family CTN_0133 putative ATP/GTP-binding protein CTN_0134 conserved hypothetical protein CTN_0147 hypothetical protein CTN_0148 putative lipoprotein CTN_0154 putative membrane protein CTN_0155 clostripain family protein CTN_0172 conserved hypothetical protein CTN_0182 conserved hypothetical protein CTN_0201 glutamine amidotransferase subunit PdxT CTN_0204 conserved hypothetical protein CTN_0208 conserved hypothetical protein CTN_0235 trap dicarboxylate transporter, dctm subunit CTN_0236 TRAP transporter, DctQ-like membrane protein

243

Table A2 continued CTN_0237 C4-dicarboxylate-binding protein CTN_0238 ATP-binding protein of sugar ABC transporter CTN_0239 sugar ABC transporter permease CTN_0240 ABC sugar transporter, periplasmic sugar binding protein CTN_0241 spermidine/putrescine import ATP-binding protein PotA CTN_0242 ribonuclease Z CTN_0243 ABC transporter permease protein CTN_0244 ABC transporter, permease protein CTN_0245 ABC transporter periplasmic binding protein CTN_0248 putative transporter protein CTN_0276 transcriptional regulator, XylR-related CTN_0309 ABC-2 type transporter family CTN_0321 conserved protein CTN_0329a hypothetical protein CTN_0355 α-xylosidase CTN_0356 binding-protein-dependent transport systems inner membrane component CTN_0357 binding-protein-dependent transport systems inner membrane component CTN_0358 sugar ABC transporter substrate-binding protein, putative CTN_0359 conserved hypothetical protein CTN_0360 hypothetical protein CTN_0361 ribose transport ATP-binding protein RbsA CTN_0362 xylose transport system permease protein XylH CTN_0363 ranched-chain amino acid transport system - permease component CTN_0364 putative periplasmic binding protein CTN_0365 oxidoreductase, short chain dehydrogenase/reductase family CTN_0366 ribose transport ATP-binding protein RbsA CTN_0367 ribose transport system permease protein RbsC CTN_0368 hypothetical oxidoreductase YisS CTN_0369 oxidoreductase, zinc-binding dehydrogenase family CTN_0370 sugar kinase, fggy family CTN_0371 transcriptional regulator, GntR family CTN_0372 succinate-semialdehyde dehydrogenase CTN_0373 glucose-6-phosphate isomerase CTN_0383 putative aldolase CTN_0415 conserved hypothetical protein

244

Table A2 continued CTN_0430 conserved hypothetical protein CTN_0444 hypothetical protein CTN_0452 UDP-N-acetylmuramoylalanine--D-glutamate ligase CTN_0466 flagellar export/assembly protein CTN_0475 conserved hypothetical protein CTN_0481 taurine import ATP-binding protein TauB CTN_0495 ABC-type iron(III)-siderophore transport system, ATPase component CTN_0496 ABC-type iron(III)-siderophore transport system, permease component CTN_0499 sensor histidine kinase CTN_0500 sensor histidine kinase CTN_0509 primosomal protein N' CTN_0522 putative membrane protein CTN_0524 conserved hypothetical protein CTN_0525 conserved hypothetical protein CTN_0526 conserved hypothetical protein CTN_0545 putative membrane protein CTN_0558 flagellin, putative CTN_0566 putative periplasmic metal-binding protein CTN_0567 ferric uptake regulation protein CTN_0586a conserved hypothetical protein CTN_0590 putative membrane protein CTN_0592 conserved hypothetical protein CTN_0599 conserved hypothetical protein CTN_0601 conserved hypothetical protein CTN_0602 conserved hypothetical protein CTN_0603 conserved hypothetical protein CTN_0608 conserved hypothetical protein CTN_0617 endo-1,3-β-xylanase CTN_0633 transposase, IS605 OrfB family CTN_0640 putative lipoprotein CTN_0649 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase CTN_0655 HD domain protein CTN_0660 ABC transporter CTN_0661 glucose transport system permease protein CTN_0662 ABC transporter CTN_0667a oligopeptide ABC transporter, ATP-binding protein

245

Table A2 continued CTN_0673 hypothetical protein CTN_0674 iron-sulfur flavoprotein CTN_0679 oxidoreductase, short chain dehydrogenase/reductase family CTN_0690 phosphatidate cytidylyltransferase, fusion protein CTN_0696 hypothetical protein CTN_0697 conserved domain protein CTN_0699 conserved hypothetical protein CTN_0701 CRISPR-associated protein family, putative CTN_0703 conserved hypothetical protein CTN_0704 putative membrane protein CTN_0708 CRISPR-associated CXXC_CXXC protein Cst1 CTN_0710 CRISPR-associated protein Cas5, tneap subtype CTN_0711 CRISPR-associated helicase Cas3 CTN_0718 sensory box/ggdef family protein CTN_0745 L-aspartate dehydrogenase CTN_0799 conserved hypothetical protein CTN_0822 probable DNA double-strand break repair Rad50 ATPase CTN_0823 exonuclease, putative CTN_0833 UvrB/UvrC motif domain protein CTN_0836 putative lipoprotein CTN_0866 diguanylate cyclase (ggdef) domain protein CTN_0886 HD domain protein CTN_0904 PASTA domain protein CTN_0909 conserved hypothetical protein CTN_0914 outer dense fiber ODF3, putative CTN_0916 putative A-ATPase I-subunit CTN_0919 putative A-ATPase E-subunit CTN_0924 putative ATP/GTP-binding protein CTN_0937 conserved hypothetical protein CTN_0943 conserved hypothetical protein CTN_0945 phosphate acetyltransferase CTN_0958 exodeoxyribonuclease VII, large subunit CTN_0965 competence protein CTN_0979 putative membrane protein CTN_0987 conserved hypothetical protein CTN_1044 tetratricopeptide repeat domain protein

246

Table A2 continued CTN_1053 translation initiation factor, eIF-2B α subunit-related CTN_1080 putative membrane protein CTN_1081 conserved hypothetical protein CTN_1082a conserved hypothetical protein CTN_1083 conserved hypothetical protein CTN_1084 possible cysteinyl-tRNA synthetase CTN_1086 conserved hypothetical protein CTN_1087a conserved hypothetical protein CTN_1091 metallo-β-lactamase superfamily, putative CTN_1095 conserved hypothetical protein CTN_1102a hypothetical protein CTN_1105 hypothetical protein CTN_1105a hypothetical protein CTN_1124 flagellar protein FlgA, putative CTN_1129 single stranded DNA-specific exonuclease, putative CTN_1133a methicillin resistance protein CTN_1156 PQQ enzyme repeat domain protein CTN_1158 conserved hypothetical protein CTN_1160 conserved hypothetical protein CTN_1167 conserved hypothetical protein CTN_1169a conserved hypothetical protein CTN_1173 conserved hypothetical protein CTN_1175 hypothetical protein CTN_1175a hypothetical protein CTN_1176 integral membrane protein DUF6 CTN_1201 putative membrane protein CTN_1202 ubiquinone/menaquinone biosynthesis methyltransferase-related protein CTN_1233 conserved hypothetical protein CTN_1243 conserved hypothetical protein CTN_1259a hypothetical protein CTN_1281a conserved hypothetical protein CTN_1285 multidrug resistance protein CTN_1293 conserved hypothetical protein CTN_1322 putative membrane protein CTN_1341 α-mannosidase-related protein CTN_1372 putative extracellular solute-binding protein

247

Table A2 continued CTN_1373 ABC transporter, permease protein CTN_1374 permease CTN_1387 ggdef domain protein CTN_1392 PleD-related protein CTN_1412 ggdef domain protein CTN_1436 putative membrane protein CTN_1461 conserved hypothetical protein CTN_1462 NADH dehydrogenase subunit f2 CTN_1540 membrane protein, putative CTN_1541 bacterial extracellular solute-binding protein CTN_1542 trehalose/maltose transport inner membrane protein CTN_1543 ABC transporter CTN_1544 transaldolase CTN_1545 altronate hydrolase CTN_1546 altronate dehydratase CTN_1547 conserved hypothetical protein CTN_1548 aldehyde dehydrogenase CTN_1549 conserved hypothetical protein CTN_1550 glycosidase CTN_1551 transcriptional regulator, putative CTN_1553 von Willebrand factor type A domain protein CTN_1554 methyl-accepting chemotaxis protein CTN_1555 hypothetical protein CTN_1574 ISTma3, transposase CTN_1601a hypothetical protein CTN_1645 conserved hypothetical protein CTN_1648a hypothetical protein CTN_1652 conserved hypothetical protein CTN_1674 conserved hypothetical protein CTN_1676 conserved hypothetical protein CTN_1677 conserved hypothetical protein CTN_1700 conserved hypothetical protein CTN_1701 glycoprotease family CTN_1704 putative membrane protein CTN_1735 conserved hypothetical protein CTN_1738 conserved hypothetical protein

248

Table A2 continued CTN_1742 conserved hypothetical protein CTN_1744 conserved hypothetical protein CTN_1769 β-N-acetylglucosaminidase CbsA CTN_1770 transcriptional regulator XylR CTN_1773 lipophilic protein, putative CTN_1784 putative membrane protein CTN_1784a sugar kinase, PfkB family CTN_1794 hypothetical protein CTN_1795 conserved hypothetical protein CTN_1796 BchE CTN_1813 transposase, IS605 OrfB family CTN_1820 putative carbamoylphosphate synthase large subunit short form CTN_1821 phosphoglycolate phosphatase, putative CTN_1825 TPR/glycosyl transferase/methyltansferase domains protein CTN_1831 uridine kinase CTN_1833 conserved hypothetical protein CTN_1838 conserved hypothetical protein CTN_1839 putative membrane protein CTN_1852 conserved hypothetical protein CTN_1868 hypothetical protein CTN_1871 conserved hypothetical protein CTN_1872 conserved hypothetical protein CTN_1874 S-layer homology domain protein CTN_1877 lipoate protein ligase CTN_1879 efflux ABC transporter, permease protein CTN_1892 conserved hypothetical protein CTN_1914 flagellar hook-length control protein, putative CTN_1915a hypothetical protein CTN_1916 conserved hypothetical protein CTN_1925 conserved hypothetical protein CTN_1930 hypothetical protein CTN_1931 hypothetical protein CTN_1932 hypothetical protein CTN_1933 general secretion pathway protein D, putative CTN_1934 secretin and TonB N terminus short domain protein CTN_1934a hypothetical protein

249

Table A2 continued CTN_1935 hypothetical protein CTN_1937 bacterial transcriptional activator domain family

250

Table A3. T. petrophila unique ORFs by 70% nt identity

ID Annotation Tpet_0257 DUF1078 domain protein Tpet_0286 peptidase C11, clostripain Tpet_0297 glycosyl transferase, group 1 Tpet_0298 hypothetical protein Tpet_0299 glycosyl transferase, group 1 Tpet_0306 polysaccharide biosynthesis protein Tpet_0307 glycosyl transferase, group 1 Tpet_0485 extracellular solute-binding protein, family 5 Tpet_0486 binding-protein-dependent transport systems inner membrane component Tpet_0487 binding-protein-dependent transport systems inner membrane component Tpet_0534 nitroreductase Tpet_0943 hypothetical protein Tpet_0944 hypothetical protein Tpet_1075 regulatory protein, ArsR Tpet_1084 CRISPR-associated helicase Cas3 Tpet_1085 CRISPR-associated protein Cas5 family Tpet_1087 hypothetical protein Tpet_1500 hypothetical protein Tpet_1621 hypothetical protein Tpet_1678 FG-GAP repeat protein Tpet_1679 extracellular solute-binding protein, family 1 Tpet_1680 binding-protein-dependent transport systems inner membrane component Tpet_1681 binding-protein-dependent transport systems inner membrane component Tpet_1682 α-L-rhamnosidase Tpet_1723 hypothetical protein Tpet_1728 hypothetical protein Tpet_1729 hypothetical protein Tpet_1735 hypothetical protein Tpet_1736 hypothetical protein Tpet_1737 hypothetical protein Tpet_1741 Esterase/lipase-like protein Tpet_1750 hypothetical protein Tpet_1751 phosphoadenosine phosphosulfate reductase Tpet_1752 hypothetical protein Tpet_1753 hypothetical protein Tpet_1754 hypothetical protein

251

Table A3 continued Tpet_1765 metal dependent phosphohydrolase Tpet_1766 methyl-accepting chemotaxis sensory transducer Tpet_1767 ATPase Tpet_1768 hypothetical protein Tpet_1769 type III restriction enzyme, res subunit Tpet_1770 hypothetical protein Tpet_1771 hypothetical protein Tpet_1772 Adenine-specific DNA methylase containing a Zn-ribbon-like protein Tpet_1773 hypothetical protein Tpet_1775 PglZ domain Tpet_1776 ATPase (AAA+ superfamily)-like protein Tpet_1778 α-2-macroglobulin domain protein Tpet_1779 diguanylate cyclase Tpet_1790 LacI transcriptional regulator Tpet_1791 RbsD or FucU transport Tpet_1792 ABC transporter related Tpet_1793 Monosaccharide-transporting ATPase Tpet_1794 Monosaccharide-transporting ATPase

252

Table A4. T. sp RQ2 unique ORFs by 70% nt identity

ID Annotation TRQ2_0168 flagellin domain protein TRQ2_0171 conserved hypothetical protein TRQ2_0259 hypothetical protein TRQ2_0296 glycosyl transferase group 1 TRQ2_0297 glycosyl transferase group 1 TRQ2_0298 hypothetical protein TRQ2_0299 rhamnosyltransferase, putative TRQ2_0300 CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase TRQ2_0301 glycerol-3-phosphate cytidylyltransferase TRQ2_0302 glycosyl transferase family 2 TRQ2_0310 polysaccharide biosynthesis protein TRQ2_0311 asparagine synthase TRQ2_0312 excinuclease ABC C subunit domain protein TRQ2_0313 CRISPR-associated RAMP protein, Cmr1 family TRQ2_0314 protein of unknown function DUF324 TRQ2_0315 CRISPR-associated protein, Csx11 family TRQ2_0316 CRISPR-associated RAMP protein, Cmr4 family TRQ2_0317 conserved hypothetical protein TRQ2_0318 protein of unknown function DUF324 TRQ2_0319 PilT protein domain protein TRQ2_0320 conserved hypothetical protein TRQ2_0459 conserved hypothetical protein TRQ2_0500 extracellular solute-binding protein family 5 TRQ2_0501 Pectinesterase TRQ2_0503 conserved hypothetical protein TRQ2_0510 extracellular solute-binding protein family 5 TRQ2_0512 extracellular solute-binding protein family 1 TRQ2_0513 binding-protein-dependent transport systems inner membrane component TRQ2_0637 phosphoenolpyruvate-protein phosphotransferase TRQ2_0638 Phosphotransferase system, phosphocarrier protein HPr TRQ2_0639 PTS system, fructose subfamily, IIC subunit TRQ2_0640 putative PTS IIA-like nitrogen-regulatory protein PtsN TRQ2_0641 1-phosphofructokinase TRQ2_0642 transcriptional regulator, DeoR family TRQ2_1025 CRISPR-associated RAMP protein, Cmr1 family TRQ2_1029 CRISPR-associated protein, Cmr5 family

253

Table A4 continued TRQ2_1100 V-type ATPase, D subunit TRQ2_1103 H+transporting two-sector ATPase E subunit TRQ2_1106 V-type ATPase 116 kDa subunit TRQ2_1108 hypothetical protein TRQ2_1281 conserved hypothetical protein TRQ2_1311 CDP-alcohol phosphatidyltransferase TRQ2_1418 conserved hypothetical protein TRQ2_1684 FG-GAP repeat protein TRQ2_1685 conserved hypothetical protein TRQ2_1686 conserved hypothetical protein TRQ2_1687 conserved hypothetical protein TRQ2_1781 conserved hypothetical protein TRQ2_1835 hypothetical protein TRQ2_1839 conserved hypothetical protein

254

APPENDIX B: ORFs differentially transcribed in microarray experiments.

255

Table B1. Fold changes of core ORFs in dye flip experiments NCBI Tma Tma Nea Nea RQ2 RQ2 Pet Pet Zoo Zoo ID Annotation Annotation (P-G) (P) (P-G) (P) (P-G) (P) (P-G) (P) (P-G) (P) TM_tRNA- TM_tRNA- Thr-3 Thr-3 TM_tRNA-Thr-3 -2.40 11.18 NC NC NC NC NC NC NC NC TM0009 TM0009 hypothetical protein NC NC -1.28 8.73 NC NC 3.29 12.65 NC NC TM0010 TM0010 NADP-reducing hydrogenase, subunit C 2.02 8.53 NC NC NC NC NC NC NC NC TM0017 TM0017 ferredoxin oxidoreductase, α subunit NC NC -2.18 18.47 NC NC NC NC NC NC TM0018 TM0018 ferredoxin oxidoreductase, β subunit NC NC -1.64 13.89 NC NC NC NC NC NC TM0023 TM0023 methyl-accepting chemotaxis protein NC NC NC NC -2.45 5.85 4.96 11.54 NC NC TM0025 TM0025 β-glucosidase NC NC NC NC NC NC 3.49 15.91 -2.05 10.83 TM0027 TM0027 ABC transporter, ATP-binding protein NC NC NC NC -2.85 12.02 7.20 14.42 -2.25 14.07 TM0029 TM0029 ABC transporter, permease protein 2.93 12.93 NC NC -2.75 10.27 4.37 8.14 -3.01 9.57 TM0030 TM0030 ABC transporter, permease protein NC NC NC NC NC NC 2.21 5.42 NC NC TM0031 TM0031 ABC transporter, periplasmic binding NC NC NC NC -2.76 11.69 NC NC -3.02 6.82 TM0032 TM0032 transcriptional regulator, XylR-related NC NC 1.03 7.68 NC NC NC NC NC NC TM0040 TM0040 dihydropteroate synthase NC NC -1.14 11.75 NC NC NC NC NC NC TM0051 TM0051 iron(II) transport protein B -5.08 14.02 NC NC NC NC NC NC NC NC TM0056 TM0056 ABC transporter, periplasmic binding 6.95 18.32 1.17 12.39 7.68 13.85 NC NC 6.60 21.05 TM0057 TM0057 ABC transporter, ATP-binding protein 4.18 13.33 1.49 8.22 5.99 10.79 3.74 11.48 5.65 9.35 TM0058 TM0058 ABC transporter, ATP-binding protein 2.38 8.62 1.11 11.07 NC NC 4.66 12.78 NC NC TM0060 TM0060 oligopeptide ABC transporter, permease NC NC 1.05 8.06 NC NC NC NC NC NC TM0061 TM0061 endo-1,4-β-xylanase A 12.09 14.63 2.56 12.72 14.32 12.63 NC NC 13.03 18.88 TM0062 TM0062 hypothetical protein NC NC NC NC NC NC 9.23 18.44 NC NC TM0063 TM0063 hypothetical protein NC NC NC NC NC NC NC NC 2.68 10.27 TM0065 TM0065 transcriptional regulator, IclR family 2.11 9.49 NC NC 2.28 10.08 NC NC NC NC

256

Table B1 continued TM0066 TM0066 4-hydroxy-2-oxoglutarate aldolase NC NC NC NC NC NC -2.18 8.80 NC NC TM0068 TM0068 D-mannonate oxidoreductase, putative NC NC NC NC 2.14 12.20 NC NC NC NC TM0070 TM0070 endo-1,4-β-xylanase B 4.13 17.66 1.19 11.35 3.22 14.18 NC NC 2.08 8.45 TM0071 TM0071 ABC transporter, periplasmic binding 10.67 16.73 1.12 8.01 13.65 13.48 3.59 12.49 10.00 18.53 TM0072 TM0072 ABC transporter, permease protein NC NC NC NC NC NC NC 2.08 8.44 TM0073 TM0073 ABC transporter, permease protein 2.26 3.04 NC NC 2.47 1.65 NC NC NC NC TM0074 TM0074 ABC transporter, ATP-binding protein NC NC 1.15 10.06 NC NC NC NC NC NC TM0075 TM0075 ABC transporter, ATP-binding protein NC NC NC NC NC NC 2.72 8.41 NC NC TM0077 TM0077 acetyl xylan esterase NC NC 1.25 12.36 2.44 10.17 NC NC 2.86 10.68 TM0095 TM0095 hypothetical protein -2.49 10.49 NC NC NC NC NC NC NC NC TM0102 TM0102 basic membrane protein NC NC -1.20 8.15 -2.19 12.57 3.66 15.33 NC NC TM0105 TM0105 sugar ABC transporter, permease protein -2.21 9.54 NC NC NC NC NC NC NC NC TM0107 TM0107 hypothetical protein -4.21 7.83 NC NC NC NC 2.44 18.13 2.31 12.19 TM0114 TM0114 ABC transporter, periplasmic binding NC NC NC NC NC NC 4.01 15.04 NC NC TM0118 TM0118 ribonucleotide reductase, B12-dependent NC NC -1.00 9.74 NC NC NC NC NC NC TM0119 TM0119 acetamidase, putative -2.05 8.10 NC NC NC NC NC NC NC NC TM0120 TM0120 oxidoreductase, putative 2.11 9.13 NC NC NC NC NC NC NC NC TM0136 TM0136 hypothetical protein -2.05 6.45 NC NC NC NC NC NC NC NC TM0153 TM0153 hypothetical protein -2.31 14.14 NC NC NC NC NC NC NC NC TM0161 TM0161 geranyltranstransferase NC NC -1.73 10.47 NC NC NC NC NC NC TM0177 TM0177 hypothetical protein NC NC NC NC NC NC NC NC -2.22 11.73 TM0180 TM0180 hypothetical protein -2.97 8.94 NC NC NC NC NC NC NC NC TM0185 TM0185 hypothetical protein 6.58 9.51 NC NC 6.19 13.47 -2.17 15.93 6.40 8.50 TM0192 TM0192 spoVS-related protein -2.38 10.55 NC NC NC NC NC NC NC NC TM0196 TM0196 hypothetical protein -2.39 7.16 NC NC NC NC NC NC NC NC TM0201 TM0201 NADP-reducing hydrogenase, subunit D NC NC NC NC NC NC 2.23 7.68 NC NC TM0202 TM0202 hypothetical protein NC NC -1.89 12.93 -6.56 12.90 5.77 21.26 -7.73 16.95

257

Table B1 continued TM0212 TM0212 glycine cleavage system H protein -5.07 17.82 NC NC NC NC NC NC 3.58 15.87 TM0216 TM0216 glycyl-tRNA synthetase subunit α 2.34 7.63 -2.13 14.91 -3.76 13.64 NC NC NC NC TM0236 TM0236 ligase NC NC -1.05 7.72 NC NC NC NC NC NC TM0239 TM0239 glucose-1-phosphate adenylyltransferase NC NC NC NC -2.11 6.25 NC NC -3.01 5.69 TM0240 TM0240 glucose-1-phosphate adenylyltransferase NC NC NC NC NC NC NC NC -2.14 10.24 TM0255 TM0255 ribosomal protein L28 -2.08 6.76 NC NC NC NC -2.24 11.71 NC NC TM0272 TM0272 pyruvate phosphate dikinase NC NC NC NC -2.09 7.06 NC NC NC NC TM0290 TM0290 citrate synthase -2.42 11.32 NC NC NC NC NC NC NC NC TM0295 TM0295 putative translaldolase NC NC NC NC 2.06 8.90 NC NC 2.24 6.15 TM0308 TM0308 α-xylosidase YicI 5.09 15.06 NC NC 2.99 7.73 NC NC 4.76 9.95 TM0309 TM0309 ABC transporter, periplasmic binding 5.80 14.94 1.75 16.02 10.29 17.48 NC NC 10.24 13.45 TM0310 TM0310 β-D-galactosidase NC NC NC NC 3.33 12.32 NC NC 3.16 9.72 TM0312 TM0312 hypothetical protein 2.05 8.98 NC NC NC NC NC NC NC NC TM0322 TM0322 ABC transporter, binding protein NC NC 1.67 12.78 3.70 11.42 NC NC NC NC TM0324 TM0324 hypothetical protein NC NC NC NC 4.65 13.09 NC NC 3.74 10.06 TM0325 TM0325 oxidoreductase, dehydrogenase family 23.85 18.01 1.49 10.54 11.26 17.18 -2.51 11.82 11.27 18.35 TM0326 TM0326 transcriptional regulator, RpiR family 2.21 8.94 NC NC 2.91 8.70 NC NC 2.22 6.15 TM0327 TM0327 phosphoglycerate dehydrogenase 7.54 21.49 NC NC 4.85 13.58 NC NC 4.85 16.56 TM0341 TM0341 hypothetical protein NC NC -1.09 12.98 NC NC NC NC NC NC TM0343 TM0343 3-deoxy-7-phosphoheptulonate synthase -2.01 7.83 -1.01 5.63 NC NC NC NC NC NC TM0358 TM0358 hypothetical protein 2.57 13.06 NC NC NC NC NC NC NC NC TM0359 TM0359 hypothetical protein -3.08 12.97 NC NC NC NC NC NC 2.31 5.99 TM0360 TM0360 mazG-related protein -2.35 6.93 NC NC NC NC NC NC NC NC TM0371 TM0371 arginine repressor -2.58 10.02 NC NC NC NC NC NC NC NC

258

Table B1 continued TM0373 TM0373 dnaK protein -3.07 13.36 NC NC NC NC NC NC 2.05 10.10 TM0374 TM0374 heat shock protein, class I NC NC 1.05 6.60 NC NC NC NC NC NC TM0375 TM0375 hypothetical protein -3.95 13.49 -1.75 16.01 NC NC NC NC NC NC TM0376 TM0376 hypothetical protein -2.62 12.51 NC NC NC NC NC NC NC NC TM0379 TM0379 NADH oxidase -2.10 8.92 NC NC NC NC NC NC 2.13 8.53 TM0385 TM0385 ribonucleoside triphosphate reductase 2.90 8.44 NC NC NC NC NC NC NC NC TM0392 TM0392 glycosyl transferase group 1 -7.44 16.10 1.01 11.46 -2.34 10.67 NC NC 8.79 20.89 TM0393 TM0393 transcriptional regulator, XylR-related -2.11 10.70 NC NC NC NC NC NC NC NC TM0394 TM0394 hypothetical protein -3.28 12.72 NC NC NC NC NC NC NC NC TM0395 TM0395 NADH oxidase, putative -2.46 11.01 NC NC NC NC NC NC NC NC TM0397 TM0397 glutamate synthase, α subunit -2.55 13.21 NC NC NC NC -2.36 6.58 NC NC TM0401 TM0401 thymidine kinase 2.90 12.61 NC NC NC NC NC NC NC NC TM0403 TM0403 nitrogen regulatory protein P-II NC NC NC NC NC NC NC NC 5.08 8.89 TM0405 TM0405 2-oxoglutarate ferredoxin oxidoreductase NC NC NC NC NC NC NC NC -2.17 8.80 TM0429 TM0429 methyl-accepting chemotaxis protein NC NC NC NC NC NC 4.17 16.80 NC NC TM0438 TM0438 6-phosphogluconate dehydrogenase 2.08 10.93 NC NC NC NC NC NC NC NC TM0440 TM0440 hypothetical protein 2.70 11.82 NC NC NC NC NC NC NC NC TM0441 TM0441 oxidoreductase, reductase family 3.24 13.40 NC NC NC NC NC NC NC NC TM0445 TM0445 hypothetical protein NC NC -1.23 9.16 NC NC NC NC NC NC TM0448 TM0448 hypothetical protein NC NC -1.27 7.41 NC NC NC NC NC NC TM0451 TM0451 ribosomal protein L33 -3.40 12.51 NC NC NC NC NC NC NC NC TM0460 TM0460 ABC transporter, periplasmic binding NC NC -1.57 11.32 NC NC NC NC NC NC TM0465 TM0465 hypothetical protein NC NC NC NC NC NC NC NC -2.09 12.05 TM0468 TM0468 response regulator 2.65 7.75 NC NC NC NC NC NC NC NC

259

Table B1 continued TM0491 TM0491 hypothetical protein NC NC -1.76 8.67 NC NC NC NC NC NC TM0506 TM0506 groEL protein -3.25 13.53 NC NC NC NC NC NC NC NC TM0510 TM0510 iron-dependent transcriptional repressor NC NC -1.54 12.19 NC NC NC NC NC NC TM0511 TM0511 hypothetical protein NC NC -1.43 13.34 NC NC NC NC NC NC TM0514 TM0514 prolyl-tRNA synthetase -2.58 9.46 NC NC NC NC NC NC NC NC TM0522 TM0522 ATP-dependent protease ATP-binding NC NC -1.31 12.00 NC NC NC NC -2.05 10.13 TM0531 TM0531 ABC transporter, periplasmic binding NC NC -1.18 14.62 NC NC NC NC NC NC TM0535 TM0535 hypothetical protein -2.55 10.42 -1.20 11.53 NC NC NC NC NC NC TM0552 TM0552 α-isopropylmalate synthase transferase -2.41 12.87 NC NC NC NC NC NC NC NC TM0557 TM0557 carbamoyl-phosphate synthase NC NC -1.17 15.19 NC NC NC NC NC NC TM0560 TM0560 hypothetical protein NC NC -1.63 11.21 NC NC NC NC NC NC TM0565 TM0565 sugar fermentation stimulation protein NC NC 1.02 7.98 NC NC NC NC NC NC TM0566 TM0566 hypothetical protein NC NC NC NC NC NC 3.81 14.40 NC NC TM0567 TM0567 hypothetical protein NC NC -1.43 11.47 NC NC NC NC NC NC TM0571 TM0571 heat shock serine protease, periplasmic NC NC -1.33 14.28 NC NC 3.03 12.48 NC NC TM0593 TM0593 ABC transporter, periplasmic binding protein NC NC -1.27 12.64 NC NC NC NC NC NC TM0596 TM0596 sugar ABC transporter, permease protein NC NC -1.30 9.03 NC NC NC NC NC NC TM0603 TM0603 ribosomal protein S6 NC NC -1.15 13.05 NC NC NC NC NC NC TM0605 TM0605 ribosomal protein S18 -2.59 11.27 NC NC NC NC NC NC NC NC TM0606 TM0606 hypothetical protein -2.27 11.96 NC NC NC NC NC NC NC NC TM0609 TM0609 hypothetical protein NC NC -1.43 12.68 NC NC NC NC NC NC TM0636 TM0636 hypothetical protein NC NC NC NC NC NC NC NC -2.11 9.10 TM0640 TM0640 hypothetical protein NC NC NC NC NC NC NC NC -2.21 8.74

260

Table B1 continued TM0644 TM0644 hypothetical protein -2.41 12.08 NC NC NC NC NC NC NC NC TM0645 TM0645 NH(3)-dependent NAD(+) synthetase -2.40 11.58 NC NC NC NC NC NC NC NC TM0654 TM0654 spermidine synthase -2.79 12.88 NC NC NC NC NC NC NC NC TM0656 TM0656 hypothetical protein NC NC -1.27 7.38 NC NC 2.93 7.24 NC NC TM0665 TM0665 cysteine synthase -3.13 11.88 NC NC 2.56 10.02 NC NC 2.16 10.24 TM0669 TM0669 hypothetical protein 2.21 10.35 NC NC NC NC NC NC NC NC TM0686 TM0686 DNA polymerase III, γ and η subunit NC NC -1.26 9.91 NC NC NC NC NC NC TM0690 TM0690 hypothetical protein -2.52 10.50 NC NC NC NC NC NC NC NC TM0702 TM0702 chemotaxis histidine kinase CheA NC NC -1.13 3.72 NC NC NC NC NC NC TM0705 TM0705 ABC transporter, ATP-binding protein 2.68 10.45 NC NC -2.45 8.75 NC NC NC NC TM0718 TM0718 purine-binding chemotaxis protein NC NC -1.48 15.20 NC NC NC NC NC NC TM0738 TM0738 hypothetical protein NC NC NC NC NC NC 2.47 15.48 NC NC TM0743 TM0743 hypothetical protein -2.24 9.62 NC NC NC NC NC NC NC NC TM0752 TM0752 α-glucosidase, putative NC NC NC NC 2.59 11.44 NC NC 2.10 8.94 TM0762 TM0762 30S ribosomal protein S2 NC NC -1.18 4.91 NC NC NC NC NC NC TM0767 TM0767 maltodextrin glycosyltransferase -7.71 18.00 -1.78 12.42 -6.41 18.32 NC NC -3.79 13.52 TM0778 TM0778 hypothetical protein NC NC NC NC NC NC NC NC -2.86 11.95 TM0788 TM0788 thiamine biosynthesis protein ThiC 3.93 14.90 -3.15 14.97 -3.86 12.57 NC NC NC NC TM0789 TM0789 Xylose isomerase domain TIM barrel 2.55 11.10 NC NC -4.07 16.63 NC NC NC NC TM0790 TM0790 hypothetical protein -3.43 14.51 -1.18 8.87 NC NC 2.67 18.11 2.17 10.49 TM0791 TM0791 7-cyano-7-deazaguanine reductase NC NC NC NC -2.18 14.36 NC NC NC NC TM0793 TM0793 ABC transporter, ATP-binding 2.06 11.37 NC NC -2.14 10.12 NC NC NC NC TM0798 TM0798 malonyl CoA-acyl carrier transacylase NC NC -1.19 13.40 NC NC NC NC NC NC

261

Table B1 continued TM0799 TM0799 bioY protein -3.28 14.56 -1.10 11.01 NC NC 2.42 14.00 NC NC TM0801 TM0801 (3R)-hydroxymyristoyl dehydratase -2.92 10.57 NC NC NC NC NC NC 3.59 11.27 TM0807 TM0807 peroxiredoxin -3.31 11.46 NC NC NC NC NC NC NC NC TM0824 TM0824 astB/chuR-related protein NC NC -1.76 11.35 NC NC NC NC NC NC TM0839 TM0839 rod shape-determining protein RodA -4.42 10.85 -1.20 9.71 NC NC NC NC 4.36 10.29 TM0841 TM0841 S-layer-like array protein NC NC 1.10 6.39 NC NC NC NC NC NC TM0845 TM0845 hemolysin-related protein NC NC -1.03 11.26 NC NC NC NC NC NC TM0846 TM0846 cytidine/deoxycytidine deaminase NC NC -1.21 9.59 NC NC NC NC NC NC TM0849 TM0849 dnaJ protein -2.65 10.93 NC NC NC NC NC NC NC NC TM0851 TM0851 heat shock operon repressor HrcA -4.05 12.47 NC NC NC NC NC NC NC NC TM0856 TM0856 tRNA pseudouridine 55 synthase NC NC -1.10 12.68 NC NC NC NC NC NC TM0863 TM0863 ribosomal protein L9 -2.51 7.93 NC NC NC NC NC NC NC NC TM0865 TM0865 hypothetical protein NC NC -1.12 11.38 NC NC NC NC NC NC TM0873 TM0873 ATP-dependent Clp protease, ATPase -3.02 11.04 NC NC NC NC NC NC NC NC TM0881 TM0881 homoserine O-succinyltransferase NC NC -2.67 12.89 NC NC NC NC NC NC TM0882 TM0882 O-acetylhomoserine sulfhydrylase -2.10 12.91 NC NC NC NC NC NC NC NC TM0896 TM0896 galactose-1-P uridylyltransferase NC NC -1.05 9.77 NC NC NC NC NC NC TM0905 TM0905 hypothetical protein NC NC NC NC -2.50 7.17 NC NC NC NC TM0922 TM0922 hypothetical protein NC NC -1.10 13.86 NC NC NC NC NC NC TM0930 TM0930 hypothetical protein 2.96 14.87 NC NC NC NC NC NC -2.41 7.06 TM0932 TM0932 hypothetical protein -3.41 9.09 NC NC NC NC NC NC 2.24 9.66 TM0938 TM0938 hypothetical protein -2.28 11.53 NC NC NC NC NC NC NC NC TM0940 TM0940 ribosomal pseudouridine synthase C -2.14 12.67 -1.07 14.73 NC NC NC NC NC NC

262

Table B1 continued TM0961 TM0961 lemA protein -3.38 11.74 -1.71 15.67 NC NC NC NC NC NC TM0963 TM0963 oligoendopeptidase, putative NC NC -2.18 17.75 -2.49 11.76 NC NC NC NC TM0964 TM0964 hypothetical protein NC NC -1.96 16.19 NC NC NC NC -2.70 15.51 TM1014 TM1014 hypothetical protein NC NC -1.01 15.04 NC NC NC NC NC NC TM1028 TM1028 ABC transporter, ATP-binding protein NC NC -1.39 10.43 NC NC NC NC -2.10 11.65 TM1029 TM1029 ABC transporter, permease protein, -2.43 10.44 NC NC NC NC NC NC NC NC TM1031 TM1031 glutaredoxin -2.27 8.52 NC NC NC NC NC NC NC NC TM1068 TM1068 α-glucosidase, putative -2.39 11.73 NC NC NC NC NC NC NC NC TM1072 TM1072 rhamnulose-1-phosphate aldolase 2.02 7.25 NC NC NC NC NC NC NC NC TM1073 TM1073 sugar kinase 2.87 11.31 NC NC NC NC NC NC -2.38 10.10 TM1083 TM1083 hypothetical protein -2.24 10.02 NC NC NC NC NC NC NC NC TM1087 TM1087 hypothetical protein NC NC NC NC NC NC NC NC -2.01 6.27 TM1092 TM1092 hypothetical protein NC NC NC NC NC NC 2.16 5.62 -2.08 9.77 TM1102 TM1102 ribonuclease III -3.56 15.68 NC NC NC NC NC NC NC NC TM1107 TM1107 hypothetical protein -2.61 9.28 NC NC NC NC NC NC 2.46 7.98 TM1124 TM1124 hypothetical protein -2.74 12.96 -1.12 12.63 NC NC NC NC NC NC TM1142 TM1142 hypothetical protein NC NC NC NC NC NC NC NC -2.16 6.22 TM1143 TM1143 methyl-accepting chemotaxis protein -2.69 11.82 NC NC NC NC NC NC NC NC TM1155 TM1155 glucose-6-phosphate 1-dehydrogenase NC NC -1.21 10.97 -2.25 10.37 NC NC -2.77 11.27 TM1161 TM1161 Mg2+ transporter MgtE, putative NC NC NC NC NC NC NC NC -2.05 9.10 TM1167 TM1167 hypothetical protein NC NC NC NC NC NC NC NC -2.10 6.32 TM1168 TM1168 α-glucan phosphorylase -11.3 16.79 -1.40 11.07 NC NC NC NC NC NC TM1169 TM1169 3-oxoacyl-acyl carrier protein reductase -2.09 10.24 NC NC NC NC NC NC NC NC

263

Table B1 continued TM1172 TM1172 hydroxylamine reductase NC NC -1.30 11.52 -2.09 11.54 NC NC NC NC TM1182 TM1182 chromosome segregation SMC protein -2.03 1.16 NC NC NC NC NC NC NC NC TM1195 TM1195 β-galactosidase NC NC NC NC NC NC NC NC 2.11 10.29 TM1202 TM1202 maltose ABC transporter, permease 2.02 8.13 NC NC NC NC NC NC NC NC TM1204 TM1204 maltose ABC transporter, periplasmic 2.99 4.79 -1.82 12.34 NC NC 2.10 6.26 NC NC TM1218 TM1218 transcriptional regulator, LacI family NC NC NC NC NC NC -2.32 13.67 NC NC TM1219 TM1219 ABC transporter, ATP-binding protein 3.23 12.62 1.31 13.60 2.45 10.99 -2.71 15.95 3.07 11.91 TM1220 TM1220 ABC transporter, ATP-binding protein NC NC 1.05 8.24 NC NC -2.43 8.31 NC NC TM1221 TM1221 oligopeptide ABC transporter, permease NC NC NC NC NC NC -2.91 20.23 NC NC TM1222 TM1222 oligopeptide ABC transporter, permease 3.20 13.04 NC NC NC NC NC NC NC NC TM1223 TM1223 ABC transporter, periplasmic binding 38.28 19.52 3.76 18.16 15.46 15.33 -5.37 27.45 10.92 23.69 TM1224 TM1224 transcriptional regulator, XylR-related 20.03 14.47 1.76 10.80 8.89 12.92 -7.51 18.18 7.38 14.80 TM1226 TM1226 ABC transporter, periplasmic binding 24.54 20.13 NC NC 16.90 11.22 NC NC NC NC TM1227 TM1227 endo-1,4-β-mannosidase 27.77 17.89 3.63 16.14 15.99 17.02 -4.21 36.47 10.52 14.56 TM1229 TM1229 glycosyl transferase family 2 2.47 9.44 NC NC NC NC NC NC NC NC TM1235 TM1235 extracellular binding protein family 1 3.20 9.31 NC NC NC NC NC NC NC NC TM1248 TM1248 formyltransferase NC NC NC NC -3.31 16.58 NC NC -3.29 11.98 TM1259 TM1259 phosphate transcriptional regulator PhoB -2.01 10.11 NC NC NC NC NC NC NC NC TM1260 TM1260 phosphate transport regulator PhoU -2.61 13.41 NC NC NC NC NC NC 2.73 14.09 TM1263 TM1263 phosphate ABC transporter, permease 2.66 10.20 NC NC NC NC NC NC NC NC TM1267 TM1267 thiamine biosynthesis protein ThiH -4.25 11.94 -2.95 16.14 NC NC NC NC NC NC TM1269 TM1269 biotin synthase NC NC -2.34 12.47 NC NC NC NC NC NC TM1272 TM1272 glutamyl tRNA-Gln amidotransferase A -3.23 12.18 NC NC NC NC NC NC 2.29 11.88

264

Table B1 continued TM1273 TM1273 glutamyl-tRNA amidotransferase B -2.79 10.48 -1.20 10.23 -2.02 9.67 NC NC NC NC TM1274 TM1274 hypothetical protein NC NC -2.11 17.27 NC NC NC NC NC NC TM1275 TM1275 hypothetical protein NC NC -1.14 8.06 NC NC NC NC NC NC TM1281 TM1281 6-phospho-β-glucosidase 2.09 8.63 NC NC NC NC NC NC -2.10 11.76 TM1302 TM1302 ABC transporter, ATP-binding NC NC -1.07 10.25 NC NC NC NC NC NC TM1312 TM1312 HicA Toxin NC NC NC NC NC NC -2.13 6.25 NC NC TM1317 TM1317 Iron-sulfur protein NC NC -1.33 13.95 NC NC NC NC NC NC TM1334 TM1334 Iron-sulfur protein NC NC -1.50 13.26 NC NC NC NC NC NC TM1335 TM1335 Iron-sulfur protein 2.32 8.35 -1.48 11.83 NC NC NC NC NC NC TM1336 TM1336 permease, putative -2.73 7.90 NC NC NC NC NC NC NC NC TM1339 TM1339 hypothetical protein NC NC -1.02 12.23 NC NC NC NC NC NC TM1352 TM1352 hypothetical protein NC NC -1.94 12.21 NC NC NC NC NC NC TM1353 TM1353 hypothetical protein NC NC -1.10 11.02 NC NC NC NC NC NC TM1368 TM1368 ABC transporter, ATP-binding NC NC -1.06 6.25 NC NC NC NC NC NC TM1369 TM1369 hypothetical protein NC NC NC NC -2.27 9.69 NC NC -2.06 8.44 TM1384 TM1384 adenine phosphoribosyltransferase NC NC -1.01 11.35 NC NC NC NC NC NC TM1387 TM1387 dephospho-CoA kinase 2.23 6.93 NC NC NC NC NC NC NC NC TM1400 TM1400 aspartate aminotransferase, putative -2.81 14.99 NC NC NC NC NC NC NC NC TM1401 TM1401 D-3-phosphoglycerate dehydrogenase -2.02 6.69 NC NC NC NC NC NC NC NC TM1427 TM1427 redox transcriptional repressor Rex NC NC NC NC NC NC NC NC -2.02 10.52 TM1428 TM1428 methyl-accepting chemotaxis protein NC NC -1.25 9.67 NC NC NC NC NC NC TM1430 TM1430 glycerol kinase NC NC -1.82 10.16 NC NC NC NC NC NC TM1435 TM1435 hypothetical protein NC NC -1.60 11.42 NC NC NC NC NC NC

265

Table B1 continued TM1439 TM1439 hypothetical protein 2.36 8.56 NC NC NC NC NC NC NC NC TM1458 TM1458 ribosomal protein L21 NC NC -1.28 12.75 NC NC NC NC NC NC TM1464 TM1464 hypothetical protein NC NC NC NC NC NC NC NC -2.07 11.76 TM1470 TM1470 transcription termination factor Rho NC NC -1.15 7.88 NC NC NC NC NC NC TM1471 TM1471 ribosomal protein L17 NC NC -1.00 10.37 NC NC NC NC NC NC TM1476 TM1476 50S ribosomal protein L36 NC NC -1.21 8.98 NC NC NC NC -3.24 10.93 TM1477 TM1477 translation initiation factor IF-1 NC NC -1.06 10.96 NC NC NC NC NC NC TM1486 TM1486 ribosomal protein S8 NC NC -1.02 7.78 NC NC NC NC -2.23 6.29 TM1491 TM1491 30S ribosomal protein S17 NC NC -1.14 11.36 NC NC NC NC NC NC TM1493 TM1493 50S ribosomal protein L16 NC NC -2.04 14.81 NC NC NC NC NC NC TM1503 TM1503 translation elongation factor G NC NC -1.44 15.32 NC NC NC NC NC NC TM1504 TM1504 30S ribosomal protein S7 NC NC NC NC NC NC NC NC -2.34 10.60 TM1505 TM1505 30S ribosomal protein S12 NC NC -1.17 11.40 NC NC NC NC NC NC TM1507 TM1507 phoH-related protein NC NC -1.02 14.99 NC NC NC NC NC NC TM1524 TM1524 endoglucanase 2.34 13.08 1.11 10.30 4.66 14.36 NC NC NC NC TM1525 TM1525 endoglucanase NC NC 1.50 12.41 NC NC NC NC NC NC TM1530 TM1530 electron transfer flavoprotein, β-subunit NC NC -1.20 14.00 NC NC NC NC NC NC TM1558 TM1558 hypothetical protein NC NC -1.44 10.29 NC NC NC NC NC NC TM1559 TM1559 deoxyribose-phosphate aldolase 2.14 10.32 NC NC NC NC NC NC NC NC TM1566 TM1566 ribosomal protein S16 NC NC -1.39 8.68 NC NC NC NC NC NC TM1571 TM1571 50S ribosomal protein L19 NC NC -1.05 6.84 NC NC NC NC NC NC TM1572 TM1572 signal peptidase I, putative NC NC 1.04 10.44 NC NC NC NC NC NC TM1590 TM1590 translation initiation factor IF-3 NC NC -1.12 9.34 NC NC NC NC NC NC

266

Table B1 continued TM1595 TM1595 hypothetical protein NC NC -1.15 14.64 NC NC NC NC NC NC TM1610 TM1610 F0F1 ATP synthase subunit β NC NC -1.06 12.41 NC NC NC NC NC NC TM1615 TM1615 ATP synthase F0, subunit c NC NC -1.01 9.63 NC NC NC NC NC NC TM1618 TM1618 cheX protein NC NC -1.15 9.14 NC NC NC NC NC NC TM1624 TM1624 β-mannosidase, putative 4.52 13.15 NC NC NC NC NC NC NC NC TM1634 TM1634 hypothetical protein NC NC -1.54 14.99 NC NC NC NC NC NC TM1641 TM1641 dihydrofolate reductase NC NC -3.09 14.79 NC NC NC NC NC NC TM1645 TM1645 nicotinate-nucleotide pyrophosphorylase -2.19 8.19 NC NC NC NC NC NC NC NC TM1655 TM1655 response regulator DrrA NC NC -1.10 11.68 NC NC NC NC NC NC TM1657 TM1657 ribosomal protein S20 NC NC -1.36 12.60 NC NC NC NC NC NC TM1658 TM1658 S-adenosylmethionine synthetase NC NC -1.32 12.79 NC NC NC NC NC NC TM1661 TM1661 polypeptide deformylase -2.76 12.85 NC NC NC NC NC NC NC NC TM1664 TM1664 hypothetical protein NC NC -1.01 5.01 NC NC NC NC NC NC TM1667 TM1667 xylose isomerase -2.19 9.30 NC NC NC NC NC NC NC NC TM1668 TM1668 hypothetical protein NC NC NC NC 2.08 7.90 NC NC NC NC TM1675 TM1675 hypothetical protein NC NC -1.09 12.99 NC NC NC NC NC NC TM1722 TM1722 hypothetical protein NC NC -2.43 15.12 NC NC NC NC -2.33 6.71 TM1760 TM1760 hypothetical protein NC NC NC NC NC NC 2.13 8.31 NC NC TM1762 TM1762 transketolase NC NC NC NC NC NC NC NC 2.24 6.42 TM1765 TM1765 transcription antitermination protein NusB NC NC -1.27 7.04 NC NC 2.31 5.49 NC NC TM1779 TM1779 hypothetical protein -2.11 8.91 NC NC NC NC NC NC 3.38 12.06 TM1780 TM1780 argininosuccinate synthase -3.26 14.82 NC NC NC NC NC NC NC NC TM1782 TM1782 N-acetyl-γ-glutamyl-phosphate reductase -2.61 14.09 NC NC NC NC NC NC NC NC

267

Table B1 continued TM1784 TM1784 acetylglutamate kinase NC NC -1.44 12.37 NC NC NC NC NC NC TM1785 TM1785 acetylornithine aminotransferase NC NC -1.16 9.53 NC NC NC NC NC NC TM1786 TM1786 hypothetical protein -4.36 14.28 NC NC NC NC NC NC 2.26 7.33 TM1825 TM1825 6,7-dimethyl-8-ribityllumazine synthase NC NC -1.69 10.94 NC NC NC NC NC NC TM1826 TM1826 GTP cyclohydrolase II protein -3.38 15.48 NC NC NC NC NC NC NC NC TM1828 TM1828 riboflavin-specific deaminase -2.63 10.99 NC NC NC NC NC NC NC NC TM1834 TM1834 α-glucosidase 3.08 12.13 NC NC -3.86 11.63 NC NC -4.44 13.71 TM1836 TM1836 maltose ABC transporter, permease -2.63 9.28 NC NC -2.77 5.41 NC NC -2.90 5.54 TM1837 TM1837 maltose ABC transporter, permease -2.33 10.26 NC NC NC NC NC NC NC NC TM1839 TM1839 maltose ABC transporter, binding -8.22 15.83 -2.96 12.94 -8.78 13.40 NC NC -2.98 7.53 TM1841 TM1841 hypothetical protein NC NC NC NC -2.72 11.96 NC NC -2.96 12.29 TM1843 TM1843 hypothetical protein NC NC NC NC -3.61 13.74 2.15 8.79 -3.68 14.65 TM1844 TM1844 hypothetical protein NC NC NC NC -5.34 17.05 2.82 8.84 -4.06 13.36 TM1845 TM1845 pullulanase NC NC NC NC NC NC 3.21 11.25 NC NC TM1847 TM1847 ROK family protein -3.80 11.18 NC NC -3.01 13.54 NC NC NC NC TM1848 TM1848 cellobiose-phosphorylase 13.62 18.75 2.23 12.66 9.35 19.30 -3.07 18.94 5.05 13.78 TM1849 TM1849 hypothetical protein -2.42 11.10 NC NC NC NC 2.23 7.43 NC NC TM1863 TM1863 hypothetical protein 2.28 10.12 NC NC NC NC NC NC NC NC TM1876 TM1876 hypothetical protein 2.12 12.73 NC NC NC NC NC NC NC NC

268

Table B2. LS means differences of non-core ORFs in dye flip experiments. NCBI Tma Tma Nea Nea RQ2 RQ2 Pet Pet Zoo Zoo ID Annotation (P-G) (P) (P-G) (P) (P-G) (P) (P-G) (P) (P-G) (P) 0035GTN_00 CTN_0660 ABC transporter -3.16 14.7 NC NC 0062GTN_00 CTN_0632 endo-1,4-β-xylanase A 4.74 14.0 NC NC 0160GTN_01 CTN_0526 conserved hypothetical protein -2.08 10.1 NC NC 0230GTN_02 CTN_0459 NADH-ubiquinone oxidoreductase -2.80 15.5 NC NC 0260GTN_02 CTN_0429 3'-5' exoribonuclease YhaM NC NC 4.24 8.3 0316GTN_03 CTN_0370 sugar kinase, fggy family -2.25 12.0 NC NC 0326GTN_03 CTN_0360 hypothetical protein -2.67 11.8 NC NC 0333GTN_03 CTN_0353 conserved hypothetical protein -3.13 15.7 -2.05 9.9 0368GTN_03 CTN_0320 3-dehydroquinate dehydratase, type II -2.25 14.0 NC NC 0371GTN_03 CTN_0316 carboxymuconolactone decarboxylase -2.42 15.9 NC NC 0439GTN_04 CTN_0245 ABC transporter periplasmic binding -2.37 10.6 NC NC 0440GTN_04 CTN_0244 ABC transporter, permease NC NC -6.06 17.5 0523GTN_05 CTN_0158 flagellar export protein FliJ -2.23 11.6 NC NC 0638GTN_06 CTN_0044 HEPN domain protein -3.66 15.0 NC NC 0650GTN_06 CTN_0032 dTDP-4-dehydrorhamnose 3,5-epimerase -2.07 8.3 NC NC 0684GTN_06 CTN_1934 TonB N terminus short domain -2.03 6.2 NC NC 0687GTN_06 CTN_1931 hypothetical protein -3.08 14.5 NC NC 0688GTN_06 CTN_1930 hypothetical protein -2.44 14.3 NC NC 0745GTN_07 CTN_1874 S-layer homology domain -2.29 10.9 NC NC 0751GTN_07 CTN_1868 hypothetical protein -2.21 10.7 NC NC 0786GTN_07 CTN_1831 uridine kinase -2.21 10.1 NC NC 0823GTN_08 CTN_1794 hypothetical protein -3.20 18.9 NC NC 1043GTN_10 CTN_1574 ISTma3, transposase NC NC 2.02 5.9

269

Table B2 continued 1064GTN_10 CTN_1554 methyl-accepting chemotaxis protein -2.28 11.0 NC NC 1076GTN_10 CTN_1542 trehalose/maltose transport membrane -2.10 10.6 NC NC 1077GTN_10 CTN_1541 extracellular solute-binding protein -2.38 14.8 NC NC 1211GTN_12 CTN_1407 α-glucan phosphorylase -3.22 14.0 -3.00 7.7 1244GTN_12 CTN_1372 putative extracellular solute-binding -2.32 10.6 NC NC 1406GTN_14 CTN_1211 conserved hypothetical protein -2.03 7.1 NC NC 1520GTN_15 CTN_1093 membrane protein, putative -2.21 8.0 NC NC 1599GTN_15 CTN_1016 translation initiation factor IF-1 -2.17 6.7 NC NC 1624GTN_16 CTN_0991 translation elongation factor Tu NC NC -2.48 14.9 1694GTN_16 CTN_0919 putative A-ATPase E-subunit -2.34 13.2 NC NC 1695GTN_16 CTN_0918 ATP synthase (F/14-kDa) subunit -2.32 9.1 NC NC 1715GTN_17 CTN_0898 putative ATP-binding protein -2.46 16.0 NC NC 1752GTN_17 CTN_0859 conserved hypothetical protein -2.00 12.1 NC NC 1827GTN_18 CTN_0782 hypothetical protein NC NC -2.40 7.3 1828GTN_18 CTN_0781 α-amylase NC NC 5.20 12.5 1829GTN_18 CTN_0780 trehalose/maltose binding protein -3.65 15.8 NC NC 1896GTN_18 CTN_0707 CRISPR-associated protein Cas6 NC NC NC NC 2.87 11.0 1903GTN_19 CTN_0699 conserved hypothetical protein NC NC NC NC 3.28 10.6 2A1 TRQ2_0974 TRQ2_0974 ATPase -2.41 7.97 -3.92 10.6 -3.72 13.7 2A6 TRQ2_0641 TRQ2_0641 1-phosphofructokinase 3.33 11.6 -3.02 9.5 2C9 TRQ2_1647 TRQ2_1647 α-glucan phosphorylase -2.38 6.8 -3.29 6.6 3A4 TRQ2_0974 TRQ2_0974 ATPase, -2.12 6.5 -3.01 11.8 -5.81 15.2

270

Table B2 continued 3A4 TRQ2_0975 TRQ2_0975 ABC transporter -2.12 6. 5 -3.01 11.8 -5.81 15.2 3F3 TRQ2_0970 TRQ2_0970 extracellular binding -2.60 6.1 -16.80 21.5 NC NC A10 TRQ2_1648 TRQ2_1648 hypothetical NC NC NC NC -2.75 7.5 A10 TRQ2_1649 TRQ2_1649 oxidase NC NC NC NC -2.75 7.5 A12 TRQ2_0666 TRQ2_0666 hypothetical NC NC NC NC 2.42 9.6 A12 TRQ2_0667 TRQ2_0667 LamG domain protein NC NC NC NC 2.42 9.6 B5 TRQ2_1838 TRQ2_1838 hypothetical NC NC NC NC 2.44 9.7 E9 TRQ2_0303 TRQ2_0303 G-1-P thymidylyltransferase NC NC NC NC -2.33 12.7 E9 TRQ2_0304 TRQ2_0304 epimerase NC NC NC NC -2.33 12.7 KJ3D10 TRQ2_1107 TRQ2_1107 two-sector ATPase NC NC NC NC -2.04 9.3 KJ3D10 TRQ2_1108 TRQ2_1108 hypothetical NC NC NC NC -2.04 9.3 KJB3 TRQ2_1842 TRQ2_1842 Methyltransferase 2.49 14.7 NC NC KJB3 TRQ2_1843 TRQ2_1843 regulator, PadR family 2.49 14.7 NC NC ORF12 TRQ2_0662 TRQ2_0662 glycoside hydrolase 43 NC NC 2.49 10.7 ORF13 TRQ2_0663 TRQ2_0663 hypothetical NC NC 2.43 9.1 ORF14 TRQ2_0664 TRQ2_0664 glycoside hydrolase 43 NC NC 2.39 10.7 ORF15 TRQ2_0665 TRQ2_0665 hypothetical NC NC 3.09 10.9 ORF17 TRQ2_0667 TRQ2_0667 LamG domain protein NC NC 3.39 13.7 RMLB TRQ2_0305 TRQ2_0305 dTDP-G 4,6-dehydratase NC NC -2.13 6.2 RMLC TRQ2_0304 TRQ2_0304 epimerase NC NC -2.28 6.7 TAA71 TRQ2_0512 TRQ2_0512 extracellular binding 2.24 7.21 NC NC TAB16 TRQ2_0514 TRQ2_0514 transport inner membrane NC NC -2.13 13.0 TAB60 TRQ2_0975 TRQ2_0975 ABC transporter related -2.02 11.9 -3.44 13.5 -5.79 19.9 TAB60 TRQ2_0976 TRQ2_0976 ROK family protein -2.02 11.9 -3.44 13.5 -5.79 19.9

271

Table B2 continued TAB89 TRQ2_0071 TRQ2_0071 pseudouridine synthase NC NC NC NC -2.11 8.7 TAB89 TRQ2_0072 TRQ2_0072 ribosome factor A NC NC NC NC -2.11 8.7 TAC08 TRQ2_0661 TRQ2_0661 solute-binding protein NC NC NC NC 4.74 16.2 TAC19 TRQ2_1829 TRQ2_1829 DNA repair protein MutS NC NC NC NC -2.34 10.9 TAC20 TRQ2_0970 TRQ2_0970 binding protein family 1 -3.33 12.0 -22.21 24.1 NC NC TAC44 TRQ2_0662 TRQ2_0662 glycoside hydrolase 43 NC NC NC NC 2.10 8.7 TAC44 TRQ2_0663 TRQ2_0663 hypothetical NC NC NC NC 2.10 8.7 TAC62 TRQ2_0313 TRQ2_0313 Cmr1 family NC NC NC NC -2.02 9.6 TAC74 TRQ2_0303 TRQ2_0303 G-1-P thymidylyltransferase NC NC NC NC -2.07 7.1 TAC78 TRQ2_0974 TRQ2_0974 ATPase NC NC -2.50 12.7 -4.69 16.0 TAC83 TRQ2_0665 TRQ2_0665 hypothetical NC NC NC NC 2.10 9.2 TAC83 TRQ2_0666 TRQ2_0666 hypothetical NC NC NC NC 2.10 9.2 TAD18 TRQ2_0658 TRQ2_0658 α-N-arabinofuranosidase NC NC NC NC 2.13 11.2 TAD18 TRQ2_0659 TRQ2_0659 transport inner membrane NC NC NC NC 2.13 11.2 TAD19 TRQ2_0973 TRQ2_0973 periplasmic binding -4.17 17.4 -18.71 20.8 -3.82 21.8 TAD39 TRQ2_0086 TRQ2_0086 PEGA domain protein NC NC NC NC -2.52 12.4 TAD39 TRQ2_0087 TRQ2_0087 hypothetical NC NC NC NC -2.52 12.4 TAD49 TRQ2_0578 TRQ2_0578 alcohol dehydrogenase NC NC NC NC -2.47 10.1 TAE12 TRQ2_1649 TRQ2_1649 oxidase NC NC -2.16 9.5 TAE94 TRQ2_0661 TRQ2_0661 binding protein family 1 NC NC 4.86 19.1 TAF05 TRQ2_0662 TRQ2_0662 glycoside hydrolase 43 -2.01 9.0 -4.77 16.7 TAF50 TRQ2_0975 TRQ2_0975 ABC transporter related NC NC -3.07 10.6 -5.85 19.2 TAF85 TRQ2_0969 TRQ2_0969 β-galactosidase NC NC NC NC NC NC NC -2.02 12.4

272

Table B2 continued TM0062 TM0062 hypothetical protein NC NC NC NC NC NC NC 2.90 11.0 TM0123 TM0123 ABC transporter, periplasmic binding NC NC 2.17 8.1 NC NC 2.18 8.5 TM0163 TM0163 hypothetical protein NC NC NC NC NC NC NC NC -2.06 5.8 TM0415 TM0415 hypothetical protein NC NC -2.83 10.9 NC NC NC NC TM0431 TM0431 sugar ABC transporter, permease NC NC NC NC NC NC NC NC 3.80 14.1 TM0433 TM0433 pectate lyase 2.19 13.0 2.27 11.8 4.32 10.0 TM0437 TM0437 exo-poly-α-D-galacturonosidase 2.90 11.7 NC NC NC NC NC NC TM0476 TM0476 hypothetical protein -2.69 12.1 NC NC -2.75 13.2 8.95 14.0 TM0622 TM0622 polysaccharide biosynthesis protein -3.38 11.3 2.76 11.9 TM0625 TM0625 hypothetical protein -2.08 9.8 NC NC TM0627 TM0627 polysaccharide biosynthesis protein -3.21 13.2 NC NC TM0628 TM0628 hypothetical protein -2.72 14.7 NC NC TM0630 TM0630 nucleotide sugar epimerase, putative -3.07 10.0 8.50 9.64 TM0631 TM0631 polysaccharide biosynthesis protein -2.25 13.0 2.31 10.9 TM0632 TM0632 polysaccharide biosynthesis protein -2.48 7.5 NC NC 3.69 9.1 TM0687 TM0687 hypothetical protein NC NC -2.98 13.7 NC NC NC NC TM0746 TM0746 hypothetical protein -2.09 4.6 NC NC NC NC NC NC TM0750 TM0750 hypothetical protein NC NC NC NC -2.18 5.4 NC NC TM0867 TM0867 hypothetical protein NC NC -2.04 10.4 NC NC NC NC TM0873 TM0873 Frame Shift NC NC NC NC NC NC 2.13 9.05 TM0898 TM0898 hypothetical protein -2.44 9.6 NC NC NC NC NC NC TM0899 TM0899 hypothetical protein -2.05 8.1 NC NC NC NC NC NC TM0951 TM0951 hypothetical protein -2.12 6.8 NC NC NC NC

273

Table B2 continued TM0955 TM0955 ribose ABC transporter, permease -3.49 10.8 NC NC NC NC TM0975 TM0975 hypothetical protein -3.77 11.9 NC NC NC NC TM0977 TM0977 hypothetical protein -5.36 11.1 NC NC 7.41 15.3 TM1001 TM1001 hypothetical protein NC NC -2.14 7.5 NC NC NC NC TM1016 TM1016 hypothetical protein NC NC NC NC NC NC -2.18 11.1 TM1062 TM1062 β-glucuronidase -2.01 8.0 NC NC NC NC NC NC TM1143 TM1143 methyl-accepting chemotaxis protein NC NC NC NC NC NC 2.25 9.4 TM1145 TM1145 hypothetical protein -2.30 9.0 NC NC TM1166 TM1166 coproporphyrinogen III oxidase -2.99 10.4 NC NC 2.51 13.2 TM1174 TM1174 hypothetical protein -4.34 12.9 -2.49 18.5 2.30 7.2 TM1176 TM1176 transcriptional regulator, metal-sensing -3.59 11.6 NC NC 2.24 9.3 TM1198 TM1198 ABC transporter, permease NC NC NC NC NC NC 2.35 10.1 TM1199 TM1199 ABC transporter, periplasmic binding NC NC -2.13 11.8 NC NC 2.94 15.5 TM1226 TM1226 ABC transporter, periplasmic binding NC NC NC NC NC NC 11.33 9.56 TM1271 TM1271 type IV pilin-related protein -3.86 18.5 3.30 11.6 TM1283 TM1283 hypothetical protein NC NC NC NC NC NC -2.39 7.2 TM1311 TM1311 HicB Antitoxin NC NC -2.27 13.4 NC NC NC NC TM1331 TM1331 hypothetical protein NC NC -2.01 8.7 NC NC NC NC TM1541 TM1541 flagellar protein FlgA, putative NC NC -2.11 0.7 NC NC NC NC TM1583 TM1583 hypothetical protein -3.30 13.6 -2.39 10.3 NC NC NC NC TM1589 TM1589 clostripain-related protein -2.48 10.6 NC NC NC NC TM1707 TM1707 hypothetical protein NC NC -2.32 9.2 NC NC NC NC TM1746 TM1746 ABC transporter, periplasmic binding 5.53 12.0 4.21 11.6 2.78 12.2

274

Table B2 continued TM1747 TM1747 ABC transporter, permease 12.63 14.8 8.88 12.1 5.06 8.3 TM1749 TM1749 ABC transporter, ATP-binding 10.53 13.6 4.56 12.8 3.58 13.3 TM1750 TM1750 ABC transporter, ATP-binding 9.96 16.9 6.32 12.0 5.21 13.8 TM1751 TM1751 endoglucanase 3.54 13.1 2.91 12.1 2.68 10.4 TM1753 TM1753 excinuclease ABC, subunit B-related NC NC -2.96 14.6 NC NC -2.54 11.6 TM1755 TM1755 phosphate butyryltransferase NC NC NC NC -2.30 5.60 NC NC TM1756 TM1756 butyrate kinase NC NC -2.20 9.3 NC NC NC NC TM1775 TM1775 hypothetical protein NC NC NC NC NC NC 2.16 7.7 TM1806 TM1806 hypothetical protein 2.66 14.4 NC NC NC NC NC NC TM1809 TM1809 hypothetical protein -3.16 9.3 NC NC NC NC 2.42 11.5 TM1812 TM1812 hypothetical protein -2.49 9.6 NC NC NC NC NC NC TM1833 TM1833 methyl-accepting chemotaxis-related NC NC NC NC NC NC -2.07 11.4 TM1838 TM1838 hypothetical protein -3.11 12.8 2.10 14.3 TM1855 TM1855 ABC transporter, periplasmic binding NC NC NC NC 2.48 11.7 Tpet_0636 Tpet_0636 extracellular solute-binding NC NC 3.50 10.9 Tpet_0642 Tpet_0642 LamG domain protein NC NC 2.12 7.9 Tpet_1768 Tpet_1768 hypothetical protein NC NC -2.21 6.5 Tpet_1794 Tpet_1794 monosaccharide-transporting ATPase NC NC 2.38 9.9 TXX2 TRQ2_0658 TRQ2_0658α-N-arabinofuranosidase NC NC 2.34 9.9 TXX5 TRQ2_0972 TRQ2_0972 transport membrane, -7.96 19.3 -5.25 16.5 TXX5 TRQ2_0973 TRQ2_0973 LacI regulator -7.96 19.3 -5.25 16.6

275

Table B3. ORFs differentially transcribed in the T. zoo vs. T. maritima in polysaccharide culture. Positive values indicate up-regulation in the zoo, negative values up-regulation in pure culture fold P- ID Annotation change value TM_rnpB TM_rnpB 2.7 12.8 TM_tRNA-Ala-1 TM_tRNA-Ala-1 4.2 13.7 TM_tRNA-Arg-3 TM_tRNA-Arg-3 3.9 13.7 TM_tRNA-Arg-4 TM_tRNA-Arg-4 2.7 11.3 TM_tRNA-Lys-2 TM_tRNA-Lys-2 4.2 12.0 TM_tRNA-Pro-1 TM_tRNA-Pro-1 3.4 11.7 TM_tRNA-Thr-1 TM_tRNA-Thr-1 2.2 10.4 TM0007 hypothetical protein 2.7 6.9 TM0017 pyruvate ferredoxin oxidoreductase, α subunit -2.2 8.3 TM0033 hypothetical protein 4.9 11.7 TM0051 iron(II) transport protein B 3.4 8.4 TM0059 oligopeptide ABC transporter, permease 3.3 13.5 TM0062 hypothetical protein -26.0 26.4 TM0067 2-keto-3-deoxygluconate kinase -2.3 12.6 TM0101 hypothetical protein 2.4 7.9 TM0107 hypothetical protein -5.5 25.0 TM0117 hypothetical protein -3.8 13.1 TM0118 ribonucleotide reductase, B12-dependent -2.0 10.1 TM0119 acetamidase, putative -2.4 12.4 TM0120 oxidoreductase, putative -2.6 10.0 TM0127 sensor histidine kinase 2.3 10.8 TM0138 tryptophan synthase subunit β 5.1 9.9 TM0141 anthranilate synthase component II 3.5 14.1 TM0143 response regulator 2.7 5.7 TM0153 hypothetical protein -3.3 14.4 TM0161 geranyltranstransferase -2.3 16.0 TM0169 redox-sensing transcriptional repressor Rex 4.1 13.9 TM0176 hypothetical protein 3.7 13.5 TM0180 hypothetical protein -2.2 12.9 TM0186 response regulator 2.9 9.5 TM0187 sensor histidine kinase 2.2 6.0 TM0193 hypothetical protein -2.2 10.8 TM0194 ABC transporter, ATP-binding protein 3.4 11.4 TM0195 guanosine pentaphosphate phosphohydrolase 2.4 13.8 TM0211 aminomethyltransferase 4.3 15.4 TM0212 glycine cleavage system H protein -3.6 21.1 TM0217 glycyl-tRNA synthetase, β subunit 4.5 16.0 TM0226 hypothetical protein 3.0 10.8 TM0229 hypothetical protein 3.7 17.4 TM0237 UDP-N-acetylmuramoylalanyl-D-glutamate ligase 2.3 8.8 TM0252 glutamyl tRNA-Gln amidotransferase, subunit C -2.0 11.0 TM0253 hypothetical protein -3.6 10.3

276

Table B3 continued TM0260 hypothetical protein 3.0 10.4 TM0272 pyruvate phosphate dikinase -2.1 12.5 TM0300 oligopeptide ABC transporter, periplasmic binding 2.1 8.4 TM0302 oligopeptide ABC transporter, permease protein 3.5 17.6 TM0328 m4C-methyltransferase 2.9 13.6 TM0335 dihydroorotase 3.5 11.0 TM0345 3-phosphoshikimate 1-carboxyvinyltransferase 3.4 14.6 TM0350 hypothetical protein 3.2 15.0 TM0376 hypothetical protein -2.7 13.7 TM0379 NADH oxidase -4.2 19.1 TM0389 ABC transporter, ATP-binding protein 2.1 10.4 TM0392 hypothetical protein -19.4 26.4 TM0393 transcriptional regulator, XylR-related -3.6 11.5 TM0394 hypothetical protein -2.4 15.5 TM0403 nitrogen regulatory protein P-II -5.8 18.8 TM0414 dehydrogenase -2.0 9.1 TM0415 hypothetical protein -2.3 14.9 TM0430 sugar ABC transporter, permease protein 2.1 8.7 TM0431 sugar ABC transporter, permease protein -4.3 12.5 TM0432 sugar ABC transporter, periplasmic sugar-binding protein -6.9 23.5 TM0433 pectate lyase -3.0 9.2 TM0458 DNA-directed RNA polymerase, β subunit -2.3 14.5 TM0467 regulatory protein, putative 2.4 12.3 TM0477 outer membrane protein α -2.1 6.9 TM0478 tyrosyl-tRNA synthetase -2.2 13.9 TM0481 hypothetical protein 2.8 12.9 TM0491 hypothetical protein -2.2 19.0 TM0493 hypothetical protein 4.2 15.8 TM0509 UDP-glucose 4-epimerase, putative 3.0 9.0 TM0519 hypothetical protein 4.3 15.1 TM0525 tRNA delta-2-isopentenylpyrophosphate transferase 5.2 12.8 TM0531 ABC transporter, periplasmic binding -4.0 27.0 TM0539 tryptophan synthase subunit β 3.7 17.0 TM0550 ketol-acid reductoisomerase 3.8 13.4 TM0551 dihydroxy-acid dehydratase -2.4 13.7 TM0562 hypothetical protein 3.5 14.3 TM0565 sugar fermentation stimulation protein, putative 3.7 16.7 TM0570 cell division protein FtsY 2.7 7.1 TM0579 hypothetical protein 3.2 11.3 TM0581 hypothetical protein 3.7 16.4 TM0608 hypothetical protein -3.6 18.8 TM0622 lipopolysaccharide biosynthesis protein, putative -18.4 27.8 TM0627 lipopolysaccharide biosynthesis protein -4.5 22.2 TM0628 hypothetical protein -3.0 8.8 TM0630 nucleotide sugar epimerase, putative -11.4 17.7

277

Table B3 continued TM0631 lipopolysaccharide biosynthesis protein -6.7 21.9 TM0639 hypothetical protein 3.5 15.7 TM0644 hypothetical protein -3.2 6.4 TM0650 hypothetical protein 4.0 15.0 TM0653 hypothetical protein 3.8 16.7 TM0662 acyl carrier protein 2.1 11.8 TM0663 hypothetical protein 3.7 14.2 TM0674 flagellar protein, putative 3.4 15.6 TM0676 motility protein B 3.2 6.6 TM0686 DNA polymerase III, γ and η subunit -2.7 21.2 TM0688 glyceraldehyde-3-phosphate dehydrogenase -2.3 5.9 TM0691 hypothetical protein 3.9 15.0 TM0693 hypothetical protein -2.1 11.2 TM0694 trigger factor, putative 4.2 15.6 TM0719 cysteinyl-tRNA synthetase 5.1 14.8 TM0720 serine hydroxymethyltransferase -2.7 17.0 TM0753 ubiquinone/menaquinone biosynthesis methyltransferase 2.0 6.1 TM0755 hypothetical protein 2.6 5.5 TM0762 30S ribosomal protein S2 -2.5 18.4 TM0764 hypothetical protein 3.0 12.1 TM0768 hypothetical protein 3.9 17.7 TM0770 hypothetical protein 3.7 11.9 TM0776 transposase, putative -3.0 17.4 TM0784 hypothetical protein 2.6 10.8 TM0788 thiamine biosynthesis protein ThiC 3.0 9.7 TM0790 hypothetical protein -3.2 21.5 TM0798 malonyl CoA-acyl carrier protein transacylase -2.4 13.8 TM0799 bioY protein -3.7 20.4 TM0803 CTP synthetase 2.5 9.4 TM0805 lipophilic protein, putative 2.0 6.8 TM0808 transcriptional regulator, XylR-related 3.0 10.5 TM0809 hydrolase, putative 2.2 14.1 TM0811 sugar ABC transporter, permease protein 2.1 8.5 TM0816 transcriptional regulator, putative, Mar family 3.1 12.0 TM0839 rod shape-determining protein RodA -2.4 7.8 TM0841 S-layer-like array protein 4.2 11.1 TM0843 formiminotetrahydrofolate cyclodeaminase 3.6 13.4 TM0848 hypothetical protein -2.3 10.9 TM0849 dnaJ protein 2.2 9.5 TM0857 riboflavin kinase/FMN adenylyltransferase 4.2 18.2 TM0880 oxaloacetate decarboxylase, β subunit 3.8 13.6 TM0881 homoserine O-succinyltransferase 2.8 10.2 TM0891 gcpE protein 2.9 10.0 TM0894 hypothetical protein 5.3 24.3 TM0898 hypothetical protein -2.0 10.6 TM0917 phosphate permease, putative 2.7 14.8 TM0923 hypothetical protein -2.0 15.1

278

Table B3 continued TM0924 hypothetical protein 4.3 15.4 TM0932 hypothetical protein -2.1 9.3 TM0940 ribosomal large subunit pseudouridine synthase C -2.5 5.7 TM0941 hypothetical protein 5.0 17.8 TM0942 hypothetical protein 2.7 11.7 TM0943 glutamine synthetase 3.2 8.3 TM0945 hypothetical protein 4.1 13.2 TM0949 transcriptional regulator, LacI family -10.6 17.0 TM0950 hypothetical protein 2.8 11.0 TM0961 lemA protein -3.0 13.7 TM0972 conserved hypothetical protein, GGDEF domain 3.2 8.3 TM0984 hypothetical protein 3.1 14.5 TM0986 hypothetical protein 3.3 16.8 TM0988 hypothetical protein 3.9 12.5 TM0989 hypothetical protein 3.7 16.4 TM0991 hypothetical protein -2.2 6.1 TM0998 heavy metal resistance transcriptional regulator 2.4 13.1 TM1002 hypothetical protein -2.1 9.8 TM1030 transcriptional regulator, TetR family 2.1 8.9 TM1031 glutaredoxin -2.6 15.8 TM1035 phosphoribosyl-ATP pyrophosphohydrolase 4.0 30.2 TM1068 α-glucosidase, putative -2.1 6.0 TM1069 transcriptional regulator, DeoR family -2.3 5.6 TM1080 sugar-phosphate isomerase -2.5 9.4 TM1098 hypothetical protein 4.4 17.1 TM1102 ribonuclease III -2.4 13.1 TM1107 hypothetical protein -4.1 17.1 TM1119 hypothetical protein -2.0 7.2 TM1134 hypothetical protein 2.7 17.6 TM1143 methyl-accepting chemotaxis protein -2.2 10.6 TM1166 oxygen-independent coproporphyrinogen III oxidase -3.5 16.2 TM1171 transcriptional regulator, crp family 3.1 11.4 TM1172 hydroxylamine reductase -2.1 9.5 TM1174 hypothetical protein -5.4 22.7 TM1188 hypothetical protein 2.1 8.4 TM1190 galactokinase 4.0 11.4 TM1206 putative monovalent cation/H+ antiporter subunit F 3.4 11.3 TM1208 hypothetical protein -2.1 9.9 TM1221 ABC transporter, permease -2.2 13.3 TM1222 ABC transporter, permease 2.1 9.2 TM1224 transcriptional regulator, XylR-related -2.3 7.8 TM1226 ABC transporter, periplasmic binding protein -3.7 7.6 TM1227 endo-1,4-β-mannosidase -2.2 16.6 TM1231 α-mannosidase-related protein 2.6 11.0 TM1240 translation-associated GTPase 3.3 9.3 TM1247 amidophosphoribosyltransferase 4.1 15.8 TM1250 phosphoribosylamine--glycine ligase -2.5 8.5

279

Table B3 continued TM1252 hypothetical protein 3.7 9.0 TM1255 aspartate aminotransferase 3.4 11.4 TM1261 phosphate ABC transporter, ATP-binding 4.1 17.2 TM1262 phosphate ABC transporter, permease 3.5 15.1 TM1263 phosphate ABC transporter, permease -3.1 8.2 TM1264 phosphate ABC transporter, phosphate-binding -2.3 15.6 TM1265 hypothetical protein 2.8 14.9 TM1271 type IV pilin-related protein -6.1 19.9 TM1272 glutamyl tRNA-Gln amidotransferase, subunit A -3.4 21.6 TM1273 aspartyl/glutamyl-tRNA amidotransferase subunit B -2.6 22.0 TM1276 sugar ABC transporter, ATP-binding protein 3.4 14.8 TM1293 hypothetical protein 3.7 15.0 TM1299 hypothetical protein 2.2 12.2 TM1359 sensor histidine kinase 2.9 13.6 TM1369 hypothetical protein 2.6 16.0 TM1371 aminotransferase, class V 2.2 6.9 TM1375 ABC transporter, periplasmic binding 3.3 16.4 TM1383 hypothetical protein -2.5 17.4 TM1388 hypothetical protein 4.4 14.7 TM1391 ATP-dependent Clp protease, ATPase subunit 2.4 8.7 TM1395 hypothetical protein 3.1 14.8 TM1402 hypothetical protein 4.1 16.5 TM1405 lipopolysaccharide biosynthesis protein-related protein 3.6 10.1 TM1406 hypothetical protein 3.6 10.2 TM1415 inositol monophosphatase family protein, putative -3.0 16.8 TM1432 hypothetical protein 3.3 16.7 TM1433 oxidoreductase, putative 4.3 16.9 TM1435 hypothetical protein -3.1 16.2 TM1442 anti-sigma factor antagonist, putative 2.0 8.7 TM1469 glucokinase 3.6 8.9 TM1470 transcription termination factor Rho -2.7 5.8 TM1472 DNA-directed RNA polymerase subunit α 3.3 14.6 TM1474 30S ribosomal protein S11 -3.2 13.9 TM1475 30S ribosomal protein S13 -2.5 10.0 TM1476 50S ribosomal protein L36 2.4 7.6 TM1485 50S ribosomal protein L6 -2.4 16.8 TM1490 ribosomal protein L14 -2.2 19.1 TM1491 30S ribosomal protein S17 -2.0 8.8 TM1492 ribosomal protein L29 -3.2 22.4 TM1493 50S ribosomal protein L16 -2.1 5.7 TM1500 ribosomal protein L3 -2.4 13.9 TM1504 30S ribosomal protein S7 -2.1 6.1 TM1505 30S ribosomal protein S12 -2.4 18.2 TM1515 ferric uptake regulation protein 2.2 6.7 TM1516 hydrolase, ama/hipO/hyuC family 2.9 9.8 TM1519 2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase 3.5 13.3 TM1522 diaminopimelate epimerase -3.5 17.2

280

Table B3 continued TM1526 hypothetical protein -2.3 8.8 TM1527 hypothetical protein -2.2 15.4 TM1536 hypothetical protein 3.6 12.8 TM1550 hypothetical protein 3.9 17.0 TM1565 signal recognition particle protein 2.3 5.8 TM1574 pseudouridylate synthase I 4.2 11.6 TM1576 hemolysin -2.8 13.4 TM1589 clostripain-related protein -2.1 12.4 TM1597 hypothetical protein 4.6 13.8 TM1600 hypothetical protein 3.8 16.9 TM1602 transcriptional regulator, biotin repressor family 2.9 12.1 TM1608 hypothetical protein 2.6 13.3 TM1610 F0F1 ATP synthase subunit β -2.1 9.2 TM1615 ATP synthase F0, subunit c -2.4 16.2 TM1629 UDP-N-acetylglucosamine pyrophosphorylase 3.8 14.7 TM1645 nicotinate-nucleotide pyrophosphorylase -2.4 14.9 TM1647 hypothetical protein 4.0 14.2 TM1648 hypothetical protein 4.6 20.2 TM1650 α-amylase, putative 2.6 9.5 TM1656 hypothetical protein 3.0 9.9 TM1661 polypeptide deformylase -2.7 13.9 TM1667 xylose isomerase -2.1 13.8 TM1676 hypothetical protein 3.7 22.2 TM1677 transposase, putative -2.6 18.7 TM1678 hypothetical protein -2.3 15.8 TM1685 hypothetical protein -2.2 11.8 TM1697 hypothetical protein 2.7 13.8 TM1703 hypothetical protein 4.1 14.1 TM1705 lysyl-tRNA synthetase 3.6 13.9 TM1715 hypothetical protein 3.0 12.7 TM1725 Frame Shift -2.6 14.2 TM1745 hypothetical protein -2.1 10.0 TM1753 excinuclease ABC, subunit B-related protein -2.2 7.4 TM1754 butyrate kinase 2.2 13.6 TM1756 butyrate kinase 2.8 12.6 TM1759 2-ketoisovalerate ferredoxin reductase 3.7 16.6 TM1761 excinuclease ABC subunit B 4.3 16.2 TM1779 hypothetical protein -4.1 22.1 TM1781 argininosuccinate lyase -2.0 7.0 TM1783 ornithine acetyltransferase/N-acetylglutamate synthase 3.8 16.2 TM1786 hypothetical protein -2.1 12.7 TM1788 conserved hypothetical protein, GGDEF domain 3.8 14.2 TM1806 hypothetical protein -2.3 7.2 TM1807 hypothetical protein -3.9 16.2 TM1809 hypothetical protein -3.5 19.4 TM1811 hypothetical protein -2.5 11.9 TM1814 hypothetical protein 4.2 15.1

281

Table B3 continued TM1827 riboflavin synthase subunit α -2.6 13.0 TM1832 transposase -2.1 7.2 TM1842 hypothetical protein -2.7 25.1 TM1847 ROK family protein -3.2 12.8 TM1856 transcriptional regulator, LacI family 3.9 11.3 TM1861 CDP-diacylglycerol-glycerol-3-phosphate 4.5 14.1 TM1870 septum site-determining protein MinD -2.2 13.8 TM1872 hypothetical protein -2.0 9.7 TM1874 cold shock protein 2.2 8.8

282

Table B4. ORFs differentially transcribed in the T. zoo vs. T. neapolitana in polysaccharide culture. Positive values indicate up-regulation in the zoo, negative values indicate up-regulation in the pure culture Fold (P) ID Annotation change value CTN_0026 putative rhamnosyltransferase 4.4 28.0 CTN_0027 hypothetical protein 2.4 14.4 CTN_0028 glycosyl transferase, group 1 family 4.0 23.1 CTN_0029 hypothetical protein 3.8 24.0 CTN_0030 glycosyl transferase, group 2 family 3.4 16.0 CTN_0032 dTDP-4-dehydrorhamnose 3,5-epimerase 3.6 14.4 CTN_0033 dTDP-glucose 4,6-dehydratase 4.7 31.7 CTN_0034 conserved hypothetical protein 4.5 35.0 CTN_0035 dTDP-4-dehydrorhamnose reductase 4.7 10.4 CTN_0038 hypothetical protein 5.3 28.6 CTN_0039 lipopolysaccharide biosynthesis protein 4.2 29.3 CTN_0039a hypothetical protein 4.4 36.9 CTN_0044 HEPN domain protein 4.4 23.7 CTN_0045 hypothetical protein 4.2 7.6 CTN_0045a hypothetical protein -1.4 12.7 CTN_0046 hypothetical protein 3.3 23.8 CTN_0046a hypothetical protein 3.5 11.3 CTN_0047 putative ATP/GTP-binding protein 5.4 29.3 CTN_0049 hypothetical protein 3.9 26.9 CTN_0049a lipopolysaccharide biosynthesis protein 4.0 32.2 CTN_0050 HD domain protein 4.3 29.5 CTN_0062 conserved hypothetical protein 2.5 16.0 CTN_0073 hypothetical protein 2.3 12.2 CTN_0094a hypothetical protein 4.1 25.9 CTN_0104 putative membrane protein 3.3 24.3 CTN_0105 conserved domain protein 3.8 21.8 CTN_0109 hypothetical protein 1.5 10.7 CTN_0130 transposase, IS605 OrfB family 4.9 31.4 CTN_0133 putative ATP/GTP-binding protein 4.0 24.7 CTN_0134 conserved hypothetical protein 3.8 12.9 CTN_0147 hypothetical protein -1.4 9.2 CTN_0158 flagellar export protein FliJ 3.0 11.7 CTN_0192 preprotein translocase, SecG subunit 4.0 7.6 CTN_0201 glutamine amidotransferase subunit PdxT 3.8 26.1 CTN_0235 trap dicarboxylate transporter, dctm subunit 3.9 16.3 CTN_0236 TRAP transporter, DctQ-like membrane protein 3.6 20.0 CTN_0237 C4-dicarboxylate-binding protein 3.4 26.6 CTN_0238 ATP-binding protein of sugar ABC transporter 2.0 15.3 CTN_0239 sugar ABC transporter permease 3.1 10.7 CTN_0240 ABC sugar transporter, periplasmic sugar binding 3.7 18.5 CTN_0241 spermidine/putrescine import ATP-binding PotA 3.3 24.2 CTN_0242 ribonuclease Z 2.2 21.2 CTN_0243 ABC transporter permease protein 4.3 23.4 CTN_0244 ABC transporter, permease protein -2.1 14.8

283

Table B4 continued CTN_0245 ABC transporter periplasmic binding protein 4.0 21.4 CTN_0288 biotin synthase 4.3 26.9 CTN_0316 carboxymuconolactone decarboxylase 4.9 26.7 CTN_0318 glycerol-3-phosphate dehydrogenase 2.5 15.9 CTN_0320 3-dehydroquinate dehydratase, type II 4.9 30.1 CTN_0321 hypothetical protine 3.0 15.2 CTN_0323 chorismate synthase 1.9 11.6 CTN_0355 α-xylosidase 3.5 29.3 CTN_0357 binding-dependent transport inner membrane 1.6 11.1 CTN_0358 sugar ABC transporter substrate-binding protein, 4.0 15.6 CTN_0359 conserved hypothetical protein 3.1 25.9 CTN_0360 hypothetical protein 2.0 14.7 CTN_0361 ribose transport ATP-binding protein RbsA 3.1 19.0 CTN_0362 xylose transport system permease protein XylH 1.7 9.4 CTN_0363 amino acid transport permease component 3.1 19.8 CTN_0364 putative periplasmic binding protein 3.6 21.7 CTN_0365 oxidoreductase, dehydrogenase/reductase family 4.0 16.8 CTN_0366 ribose transport ATP-binding protein RbsA 3.4 24.7 CTN_0367 ribose transport system permease protein RbsC 2.8 19.8 CTN_0368 hypothetical oxidoreductase YisS 4.0 24.1 CTN_0369 oxidoreductase, zinc-binding dehydrogenase family 3.5 25.9 CTN_0370 sugar kinase, fggy family 4.8 27.3 CTN_0371 transcriptional regulator, GntR family 3.7 24.1 CTN_0372 succinate-semialdehyde dehydrogenase 4.0 25.1 CTN_0373 glucose-6-phosphate isomerase 4.0 25.5 CTN_0384 fggy family of carbohydrate kiNases 3.9 25.3 CTN_0408 amino-acid ABC transporter binding protein y4oP 4.5 31.0 CTN_0432 SsrA-binding protein 4.2 28.6 CTN_0444 hypothetical protein -2.0 24.8 CTN_0459 NADH-ubiquinone oxidoreductase 24 kda subunit 3.4 20.3 CTN_0499 sensor histidine kinase 3.5 26.0 CTN_0500 sensor histidine kinase 3.5 20.0 CTN_0527 conserved hypothetical protein 4.3 36.0 CTN_0531 conserved hypothetical protein 3.5 19.6 CTN_0545 putative membrane protein 2.3 23.2 CTN_0555 metallo-β-lactamase family protein -1.2 12.8 CTN_0590 putative membrane protein 3.6 15.8 CTN_0599 conserved hypothetical protein 5.5 27.7 CTN_0617 endo-1,3-β-xylanase 3.3 21.0 CTN_0632 endo-1,4-β-xylanase A 7.8 40.3 CTN_0647 hypothetical protein 2.6 16.4 CTN_0660 ABC transporter 6.7 35.3 CTN_0661 glucose transport system permease protein 5.9 31.5 CTN_0667a oligopeptide ABC transporter, ATP-binding protein 4.6 29.2 CTN_0673 hypothetical protein 3.3 19.9 CTN_0675 iron-sulfur flavohypothetical proteinprotein 3.6 25.7

284

Table B4 continued CTN_0679 oxidoreductase, short chain dehydrogenase/reductase 4.7 34.2 CTN_0697 conserved domain protein 1.5 16.4 CTN_0709 CRISPR-associated CXXC_CXXC protein Cst1 6.1 33.1 CTN_0710 CRISPR-associated regulatory protein, DevR family 4.5 29.3 CTN_0712 CRISPR-associated helicase Cas3 3.2 22.6 CTN_0714 CRISPR-associated protein Cas1 3.2 25.5 CTN_0715 CRISPR-associated protein Cas2 4.2 28.4 CTN_0775 identified by match to protein family 1.1 17.8 CTN_0776 ribose transport system permease protein RbsC 3.1 20.6 CTN_0778 ABC transporter, permease protein, MalFG family 1.7 19.2 CTN_0779 ABC transporter, permease protein, MalFG family 1.3 7.4 CTN_0782 hypothetical protein 2.4 14.1 CTN_0784 putative lipoprotein 3.6 20.8 CTN_0822 DNA double-strand break repair Rad50 ATPase 4.4 15.8 CTN_0823 exonuclease, putative 2.8 22.0 CTN_0833 UvrB/UvrC motif domain protein 3.6 22.4 CTN_0859 conserved hypothetical protein 3.6 20.2 CTN_0898 putative ATP-binding protein 1.4 9.0 CTN_0909 conserved hypothetical protein 3.9 26.4 CTN_0913 Ser/Thr protein phosphatase family protein 1.7 21.9 CTN_0914 outer dense fiber ODF3, putative 5.2 33.1 CTN_0915 ATP synthase (C/AC39) subunit 4.2 24.5 CTN_0916 putative A-ATPase I-subunit 3.9 24.3 CTN_0917 membrane-associated ATPase C chain 3.8 26.7 CTN_0918 ATP synthase (F/14-kDa) subunit 4.7 20.6 CTN_0919 putative A-ATPase E-subunit 5.8 26.4 CTN_0921 V-type ATP synthase β chain 4.3 24.0 CTN_0979 putative membrane protein 3.1 13.1 CTN_0987 conserved hypothetical protein 3.2 23.9 CTN_0991 translation elongation factor Tu 2.9 25.3 CTN_1016 translation initiation factor IF-1 3.9 28.5 CTN_1024a conserved hypothetical protein 2.9 22.8 CTN_1029a ribosomal protein L34 3.8 28.0 CTN_1048 30S ribosomal protein S1 4.0 28.4 CTN_1056 glycosyl hydrolase, family 57 3.9 18.5 CTN_1080 putative membrane protein 3.5 20.6 CTN_1081 conserved hypothetical protein 3.5 19.2 CTN_1083 conserved hypothetical protein 3.9 23.5 CTN_1083a conserved hypothetical protein 3.8 8.9 CTN_1086 conserved hypothetical protein -1.1 8.2 CTN_1087a conserved hypothetical protein 4.1 9.5 CTN_1088 HD domain protein 2.6 24.2 CTN_1093 membrane protein, putative 5.3 18.9 CTN_1095 conserved hypothetical protein 4.3 29.9 CTN_1102a hypothetical protein 4.0 10.0 CTN_1155 hypothetical protein 2.4 8.4 CTN_1156 PQQ enzyme repeat domain protein 4.0 35.2

285

Table B4 continued CTN_1160 conserved hypothetical protein 2.9 19.4 CTN_1167 conserved hypothetical protein 3.9 12.3 CTN_1174 conserved hypothetical protein 3.5 16.3 CTN_1175 hypothetical protein 2.8 18.4 CTN_1175a hypothetical protein 3.3 25.8 CTN_1183 argininosuccinate lyase 4.5 27.4 CTN_1211 conserved hypothetical protein 3.9 22.3 CTN_1233 conserved hypothetical protein 4.5 29.6 CTN_1243 conserved hypothetical protein 3.4 18.9 CTN_1248 pyrimidine dimer DNA glycosylase 2.9 18.7 CTN_1259a hypothetical protein 3.6 18.9 CTN_1262 hypothetical protein 3.2 20.8 CTN_1263 hypothetical protein 4.1 23.2 CTN_1264 ABC transporter ATP-binding protein 3.8 22.8 CTN_1265 conserved hypothetical protein 4.4 24.5 CTN_1281a conserved hypothetical protein 3.4 23.5 CTN_1285 multidrug resistance protein 3.9 23.7 CTN_1301 type IV pilin-related protein 3.3 28.3 CTN_1372 putative extracellular solute-binding protein 4.2 26.0 CTN_1373 ABC transporter, permease protein 3.1 25.3 CTN_1374 permease 2.2 19.3 CTN_1387 ggdef domain protein 3.6 19.8 CTN_1388 putative membrane protein 2.9 19.5 CTN_1407 α-glucan phosphorylase 2.4 19.6 CTN_1415a conserved hypothetical protein 4.1 26.3 CTN_1436 putative membrane protein 4.5 32.6 CTN_1469 caax amino terminal protease family 3.1 11.8 CTN_1480 NAD-binding component of a K+ transport system 4.1 25.8 CTN_1481 TrkA 4.7 33.2 CTN_1503 oligopeptide ABC transporter, periplasmic binding 3.9 30.5 CTN_1504 conserved hypothetical protein 4.1 25.0 CTN_1540 membrane protein, putative 6.7 34.2 CTN_1542 trehalose/maltose transport inner membrane protein 2.3 16.6 CTN_1543 ABC transporter 3.1 15.1 CTN_1544 transaldolase 3.8 21.2 CTN_1545 altronate hydrolase 4.5 17.4 CTN_1546 altronate dehydratase 4.1 29.0 CTN_1547 conserved hypothetical protein 3.9 17.5 CTN_1548 identified by match to protein family 2.9 19.8 CTN_1549 conserved hypothetical protein 3.7 10.1 CTN_1550 transcriptional regulator, putative 4.8 31.4 CTN_1551 glycosidase 4.0 26.1 CTN_1552 putative lipoprotein 1.9 12.1 CTN_1553 von Willebrand factor type A domain protein 1.2 9.5 CTN_1554 methyl-accepting chemotaxis protein 4.6 30.0 CTN_1555 hypothetical protein 3.9 37.3 CTN_1570 hypothetical protein 4.1 31.1

286

Table B4 continued CTN_1574 ISTma3, transposase 2.6 6.4 CTN_1599a hypothetical protein 3.7 28.3 CTN_1622 transketolase 4.0 31.2 CTN_1623 transketolase 4.4 24.2 CTN_1648a hypothetical protein 2.6 17.5 CTN_1676 conserved hypothetical protein 5.9 28.1 CTN_1700 conserved hypothetical protein 4.4 29.7 CTN_1702 ATP-dependent Clp protease, ATPase subunit 4.1 28.8 CTN_1735 conserved hypothetical protein 4.2 36.3 CTN_1742 conserved hypothetical protein 3.9 21.5 CTN_1784 putative membrane protein 2.2 18.1 CTN_1794 hypothetical protein 6.9 38.6 CTN_1795 conserved hypothetical protein 3.3 20.7 CTN_1796 BchE 3.0 18.8 CTN_1813 transposase, IS605 OrfB family 4.7 28.0 CTN_1820 carbamoylphosphate synthase large subunit short form 4.5 32.5 CTN_1821 phosphoglycolate phosphatase, putative 3.2 25.7 CTN_1831 uridine kinase 4.8 33.2 CTN_1836 putative membrane protein -2.0 14.6 CTN_1856 integral membrane protein DUF6 2.9 21.4 CTN_1868 hypothetical protein 4.1 13.8 CTN_1905 S-layer homology domain protein 3.9 15.7 CTN_1906 FliY/N 4.6 33.4 CTN_1912 FlgE 3.8 21.4 CTN_1915 flagellar hook-length control protein, putative 2.6 20.5 CTN_1915a hypothetical protein 4.2 15.8 CTN_1916 conserved hypothetical protein 4.5 26.7 CTN_1931 hypothetical protein 5.5 36.9 CTN_1932 hypothetical protein 5.9 27.8 CTN_1933 hypothetical protein 4.2 27.6 CTN_1934 general secretion pathway protein D, putative 4.0 27.8 CTN_1935 secretin and TonB N terminus short domain protein 3.6 15.6 CTN_1935a hypothetical protein 4.0 20.8 CTN_1936 hypothetical protein 3.6 19.2 CTN_1937 bacterial transcriptional activator domain family 1.6 13.5 CTN0508a putative homing endonuclease 6.4 36.5 TM_tRNA-Ile-1 TM_tRNA-Ile-1 2.8 22.5 TM_tRNA-Lys-2 TM_tRNA-Lys-2 -1.1 5.5 TM_tRNA-Pro-1 TM_tRNA-Pro-1 -1.0 5.9 TM0007 hypothetical protein -1.2 5.5 TM0008 hypothetical protein -1.9 13.5 TM0014 methyl-accepting chemotaxis protein, putative 2.4 22.5 TM0021 hypothetical protein -1.7 6.6 TM0036 hypothetical protein -1.1 9.5 TM0037 hypothetical protein -1.8 19.1 TM0042 aminopeptidase P, putative -3.1 22.5 TM0044 hypothetical protein 1.2 11.3

287

Table B4 continued TM0045 hypothetical protein -1.2 13.2 TM0048 transposase 1.0 8.9 TM0051 iron(II) transport protein B -1.3 5.7 TM0053 esterase, putative -3.3 22.1 TM0055 α-glucuronidase -1.3 11.4 TM0056 ABC transporter, periplasmic binding protein 1.9 23.9 TM0057 oligopeptide ABC transporter, ATP-binding protein 1.6 6.3 TM0061 endo-1,4-β-xylanase A 1.1 16.1 TM0063 hypothetical protein -2.5 25.4 TM0064 glucuronate isomerase -1.2 11.1 TM0065 transcriptional regulator, IclR family -2.6 22.6 TM0066 4-hydroxy-2-oxoglutarate aldolase -1.5 6.6 TM0067 2-keto-3-deoxygluconate kinase -1.7 17.1 TM0071 ABC transporter, periplasmic binding protein -2.1 26.1 TM0072 oligopeptide ABC transporter, permease protein -1.1 6.9 TM0080 iron(III) ABC transporter, periplasmic-binding protein -1.2 9.7 TM0082 flagellar hook-associated protein 3 -1.9 13.5 TM0084 hypothetical protein -1.1 12.3 TM0091 hypothetical protein -1.4 5.5 TM0093 hypothetical protein -1.0 10.4 TM0094 general secretion pathway protein F -2.3 13.3 TM0095 hypothetical protein -2.3 7.6 TM0097 hypothetical protein -1.3 6.9 TM0107 hypothetical protein 1.6 18.4 TM0108 UDP-N-acetylglucosamine 1-carboxyvinyltransferase -1.3 14.8 TM0113 xylU-related protein -1.9 19.3 TM0114 sugar ABC transporter, periplasmic sugar-binding 1.0 14.3 TM0117 hypothetical protein 4.6 26.4 TM0120 oxidoreductase, -1.7 12.6 TM0126 response regulator -1.2 15.2 TM0127 sensor histidine kinase -1.5 13.6 TM0129 carboxypeptidase G2, putative -3.1 12.8 TM0132 flagellin, putative -2.8 22.9 TM0137 tryptophan synthase, α subunit -2.9 25.5 TM0141 anthranilate synthase component II -1.1 8.5 TM0143 response regulator -1.8 7.8 TM0144 hypothetical protein -1.2 15.6 TM0145 secreted metalloendopeptidase Gcp, putative -2.0 15.9 TM0146 ATP-dependent protease ATP-binding subunit -1.8 18.7 TM0154 hypothetical protein -1.8 13.6 TM0159 ham1 protein -1.7 12.3 TM0160 hypothetical protein -1.1 7.5 TM0167 phosphopentomutase -2.0 24.5 TM0169 redox-sensing transcriptional repressor Rex -1.2 7.9 TM0170 hypothetical protein -3.4 25.2 TM0171 hypothetical protein -1.3 10.5

288

Table B4 continued TM0176 hypothetical protein -1.2 8.2 TM0177 hypothetical protein -1.8 14.6 TM0180 hypothetical protein -1.9 20.3 TM0183 hypothetical protein -1.1 12.2 TM0185 hypothetical protein -1.0 11.9 TM0192 spoVS-related protein -1.5 9.3 TM0195 guanosine pentaphosphate phosphohydrolase, putative -1.5 15.6 TM0200 hypothetical protein -1.7 16.9 TM0205 ATP-dependent DNA helicase -1.9 18.4 TM0207 hypothetical protein -2.5 16.8 TM0209 6-phosphofructokinase -1.3 15.9 TM0212 glycine cleavage system H protein -2.4 25.4 TM0219 flagellar export/assembly protein -1.4 7.2 TM0222 ABC transporter, ATP-binding protein -1.1 7.9 TM0223 hypothetical protein 1.3 12.6 TM0227 frame shift -1.4 14.8 TM0229 hypothetical protein -1.1 9.8 TM0232 N-acetylglucosamine transferase -1.2 14.3 TM0233 cell division protein, rodA/ftsW/spoVE family 1.3 17.8 TM0246 hypothetical protein -1.2 8.9 TM0248 Na-translocating NADH-quinone reductase, Nqr5 1.5 12.8 TM0250 DNA processing chain A 1.1 12.2 TM0251 carbon storage regulator -2.0 19.4 TM0258 DNA topoisomerase -1.6 11.1 TM0261 phosphate permease, putative -3.4 21.9 TM0264 16S pseudouridylate synthase -2.5 16.0 TM0268 S-homocysteine methyltransferase -1.3 10.3 TM0277 frame shift 1.5 19.9 TM0279 sugar ABC transporter, permease protein 1.2 15.5 TM0280 hypothetical protein 2.6 14.9 TM0289 6-phosphofructokinase, pyrophosphate-dependent 1.3 16.2 TM0299 transcriptional regulator, LacI family -1.2 14.6 TM0300 ABC transporter, periplasmic binding protein -1.2 9.2 TM0301 oligopeptide ABC transporter, permease -1.5 15.0 TM0303 oligopeptide ABC transporter, ATP-binding -1.4 13.7 TM0304 oligopeptide ABC transporter, ATP-binding -1.6 6.8 TM0306 α-L-fucosidase, putative -1.4 10.8 TM0309 ABC transporter, periplasmic binding protein -1.5 12.0 TM0310 β-D-galactosidase -1.8 7.1 TM0311 hypothetical protein -2.9 22.5 TM0312 hypothetical protein 1.9 14.6 TM0313 K+ channel, β subunit 2.5 25.6 TM0316 h hypothetical protein ypothetical protein TM0316 1.7 18.9 TM0322 ABC transporter, periplasmic substrate-binding protein 2.0 15.9 TM0323 frame shift -2.0 18.3 TM0324 hypothetical protein -1.7 11.7 TM0326 transcriptional regulator, RpiR family -2.7 12.6

289

Table B4 continued TM0329 hypothetical protein -2.3 24.7 TM0330 hypothetical protein 2.1 23.5 TM0333 dihydroorotate dehydrogenase -1.9 19.2 TM0334 dihydroorotate dehydrogenase electron transfer protein 1.3 10.1 TM0338 hypothetical protein -1.5 13.2 TM0340 hypothetical protein -2.6 13.8 TM0343 3-deoxy-7-phosphoheptulonate synthase 1.7 7.5 TM0345 3-phosphoshikimate 1-carboxyvinyltransferase -1.0 7.9 TM0347 chorismate synthase 1.6 8.2 TM0350 hypothetical protein -1.7 15.6 TM0361 hypothetical protein 2.3 15.2 TM0365 putative aminopeptidase 1 -1.5 8.3 TM0369 hypothetical protein -2.4 13.9 TM0371 arginine repressor -1.7 16.6 TM0373 dnaK protein 1.2 8.3 TM0384 anaerobic ribonucleoside-triphosphate reductase-related -3.0 25.8 TM0395 NADH oxidase, putative -1.7 14.8 TM0396 iron-sulfur cluster-binding protein 1.1 8.0 TM0400 sensor histidine kinase -2.2 20.9 TM0403 nitrogen regulatory protein P-II 1.1 7.8 TM0405 2-oxoglutarate ferredoxin oxidoreductase subunit β -2.4 21.3 TM0407 diacylglycerol kinase, putative -4.5 30.9 TM0408 chemotaxis-specific methylesterase -1.2 6.9 TM0409 hypothetical protein -3.0 18.4 TM0410 hypothetical protein -2.1 11.3 TM0412 alcohol dehydrogenase, zinc-containing 1.3 14.6 TM0413 creatinine amidohydrolase, putative 2.4 13.3 TM0415 hypothetical protein -1.1 13.3 TM0420 sugar ABC transporter, permease protein -2.2 18.5 TM0427 oxidoreductase, putative -2.2 16.7 TM0428 oxidoreductase, putative -1.2 15.8 TM0433 pectate lyase -3.0 17.2 TM0434 α-glucosidase, putative 1.5 15.8 TM0436 alcohol dehydrogenase, zinc-containing -2.1 17.9 TM0437 exo-poly-α-D-galacturonosidase, putative -1.9 16.7 TM0438 6-phosphogluconate dehydrogenase -1.6 19.9 TM0441 oxidoreductase, short chain dehydrogenase/reductase 1.3 14.3 TM0447 phosphoribosylaminoimidazole carboxylase ATPase 1.7 13.2 TM0448 hypothetical protein 2.9 11.7 TM0450 hypothetical protein -1.2 11.3 TM0451 ribosomal protein L33 -3.5 23.1 TM0454 ribosomal protein L11 -1.6 17.8 TM0455 50S ribosomal protein L1 -3.8 26.2 TM0456 ribosomal protein L10 -2.5 19.6 TM0457 ribosomal protein L7/L12 -1.5 14.1 TM0459 DNA-directed RNA polymerase, β' subunit -2.0 15.6 TM0463 lipoprotein signal peptidase -2.5 23.5

290

Table B4 continued TM0465 hypothetical protein -1.9 15.2 TM0479 hypothetical protein -2.0 12.1 TM0481 hypothetical protein -1.2 9.8 TM0502 oligopeptide ABC transporter, permease protein -1.4 17.2 TM0503 oligopeptide ABC transporter, permease protein -1.6 10.1 TM0508 recombination factor protein RarA -1.8 16.6 TM0509 UDP-glucose 4-epimerase, putative -1.1 6.1 TM0513 comM protein -1.4 7.1 TM0519 hypothetical protein -1.0 6.6 TM0531 ABC transporter, periplasmic binding protein 3.5 36.1 TM0534 RNA polymerase factor ζ70 -1.6 13.3 TM0540 fumarate hydratase, N-terminal subunit -2.0 11.3 TM0541 fumarate hydratase, C-terminal subunit -1.2 13.5 TM0560 hypothetical protein 1.5 8.7 TM0565 sugar fermentation stimulation protein, putative -1.1 9.7 TM0566 hypothetical protein 1.2 18.5 TM0571 heat shock serine protease, periplasmic -1.9 26.4 TM0579 hypothetical protein -1.1 6.5 TM0580 cell division protein FtsH -1.1 6.1 TM0581 hypothetical protein -1.0 8.8 TM0590 penicillin-binding protein 2 -2.3 17.7 TM0593 ABC transporter, periplasmic binding protein 1.0 10.1 TM0594 hypothetical protein -1.4 7.2 TM0596 sugar ABC transporter, permease protein -1.5 19.1 TM0597 hypothetical protein -1.0 12.2 TM0602 iron-dependent transcriptional repressor, putative 1.5 15.9 TM0603 ribosomal protein S6 1.4 15.6 TM0604 single stranded DNA-binding protein, putative -1.8 16.4 TM0606 hypothetical protein -1.1 12.5 TM0616 hypothetical protein 1.3 11.9 TM0619 hypothetical protein 1.3 15.8 TM0636 hypothetical protein -1.6 9.3 TM0638 polysaccharide export protein, putative -1.5 13.1 TM0640 hypothetical protein -2.1 14.7 TM0643 clostripain-related protein 1.5 9.2 TM0644 hypothetical protein 3.3 13.9 TM0645 NH(3)-dependent NAD(+) synthetase, putative 3.2 29.8 TM0646 hypothetical protein 1.1 13.5 TM0650 hypothetical protein -1.2 8.3 TM0654 spermidine synthase 2.0 8.6 TM0657 rubrerythrin -2.0 19.9 TM0658 neelaredoxin 1.5 9.7 TM0659 rubredoxin -2.5 23.5 TM0665 cysteine synthase -1.2 7.0 TM0667 hypothetical protein -1.2 13.6 TM0668 pleiotropic regulatory protein -1.7 14.9 TM0669 hypothetical protein -1.9 20.6

291

Table B4 continued TM0674 flagellar protein, putative -1.1 9.3 TM0681 dehydrase-related protein -2.1 19.1 TM0685 hypothetical protein -2.1 14.0 TM0691 hypothetical protein -1.0 7.4 TM0695 ATP-dependent Clp protease, proteolytic subunit -1.2 8.7 TM0701 purine-binding chemotaxis protein -3.7 20.0 TM0703 competence-damage inducible protein, putative -2.6 10.1 TM0705 ABC transporter, ATP-binding protein -1.1 8.5 TM0710 transcriptional regulator, MarR family -1.8 20.9 TM0714 hypothetical protein 2.0 11.2 TM0717 propionyl-CoA carboxylase, γ subunit 1.6 9.5 TM0721 uracil phosphoribosyltransferase -1.2 10.2 TM0724 hypothetical protein -1.2 13.1 TM0726 tldD protein -1.3 8.4 TM0730 D-tyrosyl-tRNA deacylase -1.4 5.5 TM0731 hypothetical protein -3.1 22.2 TM0733 sigma-B regulator, putative -1.3 5.8 TM0736 mannose-6-phosphate isomerase -1.6 15.5 TM0744 hypothetical protein -2.5 26.9 TM0750 hypothetical protein 1.6 19.1 TM0752 α-glucosidase, putative 1.2 13.0 TM0754 oxidoreductase -1.5 5.4 TM0756 galactosyltransferase-related protein -1.2 11.7 TM0759 acyltransferase, putative -1.4 13.6 TM0760 lipopolysaccharide biosynthesis protein, putative -2.0 20.6 TM0762 30S ribosomal protein S2 1.6 21.4 TM0772 hypothetical protein -1.8 17.1 TM0774 hypothetical protein -1.1 6.9 TM0776 transposase, putative -1.2 13.7 TM0777 transposase -1.8 14.9 TM0780 bacterioferritin comigratory protein, ahpC/TSA family -1.5 10.6 TM0789 hypothetical protein -2.2 24.2 TM0799 bioY protein 1.5 16.6 TM0805 lipophilic protein, putative -1.4 10.0 TM0807 peroxiredoxin -1.2 24.6 TM0813 hypothetical protein -1.5 13.8 TM0814 N-acetylglucosamine-6-phosphate deacetylase -1.4 15.2 TM0818 lipopolysaccharide biosynthesis protein, putative -1.9 9.0 TM0823 transcriptional regulator, TetR family -3.0 26.0 TM0827 ABC transporter, ATP-binding protein, putative -1.3 12.6 TM0828 sugar kinase, pfkB family -2.6 21.7 TM0829 hypothetical protein -1.3 15.0 TM0830 hypothetical protein -1.5 11.5 TM0831 branched-chain amino acid aminotransferase, putative -2.5 21.0 TM0832 hypothetical protein -2.0 17.9 TM0834 hypothetical protein -1.3 11.3 TM0835 cell division protein FtsA, putative -1.4 16.3

292

Table B4 continued TM0836 cell division protein FtsZ -1.1 9.9 TM0838 hypothetical protein -1.8 13.8 TM0839 rod shape-determining protein RodA 3.9 22.9 TM0842 response regulator -2.7 24.0 TM0847 hypothetical protein -1.5 11.2 TM0850 grpE protein, putative -1.5 9.4 TM0853 sensor histidine kinase -2.5 20.5 TM0859 hypothetical protein 1.5 14.1 TM0861 protein-export membrane protein SecF, putative -1.9 20.6 TM0862 glucose-1-phosphate thymidylyltransferase -1.8 20.0 TM0863 ribosomal protein L9 2.0 20.2 TM0864 hypothetical protein 2.2 20.6 TM0867 hypothetical protein 1.2 10.0 TM0872 hypothetical protein -1.3 8.8 TM0874 hypothetical protein -2.0 13.0 TM0875 hypothetical protein -1.3 9.9 TM0876 hypothetical protein -1.3 13.6 TM0879 ferredoxin 1.5 17.1 TM0882 O-acetylhomoserine sulfhydrylase -1.3 11.8 TM0885 hypothetical protein -1.7 17.6 TM0887 methylated-DNA-protein-cysteine methyltransferase -1.5 15.4 TM0892 hypothetical protein -1.5 10.8 TM0894 hypothetical protein -1.3 15.1 TM0895 glycogen synthase -3.0 24.2 TM0897 spoVS-related protein -1.4 15.1 TM0899 hypothetical protein -1.3 8.3 TM0913 mazG protein -1.1 11.4 TM0920 alcohol dehydrogenase, iron-containing -1.4 7.0 TM0924 hypothetical protein -1.4 10.4 TM0926 chromosomal replication initiator protein -1.2 6.4 TM0932 hypothetical protein 3.9 28.2 TM0940 ribosomal large subunit pseudouridine synthase C 3.8 18.0 TM0944 hypothetical protein 2.4 17.3 TM0949 transcriptional regulator, LacI family 5.7 25.1 TM0951 hypothetical protein 1.7 9.8 TM0952 glycerol kinase 1.3 6.5 TM0953 transketolase, C-terminal subunit 1.0 11.0 TM0954 transketolase, N-terminal subunit 2.2 19.6 TM0956 ribose ABC transporter, ATP-binding protein 1.7 17.5 TM0961 lemA protein 3.8 27.3 TM0962 hypothetical protein 1.8 9.0 TM0963 oligoendopeptidase, putative 2.2 21.9 TM0973 methyl-accepting chemoreceptor-related protein -1.5 9.6 TM0975 hypothetical protein 2.1 7.5 TM0982 hypothetical protein -1.2 11.2 TM0985 hypothetical protein -1.2 8.2 TM0988 hypothetical protein -1.0 5.7

293

Table B4 continued TM0989 hypothetical protein -1.1 9.0 TM0990 hypothetical protein 1.8 8.4 TM0991 hypothetical protein 2.3 13.6 TM0993 hypothetical protein 1.7 10.8 TM0997 hypothetical protein 1.1 19.9 TM1001 hypothetical protein -1.4 16.2 TM1024 hypothetical protein -1.5 9.0 TM1026 transposase, putative -1.1 11.9 TM1030 transcriptional regulator, TetR family -1.1 8.7 TM1031 glutaredoxin 2.9 27.6 TM1033 mannose-1-phosphate guanylyltransferase -1.5 12.6 TM1035 phosphoribosyl-AMP cyclohydrolase -1.0 13.2 TM1037 PRF-5-aminoimidazole carboxamide isomerase -2.3 14.9 TM1043 histidyl-tRNA synthetase-related protein -1.4 7.9 TM1044 transposase, IS605-TnpB family -1.2 6.9 TM1046 hypothetical protein -1.1 11.6 TM1048 endoglucanase -3.2 20.8 TM1049 endoglucanase -2.3 13.8 TM1050 endoglucanase -1.5 10.3 TM1054 ABC transporter, ATP-binding protein -1.1 12.0 TM1065 oligopeptide ABC transporter, permease protein 1.0 8.1 TM1068 α-glucosidase, putative 2.1 12.9 TM1070 hypothetical protein -1.8 16.8 TM1072 rhamnulose-1-phosphate aldolase -2.0 7.9 TM1076 hypothetical protein 1.6 14.9 TM1077 pantoate-β-alanine ligase -1.1 8.8 TM1078 hypothetical protein 3.8 24.1 TM1081 anti-sigma factor antagonist, putative -1.2 14.3 TM1082 lexA repressor -1.4 10.6 TM1084 DNA gyrase, subunit A -1.2 8.7 TM1089 TRK system potassium uptake protein TrkH -1.1 10.3 TM1094 RNA methyltransferase, putative -1.1 15.0 TM1102 ribonuclease III 2.0 19.7 TM1119 hypothetical protein 3.3 22.8 TM1123 flagellar hook-associated protein 2, putative 2.6 26.9 TM1124 hypothetical protein 3.0 14.2 TM1126 hypothetical protein 2.7 18.0 TM1130 phosphate butyryltransferase 1.4 19.8 TM1141 cytochrome C-type biogenesis protein, putative 2.8 32.5 TM1150 ABC transporter, periplasmic binding protein -1.5 9.0 TM1155 glucose-6-phosphate 1-dehydrogenase -1.2 12.7 TM1165 2-oxoacid ferredoxin oxidoreductase subunit β -1.4 13.7 TM1167 hypothetical protein -2.5 20.4 TM1174 hypothetical protein 5.5 36.2 TM1178 acetyltransferase-related protein -1.8 16.0 TM1192 α-galactosidase -1.7 17.8 TM1195 β-galactosidase -1.1 5.8

294

Table B4 continued TM1207 putative monovalent cation/H+ antiporter subunit G 1.2 10.9 TM1214 NADH dehydrogenase subunit B -2.4 22.5 TM1224 transcriptional regulator, XylR-related -3.4 21.8 TM1226 ABC transporter, periplasmic binding protein -3.8 16.0 TM1228 transcriptional regulator, RpiR family -1.4 15.5 TM1229 hypothetical protein -1.3 14.0 TM1231 α-mannosidase-related protein -1.3 10.4 TM1233 sugar ABC transporter, permease protein, putative -1.1 12.7 TM1234 sugar ABC transporter, permease protein -1.5 19.0 TM1237 hypothetical protein -1.6 9.9 TM1240 translation-associated GTPase -1.9 10.4 TM1243 phosphoribosylaminoimidazole-succinocarboxamide synthase 1.1 12.0 TM1245 phosphoribosylformylglycinamidine synthase I -3.7 25.4 TM1247 amidophosphoribosyltransferase -1.3 9.8 TM1255 aspartate aminotransferase -1.1 6.5 TM1259 phosphate transcriptional regulatory protein PhoB -1.1 9.4 TM1261 phosphate ABC transporter, ATP-binding protein -1.2 9.8 TM1262 phosphate ABC transporter, permease protein -1.1 9.0 TM1266 hypothetical protein 1.0 8.2 TM1268 hypothetical protein 1.5 6.9 TM1270 cystathionine γ-synthase -1.0 14.5 TM1271 type IV pilin-related protein 3.4 24.2 TM1272 glutamyl tRNA-Gln amidotransferase, subunit A 1.7 20.7 TM1274 hypothetical protein 1.9 12.6 TM1279 hypothetical protein -1.8 19.9 TM1281 6-phospho-β-glucosidase -2.0 13.3 TM1286 methyltransferase -2.9 29.9 TM1290 hypothetical protein 1.6 17.2 TM1293 hypothetical protein -1.1 8.5 TM1294 hypothetical protein -1.6 15.0 TM1297 oxidoreductase, putative 1.3 18.9 TM1298 hypothetical protein -1.6 11.6 TM1310 ABC transporter, ATP-binding protein 1.6 8.5 TM1312 HicA Toxin, putative -3.5 29.9 TM1316 Subtilosin A-like bacteriocin, putative 1.5 16.1 TM1330 lacI family transcriptional regulator, putative -1.2 14.8 TM1331 hypothetical protein 2.2 25.8 TM1343 frame shift -1.1 9.6 TM1345 polynucleotide phosphorylase/polyadenylase -1.8 9.6 TM1346 processing protease, putative -2.1 18.7 TM1349 hypothetical protein -1.5 14.3 TM1353 hypothetical protein 2.6 25.3 TM1356 hypothetical protein -1.4 10.7 TM1360 response regulator -1.4 11.7 TM1363 peptide chain release factor RF-1 -1.1 10.3 TM1367 hypothetical protein 1.7 13.7 TM1371 aminotransferase, class V -1.0 5.9

295

Table B4 continued TM1375 ABC transporter, periplasmic binding protein -1.3 12.7 TM1377 spermidine/putrescine ABC transporter, permease -1.7 16.4 TM1381 hypothetical protein -1.1 6.6 TM1382 hypothetical protein -2.8 19.5 TM1384 adenine phosphoribosyltransferase 1.0 9.7 TM1387 dephospho-CoA kinase -2.2 26.5 TM1388 hypothetical protein -1.4 9.4 TM1393 hypothetical protein -3.0 24.9 TM1394 hypothetical protein 1.0 7.5 TM1395 hypothetical protein -2.1 18.6 TM1396 alanyl-tRNA synthetase -1.4 14.1 TM1398 hypothetical protein 3.1 24.3 TM1402 hypothetical protein -1.2 9.8 TM1406 hypothetical protein -1.2 6.1 TM1411 helicase-related protein -1.1 6.3 TM1416 hypothetical protein 1.3 6.9 TM1419 myo-inositol-1-phosphate synthase-related protein 1.2 6.7 TM1421 hydrogenase, putative -1.8 21.8 TM1425 Fe-hydrogenase, subunit β -1.2 14.4 TM1431 glycerol uptake operon antiterminator -1.1 8.1 TM1433 oxidoreductase, putative -1.0 8.1 TM1450 transcription-repair coupling factor, putative -1.1 12.8 TM1453 ribosomal protein S9 -1.1 9.2 TM1468 hypothetical protein -1.3 7.9 TM1474 30S ribosomal protein S11 -1.0 8.3 TM1478 methionine aminopeptidase -1.2 10.0 TM1484 ribosomal protein L18 -1.6 13.6 TM1485 50S ribosomal protein L6 2.5 27.4 TM1486 ribosomal protein S8 2.9 25.9 TM1493 50S ribosomal protein L16 1.5 9.0 TM1500 ribosomal protein L3 1.4 15.3 TM1509 hypothetical protein -1.3 11.0 TM1513 hypothetical protein TM1513 -1.0 10.0 TM1515 ferric uptake regulation protein -1.8 11.3 TM1519 2,3,4,5-THP-2-carboxylate N-succinyltransferase-related -1.0 7.0 TM1524 endoglucanase -1.6 19.0 TM1528 1,4-dihydroxy-2-naphthoate octaprenyltransferase -1.3 12.3 TM1546 single stranded DNA-specific exonuclease, putative -1.8 8.1 TM1548 lipopolysaccharide biosynthesis protein -1.6 8.3 TM1551 hypothetical protein -2.8 26.2 TM1552 pyruvate formate-lyase activating enzyme, putative -1.1 14.4 TM1556 maf protein -1.0 9.4 TM1559 deoxyribose-phosphate aldolase -1.2 9.1 TM1560 serine cycle enzyme, putative 1.1 12.5 TM1563 hypothetical protein -1.4 19.9 TM1570 hypothetical protein -2.7 13.7 TM1572 signal peptidase I, putative -1.5 20.3

296

Table B4 continued TM1574 pseudouridylate synthase I -1.2 5.7 TM1579 peptide chain release factor 2 -1.9 18.0 TM1580 transcriptional regulator, putative -3.0 29.7 TM1582 hypothetical protein -1.9 18.7 TM1583 hypothetical protein 1.1 12.2 TM1591 ribosomal protein L35 -1.2 7.6 TM1596 purine nucleoside phosphorylase 2.4 23.3 TM1597 hypothetical protein -1.2 7.1 TM1598 RNA polymerase sigma-E factor -1.5 7.5 TM1600 hypothetical protein -1.2 10.2 TM1607 hypothetical protein 2.0 15.9 TM1611 ATP synthase F1, subunit γ -1.0 8.1 TM1618 cheX protein 1.8 20.4 TM1624 β-mannosidase, putative -2.6 29.4 TM1625 hypothetical protein 1.5 17.5 TM1627 general stress protein Ctc -1.4 15.7 TM1628 phosphoribosyl pyrophosphate synthetase -1.3 13.2 TM1632 hypothetical protein -1.0 5.9 TM1633 ATP-dependent protease LA -1.9 20.9 TM1634 hypothetical protein 1.3 15.3 TM1641 dihydrofolate reductase 1.3 13.5 TM1643 L-aspartate dehydrogenase -1.1 9.8 TM1647 hypothetical protein -1.3 8.7 TM1653 pyrimidine-nucleoside phosphorylase -1.1 11.8 TM1654 sensor histidine kinase HpkA -1.8 8.9 TM1656 hypothetical protein -1.1 5.9 TM1657 ribosomal protein S20 -1.1 7.4 TM1660 hypothetical protein -1.7 19.5 TM1662 stationary phase survival protein -1.3 14.0 TM1663 ABC transporter, ATP-binding protein -1.3 11.0 TM1665 hypothetical protein -1.9 5.8 TM1666 succinyl-diaminopimelate desuccinylase -1.1 14.6 TM1667 xylose isomerase 1.1 14.6 TM1676 hypothetical protein -1.5 19.0 TM1680 hypothetical protein -1.8 13.2 TM1687 DNA/pantothenate metabolism flavoprotein -1.4 16.5 TM1694 thiamin biosynthesis protein ThiI -1.6 22.1 TM1697 hypothetical protein -2.6 22.4 TM1709 hypothetical protein TM1709 -1.7 15.7 TM1718 ribulose-phosphate 3-epimerase -1.5 11.4 TM1731 hypothetical protein 2.4 18.0 TM1732 hypothetical protein 1.8 13.5 TM1733 hypothetical protein -1.2 13.2 TM1734 phosphate transport system regulator PhoU, putative -1.6 6.7 TM1739 hypothetical protein -2.1 18.8 TM1745 hypothetical protein 1.0 9.6 TM1746 ABC transporter, periplasmic binding protein -4.1 26.7

297

Table B4 continued TM1747 ABC transporter, permease protein -1.4 6.9 TM1749 ABC transporter, ATP-binding protein -3.7 27.5 TM1750 ABC transporter, ATP-binding protein -3.3 22.8 TM1751 endoglucanase -5.3 21.9 TM1752 endoglucanase -1.8 6.7 TM1760 frame shift -1.1 5.6 TM1763 translation elongation factor P -1.2 12.0 TM1772 hypothetical protein -1.1 7.6 TM1773 hypothetical protein -1.3 13.5 TM1774 cofactor-independent phosphoglycerate mutase -2.4 18.2 TM1776 ferric uptake regulation protein -1.4 12.4 TM1779 hypothetical protein 1.5 17.3 TM1782 N-acetyl-γ-glutamyl-phosphate reductase -1.7 16.9 TM1783 bifunctional ornithine acetyltransferase -1.1 9.4 TM1784 acetylglutamate kinase 1.6 21.1 TM1788 conserved hypothetical protein, GGDEF domain -1.2 8.3 TM1790 hypothetical protein -1.9 15.9 TM1793 hypothetical protein -1.6 18.9 TM1801 hypothetical protein -1.6 16.5 TM1802 hypothetical protein -1.5 16.6 TM1809 hypothetical protein -2.3 23.6 TM1814 hypothetical protein -1.2 8.7 TM1820 bifunctional GMP synthase/glutamine amidotransferase 1.7 6.3 TM1824 hypothetical protein -1.6 9.4 TM1829 hypothetical protein 1.0 11.7 TM1831 transposase, putative -1.9 18.3 TM1832 transposase -1.1 7.1 TM1834 α-glucosidase 1.4 14.7 TM1839 maltose ABC transporter, periplasmic maltose-binding 1.5 5.6 TM1842 hypothetical protein 1.2 22.4 TM1847 ROK family protein 1.6 11.9 TM1858 recX protein, putative -1.6 5.7 TM1860 hypothetical protein -1.1 9.7 TM1862 hypothetical protein -1.3 10.9 TM1863 hypothetical protein -1.0 6.6 TM1864 hypothetical protein -1.7 13.7 TM1866 membrane bound protein LytR, putative -2.3 24.9 TM1869 ATP-dependent protease LA, putative -2.4 17.9 TM1870 septum site-determining protein MinD -2.6 26.0 TM1871 hypothetical protein -2.0 12.8 TM1873 ornithine decarboxylase -1.9 24.8 TM1875 glutamyl-tRNA synthetase -1.5 17.0 TM1876 hypothetical protein -1.6 6.6 TMrrnaA16 TMrrnaA16 1.2 9.5

298

Table B5. ORFs differentially transcribed in the T. zoo vs. T. petrophila in polysaccharide culture. Positive values indicate up-regulation in the zoo, negative values indicate up-regulation in pure culture Fold P- ID Annotation change value TM_tRNA-Ala-1 TM_tRNA-Ala-1 -2.1 8.3 TM_tRNA-Arg-3 TM_tRNA-Arg-3 -2.2 9.3 TM_tRNA-Arg-4 TM_tRNA-Arg-4 -2.2 11.2 TM_tRNA-Lys-2 TM_tRNA-Lys-2 -2.2 7.3 TM0013 hypothetical protein 2.1 14.5 TM0022 DNA mismatch repair protein 2.2 14.9 TM0033 hypothetical protein -2.6 8.0 TM0044 hypothetical protein 2.6 16.0 TM0056 ABC transporter, periplasmic binding protein 2.6 21.4 TM0057 oligopeptide ABC transporter, ATP-binding protein 2.2 5.6 TM0058 oligopeptide ABC transporter, ATP-binding protein 3.0 6.3 TM0059 oligopeptide ABC transporter, permease protein -2.2 11.0 TM0061 endo-1,4-β-xylanase A 7.7 34.3 TM0062 hypothetical protein -2.4 10.1 TM0072 oligopeptide ABC transporter, permease protein -2.7 12.3 TM0075 oligopeptide ABC transporter, ATP-binding protein -2.1 10.8 TM0094 general secretion pathway protein F, putative 2.1 6.5 TM0107 hypothetical protein 2.4 17.9 TM0138 tryptophan synthase subunit β -2.4 5.7 TM0141 anthranilate synthase component II -2.1 10.2 TM0169 redox-sensing transcriptional repressor Rex -2.2 9.3 TM0170 hypothetical protein 3.2 17.1 TM0176 hypothetical protein -2.1 9.3 TM0180 hypothetical protein 2.9 19.9 TM0185 hypothetical protein 2.5 18.7 TM0194 ABC transporter, ATP-binding protein -2.1 8.2 TM0211 aminomethyltransferase -2.2 10.5 TM0212 glycine cleavage system H protein 2.7 20.0 TM0217 glycyl-tRNA synthetase, β subunit -2.5 12.3 TM0226 hypothetical protein -2.3 9.9 TM0227 frame shift 2.0 13.4 TM0229 hypothetical protein -2.2 13.3 TM0260 hypothetical protein -2.1 8.0 TM0268 5-methyltetrahydrofolate S-homocysteine methyltransferase -2.3 12.0 TM0297 oxidoreductase, short chain dehydrogenase/reductase family 2.1 9.7 TM0302 oligopeptide ABC transporter, permease protein -2.1 13.0 TM0323 frame shift 2.3 14.1 TM0335 dihydroorotase -2.5 9.8 TM0345 3-phosphoshikimate 1-carboxyvinyltransferase -2.1 10.9 TM0369 hypothetical protein 3.2 11.7 TM0373 dnaK protein 4.0 17.0 TM0394 hypothetical protein -2.3 17.3 TM0422 hypothetical protein -2.1 20.3

299

Table B5 continued TM0430 sugar ABC transporter, permease protein -2.0 10.2 TM0431 sugar ABC transporter, permease protein -2.1 7.2 TM0433 pectate lyase 2.1 7.4 TM0454 ribosomal protein L11 2.4 17.1 TM0459 DNA-directed RNA polymerase, β' subunit 2.0 9.7 TM0460 ABC transporter, periplasmic binding protein 2.2 7.0 TM0519 hypothetical protein -2.5 11.4 TM0525 tRNA delta-2-isopentenylpyrophosphate transferase -2.3 7.2 TM0539 tryptophan synthase subunit β -2.7 16.3 TM0550 ketol-acid reductoisomerase -2.1 8.7 TM0562 hypothetical protein -2.2 11.1 TM0579 hypothetical protein -2.2 8.9 TM0581 hypothetical protein -2.1 11.4 TM0650 hypothetical protein -2.2 10.4 TM0653 hypothetical protein -2.0 11.0 TM0663 hypothetical protein -2.1 9.7 TM0691 hypothetical protein -2.5 12.5 TM0694 trigger factor, putative -2.5 12.2 TM0719 cysteinyl-tRNA synthetase -2.4 9.8 TM0752 α-glucosidase, putative -2.8 18.7 TM0764 hypothetical protein -2.0 9.0 TM0768 hypothetical protein -2.2 13.4 TM0779 hypothetical protein 3.5 26.3 TM0790 hypothetical protein 2.7 22.0 TM0793 ABC transporter, ATP-binding protein -2.1 10.7 TM0808 transcriptional regulator, XylR-related -2.1 8.0 TM0823 transcriptional regulator, TetR family 2.2 14.1 TM0824 astB/chuR-related protein 2.6 12.7 TM0836 cell division protein FtsZ -2.2 13.0 TM0841 S-layer-like array protein -2.2 6.7 TM0857 riboflavin kinase/FMN adenylyltransferase -2.3 13.1 TM0891 gcpE protein -2.3 9.2 TM0924 hypothetical protein -2.2 10.4 TM0926 chromosomal replication initiator protein 3.0 10.8 TM0935 hypothetical protein -2.1 12.5 TM0941 hypothetical protein -2.3 11.6 TM0943 glutamine synthetase -2.1 6.1 TM0945 hypothetical protein -2.3 9.4 TM0950 hypothetical protein -2.2 10.5 TM0986 hypothetical protein -2.1 13.0 TM0988 hypothetical protein -2.0 7.2 TM0989 hypothetical protein -2.0 10.9 TM1027 hypothetical protein -2.0 8.4 TM1035 phosphoribosyl-AMP cyclohydrolase -2.1 19.5 TM1068 α-glucosidase, putative -2.0 7.1 TM1069 transcriptional regulator, DeoR family -2.4 7.7

300

Table B5 continued TM1073 sugar kinase 2.1 11.2 TM1098 hypothetical protein -2.2 11.4 TM1166 oxygen-independent coproporphyrinogen III oxidase, putative -2.3 13.2 TM1176 transcriptional regulator, metal-sensing -2.5 14.2 TM1178 acetyltransferase-related protein 2.1 12.1 TM1190 galactokinase -2.5 8.9 TM1199 ABC transporter, periplasmic binding protein 4.2 27.1 TM1204 maltose ABC transporter, periplasmic maltose-binding protein 2.3 9.4 TM1206 putative monovalent cation/H+ antiporter subunit F -2.1 8.3 TM1222 ABC transporter, permease protein -2.0 10.8 TM1226 ABC transporter, periplasmic binding protein 7.1 13.6 TM1227 endo-1,4-β-mannosidase 2.1 18.6 TM1243 phosphoribosylaminoimidazole-succinocarboxamide synthase -4.5 24.7 TM1252 hypothetical protein -2.2 5.9 TM1261 phosphate ABC transporter, ATP-binding protein -2.2 12.2 TM1262 phosphate ABC transporter, permease protein -2.2 11.9 TM1265 hypothetical protein -2.0 12.5 TM1271 type IV pilin-related protein 2.2 11.1 TM1276 sugar ABC transporter, ATP-binding protein -2.0 10.2 TM1286 methyltransferase 4.5 28.5 TM1294 hypothetical protein 2.7 16.7 TM1388 hypothetical protein -2.1 9.0 TM1402 hypothetical protein -2.2 11.4 TM1405 lipopolysaccharide biosynthesis protein-related protein -2.2 7.2 TM1406 hypothetical protein -2.1 6.7 TM1428 methyl-accepting chemotaxis protein 2.7 22.7 TM1429 glycerol uptake facilitator protein -2.1 8.5 TM1432 hypothetical protein -2.5 16.1 TM1433 oxidoreductase, putative -2.4 13.0 TM1465 cob(I)yrinic acid a,c-diamide adenosyltransferase 2.4 12.8 TM1469 glucokinase -2.4 7.4 TM1472 DNA-directed RNA polymerase subunit α -2.2 11.7 TM1495 ribosomal protein L22 2.0 8.8 TM1519 2,3,4,5-THP-2-carboxylate N-succinyltransferase -2.2 9.5 TM1527 hypothetical protein -2.5 20.6 TM1529 hypothetical protein -2.1 11.8 TM1531 electron transfer flavoprotein, α subunit -2.2 12.7 TM1532 fixC protein -2.9 21.5 TM1536 hypothetical protein -2.3 10.2 TM1550 hypothetical protein -2.1 11.0 TM1572 signal peptidase I, putative 2.1 17.9 TM1576 hemolysin 2.1 11.6 TM1588 conserved hypothetical protein, GGDEF domain 2.3 6.9 TM1590 translation initiation factor IF-3 2.6 10.9 TM1600 hypothetical protein -2.4 14.0 TM1643 L-aspartate dehydrogenase 4.9 22.2 TM1647 hypothetical protein -2.3 10.2

301

Table B5 continued TM1648 hypothetical protein -2.4 15.0 TM1656 hypothetical protein -2.3 8.8 TM1703 hypothetical protein -2.2 9.2 TM1705 lysyl-tRNA synthetase -2.2 10.7 TM1715 hypothetical protein -2.4 12.3 TM1716 hypothetical protein -2.0 12.4 TM1722 hypothetical protein 2.8 8.7 TM1732 hypothetical protein -3.7 17.1 TM1746 ABC transporter, periplasmic binding protein 5.2 21.5 TM1749 ABC transporter, ATP-binding protein 2.5 14.7 TM1750 ABC transporter, ATP-binding protein 2.6 13.0 TM1751 endoglucanase 8.3 16.9 TM1759 2-ketoisovalerate ferredoxin reductase -2.2 13.0 TM1761 excinuclease ABC subunit B -2.3 11.6 TM1779 hypothetical protein -3.7 24.0 TM1783 ornithine acetyltransferase/N-acetylglutamate synthase -2.1 11.3 TM1788 conserved hypothetical protein, GGDEF domain -2.0 9.0 TM1809 hypothetical protein 2.6 18.2 TM1814 hypothetical protein -2.0 8.8 TM1861 CDP-diacylglycerol--glycerol-3-P 3-phosphatidyltransferase -2.4 10.1 Tpet_0298 hypothetical protein -2.5 8.5 Tpet_0635 binding-protein-dependent transport membrane component -2.1 7.0 Tpet_0636 extracellular solute-binding protein, family 1 -3.1 13.9 Tpet_1618 hypothetical protein -2.7 8.5 Tpet_1619 hypothetical protein -2.2 7.7 Tpet_1735 hypothetical protein -3.4 17.9 Tpet_1736 hypothetical protein -3.5 17.1 Tpet_1751 phosphoadenosine phosphosulfate reductase -10.6 26.3 Tpet_1753 hypothetical protein -4.3 13.5 Tpet_1754 hypothetical protein -5.3 16.2 Tpet_1768 hypothetical protein 2.2 9.7 Tpet_1772 Adenine-specific DNA methylase containing a Zn-ribbon-like -4.2 14.3 Tpet_1773 hypothetical protein -3.0 15.3 Tpet_1775 PglZ domain -5.3 18.8 Tpet_1776 ATPase (AAA+ superfamily)-like protein -2.1 10.0 Tpet_1792 ABC transporter related -2.4 12.9 Tpet_1794 Monosaccharide-transporting ATPase -12.1 22.6

302

Table B6. ORFs differentially transcribed in the T. zoo vs. T. sp RQ2 in polysaccharide culture. Positive values indicate up-regulation in the zoo, negative values indicate up-regulation in pure culture Fold P- Probe ID Annotation Change value 2A6 TRQ2_0641 1-phosphofructokinase 2.0 12.1 2C2 TRQ2_1109 metallophosphoesterase 2.1 12.0 2H5 TRQ2_0512 extracellular solute-binding protein family 1 2.1 8.8 3F3 TRQ2_0970 extracellular solute-binding protein family 1 -6.9 23.8 TRQ2_0666 hypothetical, A12 TRQ2_0667 LamG domain protein jellyroll fold domain -2.8 17.2 GAPDH TRQ2_0241 glyceraldehyde-3-P dehydrogenase, type I 2.6 11.1 NTPE TRQ2_1103 H+ transporting two-sector ATPase E subunit 2.2 11.3 ORF12 TRQ2_0662 glycoside hydrolase family 43 -2.1 9.9 ORF13 TRQ2_0663 hypothetical -3.8 22.2 ORF14 TRQ2_0664 glycoside hydrolase family 43 -2.7 18.4 ORF6 TRQ2_0298 hypothetical 2.2 7.6 TAA11 TRQ2_1557 binding-dependent transport membrane component 2.0 7.2 TAA19 TRQ2_0662 glycoside hydrolase family 43 -2.0 8.6 TAA49 TRQ2_1833 hypothetical 2.2 13.5 TAA96 TRQ2_0503 hypothetical 2.3 14.3 TAB05 TRQ2_0973 LacI transcriptional regulator 2.0 11.2 TAB07 TRQ2_0664 glycoside hydrolase family 43 -2.0 11.9 TRQ2_0522 iron-containing alcohol dehydrogenase, TAB17 TRQ2_0524 hypothetical 2.6 11.2 TAC01 TRQ2_0657 hypothetical -2.8 15.4 TRQ2_0662 glycoside hydrolase family 43, TAC44 TRQ2_0663 hypothetical -3.6 17.6 TRQ2_0665 hypothetical, TAC83 TRQ2_0666 hypothetical -4.3 24.0 TRQ2_0414 hypothetical, TAC91, TRQ2_0415 heat shock protein Hs1VU, ATPase subunit Hs1U 2.2 12.7 TAD02 TRQ2_1840 pseudouridine synthase, RluA family 2.3 14.0 TRQ2_0658 α-N-arabinofuranosidase, TAD18 TRQ2_0659 binding-dependent transport membrane component -2.9 12.9 TAD19 TRQ2_0973 LacI transcriptional regulator -2.2 16.7 TRQ2_1106 V-type ATPase 116 kDa subunit, TAD58 TRQ2_1107 H+transporting two-sector ATPase C subunit 2.5 13.2 TAD72 TRQ2_0284 lipopolysaccharide biosynthesis protein 2.2 11.6 TAE01 TRQ2_1645 metal dependent phosphohydrolase 2.1 8.3 TAE11 TRQ2_0666 hypothetical -2.1 7.8 TAE30 TRQ2_0666 hypothetical -2.4 15.2 TAE46 TRQ2_0660 binding dependent transport membrane component -2.3 12.3 TAE55 TRQ2_0611 TRAP dicarboxylate transporter, DctP subunit 2.9 15.8 TAE56 TRQ2_0283 NAD+ synthetase 2.4 14.5 TRQ2_0666 hypothetical, TAE65 TRQ2_0667 LamG domain protein jellyroll fold domain -3.0 15.3 TAE82 TRQ2_0658 α-N-arabinofuranosidase 2.1 8.8 TAF15 TRQ2_0664 glycoside hydrolase family 43 -2.0 8.3

303

Table B6 continued TM_tRNA-Ala-1 TM_tRNA-Arg-3 2.4 10.4 TM_tRNA-Lys-2 TM_tRNA-Lys-2 2.3 8.1 TM0007 hypothetical protein 2.1 6.0 TM0033 hypothetical protein 2.6 8.3 TM0062 hypothetical protein -3.2 13.8 TM0101 hypothetical protein 2.3 8.7 TM0117 hypothetical protein -2.4 10.6 TM0127 sensor histidine kinase 2.4 13.7 TM0138 tryptophan synthase subunit β 3.0 8.0 TM0141 anthranilate synthase component II 2.2 11.0 TM0143 response regulator 2.3 5.6 TM0169 redox-sensing transcriptional repressor Rex 2.1 8.4 TM0180 hypothetical protein -2.4 17.1 TM0186 response regulator 2.3 8.8 TM0194 ABC transporter, ATP-binding protein 2.5 10.4 TM0206 hypoxanthine phosphoribosyltransferase -2.1 17.8 TM0211 aminomethyltransferase 2.6 12.6 TM0217 glycyl-tRNA synthetase, β subunit 2.6 12.8 TM0229 hypothetical protein 2.3 13.9 TM0253 hypothetical protein -2.5 9.1 TM0268 5-methyltetrahydrofolate S-homocysteine methyltransferase 2.4 12.8 TM0283 L-ribulose-5-phosphate 4-epimerase -2.1 14.7 TM0302 oligopeptide ABC transporter, permease protein 2.1 12.9 TM0316 hypothetical protein -2.3 16.5 TM0328 m4C-methyltransferase 2.2 12.1 TM0335 dihydroorotase 2.4 9.1 TM0337 hypothetical protein 2.2 15.2 TM0345 3-phosphoshikimate 1-carboxyvinyltransferase 2.3 12.1 TM0350 hypothetical protein 2.2 12.7 TM0376 hypothetical protein -2.2 13.1 TM0392 hypothetical protein -5.8 20.9 TM0393 transcriptional regulator, XylR-related -2.8 11.3 TM0403 nitrogen regulatory protein P-II -2.6 13.1 TM0431 ABC transporter, permease protein -2.4 8.8 TM0432 ABC transporter, periplasmic sugar-binding protein -2.8 16.9 TM0433 pectate lyase -2.5 9.4 TM0467 regulatory protein, putative 2.2 13.3 TM0481 hypothetical protein 2.4 13.4 TM0493 hypothetical protein 2.1 9.9 TM0509 UDP-glucose 4-epimerase, putative 2.1 7.2 TM0519 hypothetical protein 2.3 10.5 TM0525 tRNA delta-2-isopentenylpyrophosphate transferase 2.9 9.9 TM0531 ABC transporter, periplasmic binding protein -4.0 30.4 TM0539 tryptophan synthase subunit β 2.3 13.9 TM0550 ketol-acid reductoisomerase 2.5 11.1 TM0565 sugar fermentation stimulation protein, putative 2.6 15.2

304

Table B6 continued TM0570 cell division protein FtsY 2.6 8.4 TM0608 hypothetical protein -2.3 15.4 TM0622 lipopolysaccharide biosynthesis protein, putative -9.1 26.6 TM0627 lipopolysaccharide biosynthesis protein -3.4 22.2 TM0628 hypothetical protein -2.3 7.9 TM0630 nucleotide sugar epimerase, putative -6.1 16.3 TM0631 lipopolysaccharide biosynthesis protein -3.5 18.5 TM0639 hypothetical protein 2.1 11.6 TM0650 hypothetical protein 2.2 10.2 TM0653 hypothetical protein 2.7 15.5 TM0663 hypothetical protein 2.6 13.0 TM0674 flagellar protein, putative 2.1 12.0 TM0691 hypothetical protein 2.1 9.9 TM0694 trigger factor, putative 2.1 9.9 TM0719 cysteinyl-tRNA synthetase 2.9 11.8 TM0734 tRNA (uracil-5-)-methyltransferase Gid 2.4 6.9 TM0761 hypothetical protein 2.3 14.4 TM0764 hypothetical protein 2.1 9.3 TM0768 hypothetical protein 2.0 11.6 TM0770 hypothetical protein 2.8 11.3 TM0784 hypothetical protein 2.1 10.1 TM0788 thiamine biosynthesis protein ThiC 2.5 10.0 TM0801 (3R)-hydroxymyristoyl-(acyl carrier protein) dehydratase -2.0 14.5 TM0803 CTP synthetase 2.5 11.5 TM0808 transcriptional regulator, XylR-related 2.3 9.5 TM0816 transcriptional regulator, putative, Mar family 2.5 11.7 TM0839 rod shape-determining protein RodA -2.7 11.2 TM0841 S-layer-like array protein 2.6 8.9 TM0843 formiminotransferase-cyclodeaminase 2.5 11.9 TM0857 riboflavin kinase/FMN adenylyltransferase 2.7 15.7 TM0879 ferredoxin -2.0 14.5 TM0880 oxaloacetate decarboxylase, β subunit 2.7 12.2 TM0881 homoserine O-succinyltransferase 2.3 9.6 TM0891 gcpE protein 2.4 9.8 TM0894 hypothetical protein 2.1 15.3 TM0898 hypothetical protein -2.3 15.2 TM0917 phosphate permease, putative 2.1 13.5 TM0924 hypothetical protein 2.3 10.6 TM0929 hypothetical protein 2.1 7.3 TM0932 hypothetical protein -2.9 16.4 TM0933 hypothetical protein 2.1 6.8 TM0941 hypothetical protein 2.8 14.3 TM0942 hypothetical protein 2.4 12.5 TM0943 glutamine synthetase 2.7 8.9 TM0945 hypothetical protein 2.7 11.3 TM0949 transcriptional regulator, LacI family -5.4 15.0 TM0950 hypothetical protein 2.2 10.4

305

Table B6 continued TM0961 lemA protein -2.9 16.3 TM0972 conserved hypothetical protein, GGDEF domain 2.4 7.3 TM0984 hypothetical protein 2.6 14.7 TM0986 hypothetical protein 2.6 16.4 TM0988 hypothetical protein 2.7 10.8 TM1020 hypothetical protein 2.0 7.2 TM1035 phosphoribosyl-AMP cyclohydrolase 2.1 18.5 TM1069 transcriptional regulator, DeoR family -2.0 5.9 TM1098 hypothetical protein 2.3 12.2 TM1107 hypothetical protein -3.8 19.4 TM1134 hypothetical protein 2.4 18.7 TM1143 methyl-accepting chemotaxis protein -2.1 12.5 TM1166 oxygen-independent coproporphyrinogen III oxidase, putative -2.7 15.9 TM1171 transcriptional regulator, crp family 2.3 10.2 TM1174 hypothetical protein -4.8 24.7 TM1190 galactokinase 2.0 6.6 TM1195 β-galactosidase -2.0 6.8 TM1199 ABC transporter, periplasmic binding protein -3.5 24.8 TM1222 oligopeptide ABC transporter, permease protein 2.1 11.0 TM1231 α-mannosidase-related protein 2.4 12.5 TM1240 translation-associated GTPase 2.5 8.5 TM1247 amidophosphoribosyltransferase 2.4 12.2 TM1255 aspartate aminotransferase 2.3 8.9 TM1261 phosphate ABC transporter, ATP-binding protein 2.1 11.5 TM1271 type IV pilin-related protein -3.9 18.7 TM1272 glutamyl tRNA-Gln amidotransferase, subunit A -2.1 17.0 TM1276 sugar ABC transporter, ATP-binding protein 2.3 12.3 TM1293 hypothetical protein 2.1 10.5 TM1297 oxidoreductase, putative -2.0 18.6 TM1299 hypothetical protein 2.1 14.0 TM1312 hypothetical protein 2.1 15.0 TM1359 sensor histidine kinase 2.4 13.4 TM1375 ABC transporter, periplasmic binding protein 2.2 13.7 TM1388 hypothetical protein 2.3 10.0 TM1391 ATP-dependent Clp protease, ATPase subunit 2.3 10.3 TM1395 hypothetical protein 2.1 12.4 TM1402 hypothetical protein 2.2 11.3 TM1405 lipopolysaccharide biosynthesis protein-related protein 3.0 10.5 TM1432 hypothetical protein 2.1 12.9 TM1433 oxidoreductase, putative 2.1 11.0 TM1469 glucokinase 2.2 6.6 TM1472 DNA-directed RNA polymerase subunit α 2.5 13.7 TM1474 30S ribosomal protein S11 -2.0 10.1 TM1485 50S ribosomal protein L6 -2.1 17.2 TM1492 ribosomal protein L29 -2.1 18.2 TM1500 ribosomal protein L3 -2.1 14.4 TM1516 hydrolase, ama/hipO/hyuC family 2.3 9.1

306

Table B6 continued TM1522 diaminopimelate epimerase -2.4 14.6 TM1536 hypothetical protein 2.2 9.7 TM1550 hypothetical protein 2.6 14.6 TM1558 hypothetical protein -2.1 7.0 TM1574 pseudouridylate synthase I 2.3 7.6 TM1597 hypothetical protein 2.2 8.0 TM1600 hypothetical protein 2.1 11.5 TM1602 transcriptional regulator, biotin repressor family 2.2 10.9 TM1607 hypothetical protein -2.5 12.6 TM1615 ATP synthase F0, subunit c -2.1 16.4 TM1629 UDP-N-acetylglucosamine pyrophosphorylase 2.5 12.5 TM1645 nicotinate-nucleotide pyrophosphorylase -2.3 17.4 TM1647 hypothetical protein 2.4 10.9 TM1648 hypothetical protein 2.6 16.2 TM1650 α-amylase, putative 2.3 10.3 TM1661 polypeptide deformylase -2.2 13.8 TM1667 xylose isomerase -2.3 18.2 TM1676 hypothetical protein 2.3 18.8 TM1696 type IV prepilin peptidase 2.0 9.0 TM1703 hypothetical protein 2.7 12.1 TM1705 lysyl-tRNA synthetase 2.1 9.4 TM1715 hypothetical protein 2.1 10.1 TM1732 hypothetical protein -2.2 10.3 TM1747 oligopeptide ABC transporter, permease protein 2.0 5.7 TM1756 butyrate kinase 2.3 12.6 TM1759 2-ketoisovalerate ferredoxin reductase 2.3 13.1 TM1761 excinuclease ABC subunit B 2.1 9.9 TM1779 hypothetical protein -4.4 26.3 TM1783 ornithine acetyltransferase/N-acetylglutamate synthase protein 2.4 13.6 TM1807 hypothetical protein -2.7 14.6 TM1809 hypothetical protein -2.4 17.1 TM1814 hypothetical protein 2.4 11.0 TM1842 hypothetical protein -2.4 26.4 TM1847 ROK family protein -2.4 11.5 TM1856 transcriptional regulator, LacI family 2.4 8.5 TM1861 CDP-diacylglycerol--glycerol-3-P 3-phosphatidyltransferase 2.1 8.2 TM1872 hypothetical protein -2.1 12.4 TM1874 cold shock protein 2.1 10.8 TXX2 TRQ2_0658 α-N-arabinofuranosidase -2.7 11.7 TRQ2_0659 binding-dependent transport membrane component, TXX3 TRQ2_0660 binding-dependent transport membrane component -2.2 7.1 TRQ2_0659 binding-dependent transport membrane component, TXX5 TRQ2_0660 binding-dependent transport membrane component -2.2 6.2 WZX TRQ2_0307 polysaccharide biosynthesis protein 2.1 15.6

307

Table B7. ORFs differentially transcribed in the T. zoo vs. T. maritima in glucose culture. Positive values indicate up-regulation in the zoo, negative values indicate up-regulation in the pure culture

Fold P- Probe ID Annotation Change value TM_rnpB TM_rnpB -2.9 14.5 TM_tRNA-Arg-3 TM_tRNA-Arg-3 -2.9 11.9 TM_tRNA-Arg-4 TM_tRNA-Arg-4 -3.1 9.5 TM_tRNA-Glu-2 TM_tRNA-Glu-2 -2.1 9.7 TM_tRNA-Gly-2 TM_tRNA-Gly-2 -2.4 9.9 TM_tRNA-Ile-1 TM_tRNA-Ile-1 -3.6 15.1 TM_tRNA-Leu-4 TM_tRNA-Leu-4 2.0 7.2 TM_tRNA-Lys-2 TM_tRNA-Lys-2 -3.0 10.5 TM_tRNA-Met-2 TM_tRNA-Met-2 3.2 11.6 TM_tRNA-Ser-1 TM_tRNA-Ser-1 3.1 8.7 TM_tRNA-Ser-2 TM_tRNA-Ser-2 3.3 9.5 TM_tRNA-Ser-3 TM_tRNA-Ser-3 3.1 8.0 TM_tRNA-Thr-1 TM_tRNA-Thr-1 -2.1 10.6 TM_tRNA-Thr-3 TM_tRNA-Thr-3 -4.6 22.1 TM0008 hypothetical protein 2.9 9.3 TM0011 NADP-reducing hydrogenase, subunit B 2.8 10.0 TM0012 NADP-reducing hydrogenase, subunit A 4.9 18.3 TM0013 hypothetical protein 2.8 8.2 TM0015 pyruvate ferredoxin oxidoreductase, γ subunit 2.5 6.8 TM0017 pyruvate ferredoxin oxidoreductase, α subunit 2.7 14.9 TM0019 3-ketoacyl-(acyl-carrier-protein) reductase 7.2 20.9 TM0020 hypothetical protein 5.8 20.1 TM0021 hypothetical protein 2.6 5.6 TM0022 DNA mismatch repair protein 2.2 10.6 TM0023 methyl-accepting chemotaxis protein 12.5 15.0 TM0024 laminarinase 5.6 17.2 TM0025 β-glucosidase 4.6 13.7 TM0028 oligopeptide ABC transporter, ATP-binding protein 4.4 16.6 TM0030 oligopeptide ABC transporter, permease protein 7.7 19.9 TM0033 hypothetical protein -2.9 8.2 TM0037 hypothetical protein 6.5 18.5 TM0039 hypothetical protein -2.8 13.0 TM0044 hypothetical protein -2.1 8.0 TM0045 hypothetical protein 5.1 21.2 TM0052 hypothetical protein 2.2 18.4 TM0059 ABC transporter, permease protein -3.3 10.7 TM0065 transcriptional regulator, IclR family 3.7 17.4 TM0071 ABC transporter, periplasmic binding protein 2.9 17.1 TM0073 oligopeptide ABC transporter, permease protein 2.5 5.6 TM0082 flagellar hook-associated protein 3 3.9 11.2 TM0094 general secretion pathway protein F, putative 9.0 7.4 TM0096 hypothetical protein 4.1 23.7

308

Table B7 continued TM0104 sugar ABC transporter, permease protein 2.9 9.8 TM0108 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 8.7 20.3 TM0109 pyruvate formate lyase activating enzyme, putative 2.4 20.0 TM0112 sugar ABC transporter, permease protein 2.1 10.5 TM0113 xylU-related protein 2.7 12.0 TM0114 sugar ABC transporter, periplasmic sugar-binding protein 3.5 13.6 TM0120 oxidoreductase, putative 9.3 22.3 TM0123 zinc ABC transporter, periplasmic zinc-binding protein -2.0 9.1 TM0126 response regulator 2.7 14.0 TM0128 oxaloacetate decarboxylase 3.6 20.3 TM0129 carboxypeptidase G2, putative 7.2 14.7 TM0130 hypothetical protein 5.0 22.5 TM0133 isochorismatase-related protein -2.0 7.7 TM0134 thioredoxin reductase-related protein 4.1 12.1 TM0136 hypothetical protein -2.5 6.8 TM0138 tryptophan synthase subunit β -3.2 14.4 TM0141 anthranilate synthase component II -3.4 15.9 TM0144 hypothetical protein 6.6 15.9 TM0145 secreted metalloendopeptidase Gcp, putative 2.1 7.4 TM0148 glucosamine-fructose-6-P aminotransferase, isomerizing 3.8 23.0 TM0149 fatty acid/phospholipid synthesis protein 4.7 25.6 TM0152 hypothetical protein 2.4 9.9 TM0159 ham1 protein 2.1 12.6 TM0160 hypothetical protein 7.4 11.1 TM0163 hypothetical protein 2.9 11.0 TM0164 hypothetical protein 4.9 20.1 TM0165 Holliday junction DNA helicase 3.5 12.8 TM0167 phosphopentomutase 5.8 14.3 TM0169 redox-sensing transcriptional repressor Rex -2.8 11.8 TM0170 hypothetical protein 15.2 24.6 TM0175 acyl carrier protein 7.0 20.4 TM0176 hypothetical protein -2.5 12.4 TM0178 primosomal protein N' 3.9 19.5 TM0181 hypothetical protein 2.0 12.1 TM0182 hypothetical protein 2.3 15.8 TM0190 iron(III) ABC transporter, permease protein, putative -2.3 5.7 TM0194 ABC transporter, ATP-binding protein -2.6 9.7 TM0196 hypothetical protein -3.1 20.4 TM0200 hypothetical protein 4.8 10.9 TM0201 NADP-reducing hydrogenase, subunit D, putative 6.2 23.4 TM0202 hypothetical protein 9.6 22.8 TM0205 ATP-dependent DNA helicase 5.3 17.9 TM0207 hypothetical protein 17.1 23.9 TM0208 pyruvate kinase 2.1 7.6 TM0209 6-phosphofructokinase 2.3 10.0 TM0211 aminomethyltransferase -2.9 12.6

309

Table B7 continued TM0216 glycyl-tRNA synthetase subunit α 2.0 9.2 TM0220 flagellar motor switch protein G 2.3 9.6 TM0223 hypothetical protein -2.6 14.7 TM0226 hypothetical protein -2.4 7.5 TM0229 hypothetical protein -3.4 15.1 TM0234 UDP-N-acetylmuramoylalanine--D-glutamate ligase 2.1 14.8 TM0235 phospho-N-acetylmuramoyl-pentapeptide-transferase 2.1 8.5 TM0237 ligase -2.5 5.7 TM0238 hypothetical protein 2.1 12.6 TM0239 glucose-1-phosphate adenylyltransferase 2.4 7.0 TM0240 glucose-1-phosphate adenylyltransferase 4.3 9.5 TM0246 hypothetical protein 5.1 14.0 TM0250 DNA processing chain A 2.4 8.4 TM0253 hypothetical protein -6.7 6.4 TM0254 SsrA-binding protein 6.4 17.4 TM0255 ribosomal protein L28 -6.5 17.8 TM0256 hypothetical protein 2.7 17.1 TM0257 frame shift -3.2 19.4 TM0258 DNA topoisomerase 6.0 22.6 TM0260 hypothetical protein -2.1 11.0 TM0261 phosphate permease, putative 9.0 21.0 TM0264 16S pseudouridylate synthase 7.8 18.6 TM0268 5-methyltetrahydrofolate S-homocysteine methyltransferase -2.9 15.9 TM0271 hypothetical protein 3.0 9.6 TM0275 transcriptional regulator, GntR family 2.4 10.2 TM0282 aldose 1-epimerase 5.3 27.5 TM0283 L-ribulose-5-phosphate 4-epimerase 3.6 14.5 TM0285 araM protein, putative 3.1 9.2 TM0291 3-isopropylmalate dehydratase large subunit -2.1 12.0 TM0292 3-isopropylmalate dehydratase small subunit 3.7 15.9 TM0297 oxidoreductase, short chain dehydrogenase/reductase family 2.4 7.2 TM0302 oligopeptide ABC transporter, permease protein -2.8 12.1 TM0306 α-L-fucosidase, putative 2.8 15.4 TM0307 L-fucose isomerase, putative 2.0 7.5 TM0309 ABC transporter, periplasmic binding protein 2.7 9.2 TM0310 β-D-galactosidase 3.0 13.6 TM0313 K+ channel, β subunit -2.3 14.3 TM0314 hypothetical protein -3.2 19.8 TM0316 hypothetical protein 3.3 18.6 TM0318 ubiquinone/menaquinone biosynthesis-related protein 3.0 20.9 TM0321 hypothetical protein 5.8 25.8 TM0328 m4C-methyltransferase -2.8 13.6 TM0329 hypothetical protein 9.7 20.1 TM0331 orotate phosphoribosyltransferase 3.3 12.9 TM0332 orotidine 5'-phosphate decarboxylase, putative 9.6 21.0 TM0335 dihydroorotase -2.5 8.9 TM0337 hypothetical protein -2.4 9.6

310

Table B7 continued TM0338 hypothetical protein 4.6 14.9 TM0340 hypothetical protein 6.5 14.8 TM0342 permease, putative -3.3 9.2 TM0344 prephenate dehydrogenase 3.0 9.7 TM0345 3-phosphoshikimate 1-carboxyvinyltransferase -3.2 12.4 TM0349 3-dehydroquinate dehydratase 8.0 13.1 TM0351 hypothetical protein 3.7 12.6 TM0354 hypothetical protein 2.3 10.5 TM0355 hypothetical protein 2.7 12.5 TM0358 hypothetical protein 4.4 13.2 TM0359 hypothetical protein -3.2 6.2 TM0360 mazG-related protein -2.8 22.6 TM0361 hypothetical protein -26.3 20.4 TM0365 putative aminopeptidase 1 2.9 14.4 TM0369 hypothetical protein 2.2 7.1 TM0373 dnaK protein -9.9 26.5 TM0379 NADH oxidase -2.5 8.4 TM0383 NADH oxidoreductase, putative -2.2 7.3 TM0384 anaerobic ribonucleoside-triphosphate reductase 4.1 10.9 TM0392 hypothetical protein -43.1 34.9 TM0393 transcriptional regulator, XylR-related -9.3 25.1 TM0394 hypothetical protein -3.3 11.0 TM0395 NADH oxidase, putative -3.3 14.1 TM0399 response regulator 3.6 16.2 TM0400 sensor histidine kinase 2.9 14.8 TM0401 thymidine kinase 10.3 21.6 TM0402 ammonium transporter -4.7 19.7 TM0403 nitrogen regulatory protein P-II -10.1 26.6 TM0405 2-oxoglutarate ferredoxin oxidoreductase subunit β 8.9 25.5 TM0407 diacylglycerol kinase, putative 10.2 18.2 TM0409 hypothetical protein 5.1 15.4 TM0410 hypothetical protein 5.3 15.7 TM0411 transcriptional regulator, XylR-related -2.1 7.0 TM0415 hypothetical protein 3.5 8.0 TM0418 sugar ABC transporter, periplasmic binding protein -3.1 16.6 TM0419 sugar ABC transporter, permease protein -2.8 15.0 TM0420 sugar ABC transporter, permease protein 3.7 13.7 TM0421 sugar ABC transporter, ATP-binding protein -7.7 22.0 TM0425 oxidoreductase, putative 2.6 17.1 TM0426 PHT4-related protein 2.1 14.1 TM0429 methyl-accepting chemotaxis protein 4.2 7.4 TM0430 sugar ABC transporter, permease protein -3.5 9.2 TM0431 sugar ABC transporter, permease protein -3.2 15.0 TM0432 sugar ABC transporter, periplasmic sugar-binding protein -4.3 17.2 TM0433 pectate lyase 2.0 10.6 TM0434 α-glucosidase, putative -8.6 24.0 TM0435 acetyl xylan esterase-related protein -2.2 6.8

311

Table B7 continued TM0437 exo-poly-α-D-galacturonosidase, putative 3.5 16.6 TM0438 6-phosphogluconate dehydrogenase 5.3 21.2 TM0448 hypothetical protein -7.2 10.3 TM0450 hypothetical protein 4.9 15.3 TM0451 ribosomal protein L33 5.9 18.5 TM0453 N utilization substance protein G 3.4 15.1 TM0454 ribosomal protein L11 3.6 21.6 TM0457 ribosomal protein L7/L12 17.9 24.6 TM0459 DNA-directed RNA polymerase, β' subunit 2.8 10.0 TM0463 lipoprotein signal peptidase 5.3 18.4 TM0465 hypothetical protein 11.0 22.3 TM0466 hypothetical protein 2.7 9.4 TM0468 response regulator 2.2 5.8 TM0469 hypothetical protein -2.0 5.7 TM0471 hypothetical protein 3.3 17.9 TM0476 hypothetical protein -22.0 23.6 TM0481 hypothetical protein -2.1 7.5 TM0484 pyrimidine precursor biosynthesis enzyme, putative 2.4 12.0 TM0486 hypothetical protein 2.8 17.3 TM0489 hypothetical protein 6.6 18.3 TM0493 hypothetical protein -2.2 12.4 TM0495 phoH-related protein -3.4 17.2 TM0499 hypothetical protein 2.4 8.0 TM0500 oligopeptide ABC transporter, ATP-binding protein 4.7 14.7 TM0501 oligopeptide ABC transporter, ATP-binding protein 3.4 13.7 TM0502 oligopeptide ABC transporter, permease protein 3.1 16.3 TM0505 groES protein -2.2 5.7 TM0507 hypothetical protein 2.3 7.6 TM0508 recombination factor protein RarA 3.2 18.0 TM0509 UDP-glucose 4-epimerase, putative -2.0 6.3 TM0511 hypothetical protein 2.3 15.3 TM0512 hypothetical protein 7.1 16.7 TM0514 prolyl-tRNA synthetase -3.4 14.8 TM0516 clostripain-related protein -2.1 11.0 TM0519 hypothetical protein -2.9 10.8 TM0523 hypothetical protein 4.2 16.4 TM0524 hypothetical protein 2.7 6.0 TM0525 tRNA delta-2-isopentenylpyrophosphate transferase -2.7 8.8 TM0529 heavy metal binding protein 2.9 5.7 TM0531 ABC transporter, periplasmic binding protein -3.3 25.1 TM0535 hypothetical protein -2.2 12.6 TM0539 tryptophan synthase subunit β -2.4 6.8 TM0541 fumarate hydratase, C-terminal subunit 9.1 22.3 TM0543 hypothetical protein -3.2 15.7 TM0544 ABC transporter, ATP-binding protein -2.4 8.8 TM0550 ketol-acid reductoisomerase -3.0 9.3 TM0551 dihydroxy-acid dehydratase -2.2 9.0

312

Table B7 continued TM0554 3-isopropylmalate dehydratase, large subunit -3.2 6.5 TM0559 hypothetical protein 3.7 19.1 TM0560 hypothetical protein -8.1 23.3 TM0561 divalent cation transport-related protein -3.1 13.2 TM0562 hypothetical protein -2.5 9.8 TM0564 hypothetical protein 2.3 14.3 TM0565 sugar fermentation stimulation protein, putative -2.7 7.9 TM0566 hypothetical protein 11.3 26.6 TM0567 hypothetical protein -2.8 13.7 TM0568 hypothetical protein 2.2 16.0 TM0569 hypothetical protein 3.7 19.1 TM0571 heat shock serine protease, periplasmic 6.2 20.0 TM0577 hypothetical protein 2.7 15.2 TM0579 hypothetical protein -2.4 8.6 TM0581 hypothetical protein -3.7 7.4 TM0590 penicillin-binding protein 2 2.1 6.9 TM0591 amino acid ABC transporter, ATP-binding protein -2.5 10.9 TM0592 amino acid ABC transporter, permease protein 2.3 6.6 TM0593 amino acid ABC transporter, periplasmic binding protein -4.1 19.0 TM0594 hypothetical protein -2.9 12.7 TM0595 sugar ABC transporter, periplasmic sugar-binding protein 4.6 8.9 TM0598 sugar ABC transporter, permease protein 2.3 15.1 TM0600 hypothetical protein 2.0 6.5 TM0602 iron-dependent transcriptional repressor, putative -3.7 11.4 TM0604 single stranded DNA-binding protein, putative 3.4 16.6 TM0607 hypothetical protein -3.0 12.8 TM0608 hypothetical protein 2.1 6.2 TM0616 hypothetical protein -9.3 23.5 TM0619 hypothetical protein -2.6 10.2 TM0620 lipopolysaccharide biosynthesis protein -2.3 11.5 TM0622 lipopolysaccharide biosynthesis protein, putative -71.1 29.5 TM0623 hypothetical protein 3.3 20.0 TM0624 N-acetylglucosaminyl-phosphatidylinositol biosynthesis -4.1 11.6 TM0625 hypothetical protein -3.0 8.8 TM0626 hypothetical protein 6.1 14.9 TM0627 lipopolysaccharide biosynthesis protein -47.5 34.0 TM0628 hypothetical protein -4.9 20.4 TM0630 nucleotide sugar epimerase, putative -29.8 26.1 TM0631 lipopolysaccharide biosynthesis protein -13.1 25.7 TM0633 flagellar-related protein -6.9 21.9 TM0640 hypothetical protein 7.8 20.8 TM0643 clostripain-related protein -4.5 14.7 TM0644 hypothetical protein -27.7 17.1 TM0648 hypothetical protein -3.8 12.0 TM0649 hypothetical protein 2.7 9.8 TM0650 hypothetical protein -2.6 11.5 TM0652 hypothetical protein 3.6 13.6

313

Table B7 continued TM0653 hypothetical protein -3.6 12.0 TM0654 spermidine synthase -10.1 12.8 TM0659 rubredoxin 3.4 16.9 TM0663 hypothetical protein -3.0 9.7 TM0665 cysteine synthase -17.1 26.0 TM0667 hypothetical protein 2.9 9.7 TM0668 pleiotropic regulatory protein 2.2 7.9 TM0669 hypothetical protein 9.0 22.7 TM0674 flagellar protein, putative -2.9 9.4 TM0676 motility protein B -2.3 12.7 TM0680 chemotaxis protein 3.1 10.8 TM0681 dehydrase-related protein 8.7 17.9 TM0683 hypothetical protein 2.2 6.4 TM0690 hypothetical protein 4.9 19.2 TM0691 hypothetical protein -3.1 12.6 TM0694 trigger factor, putative -3.3 14.1 TM0697 flagellar biosynthesis protein FliQ -3.8 14.2 TM0698 flagellar biosynthesis protein FliP 3.4 14.2 TM0700 chemotaxis response regulator CheY 2.3 6.7 TM0703 competence-damage inducible protein, putative 4.6 7.5 TM0705 ABC transporter, ATP-binding protein 6.5 25.8 TM0707 glucose-inhibited division protein B 2.7 11.2 TM0709 hypothetical protein -3.1 13.2 TM0710 transcriptional regulator, MarR family 4.8 17.5 TM0714 hypothetical protein -6.1 16.4 TM0715 tRNA nucleotidyl transferase-related protein 2.9 12.4 TM0717 propionyl-CoA carboxylase, γ subunit -6.1 20.6 TM0719 cysteinyl-tRNA synthetase -2.2 8.1 TM0727 pmbA-related protein 3.3 12.4 TM0729 (p)ppGpp synthetase 2.3 13.4 TM0732 hypothetical protein 2.6 15.0 TM0736 mannose-6-phosphate isomerase 2.2 12.4 TM0744 hypothetical protein 6.1 20.6 TM0746 hypothetical protein -3.5 19.5 TM0752 α-glucosidase, putative -2.2 14.6 TM0753 ubiquinone/menaquinone biosynthesis methyltransferase -2.3 5.7 TM0757 hypothetical protein -2.1 9.7 TM0764 hypothetical protein -2.9 11.4 TM0767 maltodextrin glycosyltransferase -2.3 11.7 TM0768 hypothetical protein -3.1 13.2 TM0770 hypothetical protein -4.0 16.2 TM0772 hypothetical protein 2.3 7.2 TM0774 hypothetical protein 2.3 9.8 TM0775 translation initiation factor IF-2 3.9 24.0 TM0778 hypothetical protein 5.2 18.9 TM0779 hypothetical protein 5.6 16.2 TM0780 bacterioferritin comigratory protein, ahpC/TSA family 4.8 16.3

314

Table B7 continued TM0781 hypothetical protein 2.2 10.0 TM0785 bacteriocin 3.0 14.0 TM0787 putative thiazole biosynthetic enzyme -2.8 9.9 TM0788 thiamine biosynthesis protein ThiC -3.3 12.2 TM0789 hypothetical protein 5.8 11.8 TM0790 hypothetical protein -7.3 13.0 TM0791 7-cyano-7-deazaguanine reductase 3.7 11.3 TM0793 ABC transporter, ATP-binding protein 7.1 14.7 TM0797 2-phosphosulfolactate phosphatase 3.1 13.1 TM0799 bioY protein -2.6 11.6 TM0801 (3R)-hydroxymyristoyl-(acyl carrier protein) dehydratase -3.1 10.8 TM0804 hypothetical protein 7.6 21.6 TM0813 hypothetical protein 4.1 20.4 TM0816 transcriptional regulator, putative, Mar family -2.4 9.2 TM0819 uracil permease 2.4 12.1 TM0823 transcriptional regulator, TetR family 4.9 17.5 TM0824 astB/chuR-related protein 2.1 16.3 TM0827 ABC transporter, ATP-binding protein, putative 2.9 7.9 TM0828 sugar kinase, pfkB family 4.0 14.8 TM0829 hypothetical protein 2.5 16.6 TM0831 branched-chain amino acid aminotransferase, putative 2.5 11.1 TM0832 hypothetical protein 3.4 8.8 TM0833 DNA gyrase, subunit B 4.0 10.4 TM0834 hypothetical protein 3.0 11.9 TM0835 cell division protein FtsA, putative 4.8 17.8 TM0836 cell division protein FtsZ 4.8 16.3 TM0838 hypothetical protein 2.3 10.1 TM0841 S-layer-like array protein -3.4 14.7 TM0842 response regulator 6.5 18.0 TM0843 formiminotransferase-cyclodeaminase -2.7 8.1 TM0849 dnaJ protein -3.1 8.9 TM0851 heat shock operon repressor HrcA -26.1 29.9 TM0853 sensor histidine kinase 5.4 16.9 TM0857 riboflavin kinase/FMN adenylyltransferase -4.1 13.0 TM0858 hypothetical protein 2.6 8.8 TM0860 protein-export membrane protein SecD, putative 2.0 7.3 TM0863 ribosomal protein L9 -6.4 25.1 TM0865 hypothetical protein 4.1 15.0 TM0868 glutaredoxin-related protein -2.9 12.2 TM0870 penicillin-binding protein 2 2.4 11.9 TM0872 hypothetical protein 3.8 20.5 TM0873 Frame Shift -12.6 26.9 TM0876 hypothetical protein 6.9 23.0 TM0877 enolase 5.6 18.8 TM0879 ferredoxin -3.1 18.4 TM0880 oxaloacetate decarboxylase, β subunit -3.2 8.0 TM0881 homoserine O-succinyltransferase -4.0 15.6

315

Table B7 continued TM0887 methylated-DNA-protein-cysteine methyltransferase 3.5 13.7 TM0888 hypothetical protein 2.6 12.5 TM0891 gcpE protein -2.8 8.7 TM0894 hypothetical protein -2.2 9.3 TM0895 glycogen synthase 3.3 13.4 TM0896 galactose-1-phosphate uridylyltransferase, putative 2.8 13.4 TM0898 hypothetical protein -2.5 11.2 TM0900 hypothetical protein -2.6 9.7 TM0901 hypothetical protein -2.8 12.2 TM0903 chemotaxis methylation protein 2.2 7.9 TM0904 chemotaxis protein CheC 3.0 14.2 TM0905 hypothetical protein 14.6 27.7 TM0911 translation initiation factor, aIF-2B α subunit-related 2.6 12.2 TM0912 basic membrane protein, putative 3.5 10.0 TM0916 hypothetical protein 2.3 6.2 TM0917 phosphate permease, putative -2.1 11.0 TM0919 hypothetical protein 2.7 10.3 TM0923 hypothetical protein 5.8 26.3 TM0924 hypothetical protein -2.5 9.4 TM0926 chromosomal replication initiator protein 2.2 13.5 TM0932 hypothetical protein -35.7 24.0 TM0934 hypothetical protein -2.2 8.8 TM0935 hypothetical protein -4.4 21.4 TM0936 hypothetical protein -2.1 6.8 TM0937 hypothetical protein 2.4 6.6 TM0938 hypothetical protein -5.5 15.0 TM0939 aspartate α-decarboxylase -2.0 6.5 TM0941 hypothetical protein -3.1 17.9 TM0942 hypothetical protein -4.1 11.9 TM0943 glutamine synthetase -3.7 12.2 TM0944 hypothetical protein -4.3 26.9 TM0945 hypothetical protein -2.6 7.8 TM0947 hypothetical protein 2.1 7.4 TM0950 hypothetical protein -3.2 11.3 TM0951 hypothetical protein -4.8 7.5 TM0952 glycerol kinase -3.6 11.3 TM0953 transketolase, C-terminal subunit -3.5 14.8 TM0955 ribose ABC transporter, permease protein -3.9 17.1 TM0956 ribose ABC transporter, ATP-binding protein -7.3 27.4 TM0963 oligoendopeptidase, putative -3.7 13.7 TM0966 hypothetical protein 2.3 11.0 TM0968 hypothetical protein 3.7 14.3 TM0972 conserved hypothetical protein, GGDEF domain -2.5 7.3 TM0973 methyl-accepting chemoreceptor-related protein -2.9 11.5 TM0975 hypothetical protein -4.7 9.2 TM0977 hypothetical protein -28.3 21.0 TM0978 hypothetical protein -4.9 17.8

316

Table B7 continued TM0982 hypothetical protein 4.6 12.8 TM0984 hypothetical protein -3.6 10.5 TM0986 hypothetical protein -3.4 10.7 TM0988 hypothetical protein -3.0 15.0 TM0989 hypothetical protein -2.4 10.6 TM0992 hypothetical protein -3.7 10.5 TM0993 hypothetical protein -4.8 17.5 TM0998 heavy metal resistance transcriptional regulator -2.9 13.9 TM1001 hypothetical protein 3.6 16.8 TM1002 hypothetical protein 2.2 9.6 TM1003 transposase-related protein 2.6 17.2 TM1012 hypothetical protein -2.5 18.0 TM1015 glutamate dehydrogenase -2.9 8.5 TM1016 hypothetical protein 4.7 15.5 TM1017 hypothetical protein -2.6 12.3 TM1018 hypothetical protein -2.2 8.9 TM1027 hypothetical protein -2.3 9.0 TM1028 ABC transporter, ATP-binding protein 3.2 13.6 TM1035 phosphoribosyl-AMP cyclohydrolase -2.8 21.5 TM1042 ATP phosphoribosyltransferase 4.1 22.9 TM1044 transposase, IS605-TnpB family 2.5 10.5 TM1047 septum site-determining protein MinC, putative 3.5 9.2 TM1048 endoglucanase 3.2 10.5 TM1049 endoglucanase 2.4 7.5 TM1057 potassium channel, putative 4.1 21.5 TM1058 glutamate synthase-related protein -2.8 15.9 TM1062 β-glucuronidase -2.0 10.8 TM1064 ABC transporter, ATP-binding protein -3.8 12.4 TM1067 ABC transporter, periplasmic binding protein -2.5 7.0 TM1068 α-glucosidase, putative -10.7 29.7 TM1069 transcriptional regulator, DeoR family -6.5 19.1 TM1072 rhamnulose-1-phosphate aldolase 5.4 13.2 TM1073 sugar kinase 6.7 11.4 TM1082 lexA repressor 2.7 12.6 TM1083 hypothetical protein -2.5 11.2 TM1084 DNA gyrase, subunit A 3.9 24.2 TM1085 methionyl-tRNA synthetase 2.8 7.3 TM1087 hypothetical protein 3.1 13.1 TM1088 TRK system potassium uptake protein TrkA -2.3 9.8 TM1091 hypothetical protein -2.6 15.9 TM1092 hypothetical protein 4.8 11.6 TM1098 hypothetical protein -3.1 7.3 TM1102 ribonuclease III -6.9 15.4 TM1112 hypothetical protein 3.4 11.6 TM1115 hypothetical protein -2.2 10.3 TM1117 general secretion pathway protein D, putative -3.2 9.5 TM1119 hypothetical protein -5.5 20.1

317

Table B7 continued TM1123 flagellar hook-associated protein 2, putative -8.3 11.9 TM1124 hypothetical protein -11.5 22.8 TM1125 hypothetical protein 2.5 12.5 TM1126 hypothetical protein -5.1 19.7 TM1129 5-methylthioadenosine 3.5 17.4 TM1133 hypothetical protein 2.9 15.1 TM1142 hypothetical protein 6.0 17.3 TM1143 methyl-accepting chemotaxis protein -5.6 22.0 TM1145 hypothetical protein -4.3 14.7 TM1148 isocitrate dehydrogenase -3.4 12.2 TM1151 oligopeptide ABC transporter, ATP-binding protein -2.7 10.0 TM1154 oxidoreductase, sol/devB family 2.0 10.2 TM1157 hypothetical protein 2.6 14.5 TM1163 conserved hypothetical protein, GGDEF domain 2.7 5.6 TM1164 2-oxoacid ferredoxin oxidoreductase, α subunit 2.9 9.9 TM1165 2-oxoacid ferredoxin oxidoreductase subunit β 3.4 17.6 TM1166 oxygen-independent coproporphyrinogen III oxidase, -3.9 6.8 TM1168 frame shift -10.9 24.3 TM1169 3-oxoacyl-(acyl carrier protein) reductase -2.8 14.1 TM1171 transcriptional regulator, crp family -3.0 11.8 TM1176 transcriptional regulator, metal-sensing -13.3 25.9 TM1177 hypothetical protein -3.5 14.2 TM1180 hypothetical protein 2.9 15.3 TM1183 oxidoreductase, aldo/keto reductase family 2.6 11.0 TM1185 methylglyoxal synthase 2.5 10.0 TM1186 hypothetical protein 3.7 12.1 TM1188 hypothetical protein -2.0 7.0 TM1190 galactokinase -2.6 11.1 TM1192 α-galactosidase 3.2 14.1 TM1193 β-galactosidase 2.2 8.4 TM1194 ABC transporter, ATP-binding protein 2.4 15.8 TM1199 ABC transporter, periplasmic binding protein 2.1 15.5 TM1200 transcriptional regulator, LacI family 4.7 20.2 TM1201 arabinogalactan endo-1,4-β-galactosidase, putative 2.2 13.6 TM1202 maltose ABC transporter, permease protein 4.6 19.0 TM1203 maltose ABC transporter, permease protein 2.1 6.4 TM1204 maltose ABC transporter, maltose-binding protein 9.5 26.0 TM1206 putative monovalent cation/H+ antiporter subunit F -2.8 8.0 TM1207 putative monovalent cation/H+ antiporter subunit G -4.2 15.2 TM1208 hypothetical protein 3.2 18.8 TM1211 NADH dehydrogenase, putative -2.1 9.6 TM1214 NADH dehydrogenase subunit B 6.8 16.1 TM1215 NADH dehydrogenase, 30 kDa subunit, putative 2.5 13.7 TM1222 ABC transporter, permease protein -2.1 6.4 TM1223 ABC transporter, periplasmic binding protein 4.5 14.7 TM1224 transcriptional regulator, XylR-related 5.8 24.5 TM1225 hypothetical protein -4.1 11.1

318

Table B7 continued TM1228 transcriptional regulator, RpiR family 2.9 13.9 TM1231 α-mannosidase-related protein -2.1 10.0 TM1232 sugar ABC transporter, ATP-binding protein -2.7 12.7 TM1239 hypothetical protein 2.3 5.5 TM1241 hypothetical protein -2.4 11.3 TM1243 phosphoribosylaminoimidazole-succinocarboxamide synthase 3.2 24.3 TM1246 phosphoribosylformylglycinamidine synthase II 9.8 18.8 TM1247 amidophosphoribosyltransferase -2.5 12.3 TM1248 phosphoribosylglycinamide formyltransferase 8.2 26.3 TM1249 IMP cyclohydrolase -2.8 7.4 TM1252 hypothetical protein -3.4 14.1 TM1255 aspartate aminotransferase -2.8 10.8 TM1256 ABC transporter, ATP-binding protein 2.9 14.8 TM1259 phosphate regulon transcriptional regulatory protein PhoB -2.8 13.6 TM1260 phosphate transport system regulator PhoU -4.3 20.2 TM1261 phosphate ABC transporter, ATP-binding protein -2.8 12.3 TM1262 phosphate ABC transporter, permease protein -4.2 16.6 TM1265 hypothetical protein -3.5 16.9 TM1269 biotin synthase -3.7 15.4 TM1272 glutamyl tRNA-Gln amidotransferase, subunit A -19.2 21.5 TM1273 aspartyl/glutamyl-tRNA amidotransferase subunit B -5.0 19.6 TM1276 sugar ABC transporter, ATP-binding protein -2.8 13.8 TM1280 hypothetical protein 3.3 13.3 TM1281 6-phospho-β-glucosidase 15.5 26.3 TM1283 hypothetical protein 3.0 13.9 TM1284 oxidase-related protein 2.9 22.0 TM1287 hypothetical protein 5.3 15.9 TM1293 hypothetical protein -2.9 8.3 TM1294 hypothetical protein 4.0 19.6 TM1299 hypothetical protein -2.6 10.4 TM1300 hypothetical protein 8.0 24.6 TM1301 astB/chuR-related protein -2.1 7.2 TM1302 ABC transporter, ATP-binding protein -2.2 9.6 TM1310 ABC transporter, ATP-binding protein -2.7 7.1 TM1311 HicB antitoxin, purative 2.6 9.7 TM1313 HicB antitoxin, purative -2.1 12.0 TM1320 HicA toxin, putative 8.9 19.4 TM1321 HicB antitoxin, purative 11.5 21.0 TM1329 hypothetical protein -2.4 21.9 TM1332 hypothetical protein -12.0 13.9 TM1336 permease, putative -2.8 9.4 TM1337 hypothetical protein -2.4 13.6 TM1343 Frame Shift 3.5 16.6 TM1351 glutamyl-tRNA synthetase 2.4 12.2 TM1354 inosine-5-monophosphate dehydrogenase-related protein 2.4 9.3 TM1356 hypothetical protein 2.3 6.3 TM1359 sensor histidine kinase -2.9 9.5

319

Table B7 continued TM1365 flagellar basal-body rod protein FlgC 2.0 5.7 TM1366 flagellar hook-basal body complex protein FliE -2.0 5.5 TM1368 ABC transporter, ATP-binding protein -2.5 11.3 TM1369 hypothetical protein 4.8 11.1 TM1371 aminotransferase, class V 3.0 13.7 TM1373 hypothetical protein 3.8 19.0 TM1375 ABC transporter, periplasmic binding protein 2.3 11.2 TM1379 seryl-tRNA synthetase 2.9 12.6 TM1382 hypothetical protein 6.5 12.3 TM1386 hypothetical protein -2.2 9.0 TM1388 hypothetical protein -2.9 11.2 TM1392 hypothetical protein 4.5 14.8 TM1393 hypothetical protein 2.7 8.2 TM1394 hypothetical protein -5.3 14.6 TM1397 phosphatidate cytidylyltransferase, putative 4.5 12.5 TM1398 hypothetical protein -19.6 27.2 TM1399 ribosome recycling factor 8.1 22.1 TM1401 D-3-phosphoglycerate dehydrogenase -3.6 6.7 TM1402 hypothetical protein -2.4 11.2 TM1405 lipopolysaccharide biosynthesis protein-related protein -2.7 12.8 TM1406 hypothetical protein -2.5 10.8 TM1420 hypothetical protein -2.5 6.0 TM1423 hypothetical protein 2.5 7.0 TM1424 Fe-hydrogenase, subunit γ -2.3 12.3 TM1426 Fe-hydrogenase, subunit α -2.7 10.3 TM1429 glycerol uptake facilitator protein -5.8 19.2 TM1430 glycerol kinase -3.4 6.3 TM1433 oxidoreductase, putative -2.6 12.1 TM1439 hypothetical protein 6.1 20.0 TM1443 cytidylate kinase 2.4 16.5 TM1446 hypothetical protein 2.5 14.8 TM1447 hypothetical protein 3.4 18.9 TM1449 hypothetical protein 2.2 7.0 TM1451 RNA polymerase sigma-A factor 2.8 11.6 TM1453 ribosomal protein S9 4.3 20.4 TM1454 50S ribosomal protein L13 3.3 17.7 TM1456 50S ribosomal protein L27 2.1 8.6 TM1457 hypothetical protein 5.9 10.0 TM1460 jag protein, putative 5.0 25.7 TM1464 hypothetical protein 5.9 26.2 TM1465 cob(I)yrinic acid a,c-diamide adenosyltransferase 5.5 13.7 TM1466 hypothetical protein 2.9 9.7 TM1469 glucokinase -2.5 8.4 TM1472 DNA-directed RNA polymerase subunit α -2.8 11.2 TM1473 30S ribosomal protein S4 6.5 20.8 TM1474 30S ribosomal protein S11 5.0 19.5 TM1475 30S ribosomal protein S13 6.1 21.9

320

Table B7 continued TM1477 translation initiation factor IF-1 3.5 18.1 TM1479 adenylate kinase 5.3 20.5 TM1481 ribosomal protein L15 2.8 19.2 TM1484 ribosomal protein L18 4.2 17.0 TM1488 50S ribosomal protein L5 8.2 23.6 TM1491 30S ribosomal protein S17 2.2 6.4 TM1495 ribosomal protein L22 3.5 13.3 TM1496 ribosomal protein S19 3.6 18.8 TM1499 50S ribosomal protein L4 -3.8 13.6 TM1501 ribosomal protein S10 5.0 17.6 TM1504 30S ribosomal protein S7 4.9 15.6 TM1505 30S ribosomal protein S12 3.0 19.7 TM1508 hypothetical protein 2.3 12.1 TM1516 hydrolase, ama/hipO/hyuC family -2.5 10.0 TM1519 2,3,4,5-THP-2-carboxylate N-succinyltransferase -3.0 13.4 TM1522 diaminopimelate epimerase 2.6 10.7 TM1527 hypothetical protein 2.8 17.3 TM1528 1,4-dihydroxy-2-naphthoate octaprenyltransferase, putative 7.6 22.4 TM1529 hypothetical protein 3.0 8.2 TM1530 electron transfer flavoprotein, β subunit 2.3 17.1 TM1533 ferredoxin 2.9 8.2 TM1536 hypothetical protein -2.4 10.8 TM1548 lipopolysaccharide biosynthesis protein 3.3 19.2 TM1550 hypothetical protein -2.6 10.6 TM1555 hypothetical protein 3.5 12.8 TM1556 maf protein 4.9 8.4 TM1557 DNA repair protein 5.6 8.0 TM1559 deoxyribose-phosphate aldolase 3.1 15.6 TM1563 hypothetical protein 5.2 20.4 TM1567 hypothetical protein 2.8 16.6 TM1568 16S rRNA processing protein, putative 2.4 17.8 TM1570 hypothetical protein 15.2 23.6 TM1572 signal peptidase I, putative 2.6 13.5 TM1574 pseudouridylate synthase I -2.3 12.1 TM1578 preprotein translocase subunit SecA 3.2 11.4 TM1580 transcriptional regulator, putative 4.7 21.1 TM1582 hypothetical protein 3.1 10.9 TM1584 comFC protein, putative 2.1 8.0 TM1586 hypothetical protein 2.1 12.3 TM1587 hypothetical protein 2.7 14.2 TM1589 clostripain-related protein -3.1 15.0 TM1591 ribosomal protein L35 3.7 7.2 TM1593 hypothetical protein -2.3 15.8 TM1594 conserved hypothetical protein, GGDEF domain 3.4 17.9 TM1597 hypothetical protein -2.7 13.9 TM1598 RNA polymerase sigma-E factor 2.0 7.0 TM1600 hypothetical protein -3.4 16.4

321

Table B7 continued TM1602 transcriptional regulator, biotin repressor family -2.0 7.5 TM1605 elongation factor Ts 5.0 19.2 TM1608 hypothetical protein -3.6 13.2 TM1609 ATP synthase F1, subunit epsilon 6.3 23.2 TM1613 ATP synthase F1, subunit delta -2.5 9.3 TM1618 cheX protein -5.0 17.8 TM1624 β-mannosidase, putative 11.0 23.8 TM1629 UDP-N-acetylglucosamine pyrophosphorylase -2.7 11.0 TM1632 hypothetical protein 5.4 14.1 TM1633 ATP-dependent protease LA 4.8 17.7 TM1636 hypothetical protein 4.2 14.0 TM1637 hypothetical protein -2.9 10.2 TM1643 L-aspartate dehydrogenase 2.8 8.1 TM1647 hypothetical protein -2.6 9.9 TM1648 hypothetical protein -2.9 6.4 TM1650 α-amylase, putative -2.4 7.0 TM1651 translation elongation factor G 2.9 12.8 TM1653 pyrimidine-nucleoside phosphorylase 2.7 11.2 TM1660 hypothetical protein 3.6 18.2 TM1662 stationary phase survival protein 2.3 11.4 TM1663 ABC transporter, ATP-binding protein 5.8 25.7 TM1664 hypothetical protein 2.9 6.6 TM1665 hypothetical protein 4.6 10.6 TM1671 hypothetical protein 4.6 13.0 TM1674 hypothetical protein -2.7 14.7 TM1676 hypothetical protein -2.5 6.9 TM1677 transposase, putative 2.1 6.8 TM1679 hypothetical protein 4.7 21.0 TM1680 hypothetical protein 3.2 8.6 TM1681 hypothetical protein 2.8 15.6 TM1684 ribosomal protein L31 10.6 24.0 TM1688 hypothetical protein 2.6 7.0 TM1691 hypothetical protein 3.6 24.5 TM1694 thiamin biosynthesis protein ThiI 3.7 17.3 TM1696 type IV prepilin peptidase -2.5 8.4 TM1697 hypothetical protein 3.3 10.9 TM1700 hypothetical protein -2.5 10.1 TM1701 hypothetical protein 4.0 18.0 TM1702 hypothetical protein 2.6 6.2 TM1703 hypothetical protein -3.0 14.2 TM1705 lysyl-tRNA synthetase -2.8 10.7 TM1706 transcription elongation factor, greA/greB family 3.2 14.1 TM1708 hypothetical protein 2.9 14.1 TM1709 hypothetical protein 6.8 14.1 TM1710 Frame Shift -4.3 19.3 TM1711 hypothetical protein 2.3 12.6 TM1712 hypothetical protein 2.8 10.9

322

Table B7 continued TM1713 proline dipeptidase, putative -2.7 9.8 TM1715 hypothetical protein -2.9 12.7 TM1728 3-methyl-2-oxobutanoate hydroxymethyltransferase 6.9 26.7 TM1729 outer membrane protein 2.1 8.4 TM1731 hypothetical protein -7.1 15.1 TM1732 hypothetical protein 3.4 18.1 TM1733 hypothetical protein 2.5 14.5 TM1739 hypothetical protein 4.8 13.4 TM1740 hypothetical protein 2.2 10.6 TM1742 nagD protein, putative 3.8 21.7 TM1743 oxidoreductase, aldo/keto reductase family 2.0 9.4 TM1746 ABC transporter, periplasmic binding protein 3.7 19.7 TM1747 oligopeptide ABC transporter, permease protein -2.8 11.7 TM1751 endoglucanase 3.2 15.8 TM1753 excinuclease ABC, subunit B-related protein 3.0 16.3 TM1756 butyrate kinase -3.0 9.4 TM1759 2-ketoisovalerate ferredoxin reductase -2.4 9.4 TM1761 excinuclease ABC subunit B -3.7 16.0 TM1762 transketolase -3.7 21.6 TM1764 hypothetical protein 2.9 14.6 TM1765 transcription antitermination protein NusB 2.9 8.5 TM1766 formate--tetrahydrofolate ligase 6.0 22.0 TM1768 exodeoxyribonuclease VII, large subunit 2.7 7.4 TM1771 hypothetical protein 2.7 8.2 TM1772 hypothetical protein 5.7 20.4 TM1773 hypothetical protein 4.7 13.4 TM1774 cofactor-independent phosphoglycerate mutase 4.9 9.8 TM1780 argininosuccinate synthase -3.1 14.1 TM1781 argininosuccinate lyase -3.0 9.7 TM1783 bifunctional ornithine acetyltransferase -2.4 6.9 TM1784 acetylglutamate kinase -2.8 14.1 TM1786 hypothetical protein -7.2 21.2 TM1788 conserved hypothetical protein, GGDEF domain -2.7 10.1 TM1790 hypothetical protein 3.2 10.9 TM1791 hypothetical protein 2.7 15.5 TM1792 hypothetical protein 4.4 14.6 TM1793 hypothetical protein 2.7 7.3 TM1794 hypothetical protein 5.1 18.6 TM1795 hypothetical protein -2.7 11.8 TM1796 hypothetical protein 3.2 13.2 TM1799 hypothetical protein 5.3 15.9 TM1802 hypothetical protein 3.2 11.0 TM1804 hypothetical protein 3.0 9.9 TM1806 hypothetical protein 7.7 19.5 TM1807 hypothetical protein -3.2 12.9 TM1811 hypothetical protein -2.6 8.7 TM1814 hypothetical protein -2.9 11.0

323

Table B7 continued TM1815 ferredoxin 2.7 26.0 TM1816 hypothetical protein 5.1 22.0 TM1817 valyl-tRNA synthetase 2.2 11.1 TM1822 ftsH protease activity modulator HflK -2.8 6.7 TM1826 3,4-dihydroxy-2-butanone 4-phosphate synthase -4.4 11.1 TM1827 riboflavin synthase subunit α 6.8 21.0 TM1828 riboflavin-specific deaminase -2.1 7.9 TM1829 hypothetical protein -3.2 18.0 TM1831 transposase, putative 4.0 12.1 TM1832 transposase 9.1 16.5 TM1834 α-glucosidase 5.6 20.8 TM1838 hypothetical protein -10.4 16.8 TM1841 hypothetical protein 4.9 25.1 TM1843 hypothetical protein 5.8 21.4 TM1844 hypothetical protein 14.6 29.2 TM1845 pullulanase 5.1 15.5 TM1848 cellobiose-phosphorylase -2.6 10.2 TM1851 α-mannosidase, putative -4.8 17.2 TM1852 hypothetical protein -3.9 19.0 TM1855 sugar ABC transporter, periplasmic sugar-binding protein, -7.9 22.1 TM1856 transcriptional regulator, LacI family -2.1 8.2 TM1857 hypothetical protein 2.3 15.3 TM1858 recX protein, putative 7.0 11.7 TM1859 recombinase A 2.5 15.0 TM1860 hypothetical protein 5.2 13.3 TM1861 CDP-diacylglycerol--glycerol-3-P 3-phosphatidyltransferase -3.1 10.2 TM1862 hypothetical protein 3.1 5.8 TM1863 hypothetical protein 12.0 25.5 TM1866 membrane bound protein LytR, putative 3.7 15.9 TM1869 ATP-dependent protease LA, putative 4.0 15.1 TM1871 hypothetical protein 11.0 21.0 TM1872 hypothetical protein 3.6 17.7 TM1873 ornithine decarboxylase 3.2 16.5 TM1875 glutamyl-tRNA synthetase 2.5 10.6 TM1876 hypothetical protein 6.5 18.2 TM1878 UDP-sugar hydrolase 6.4 11.0

324

Table B8. ORFs differentially transcribed in the T. zoo vs. T. neapolitana in glucose culture. Positive values indicate up-regulation in the zoo, negative values indicate up-regulation in the pure culture Fold P- Probe ID Annotation Change value CTN_0026 putative rhamnosyltransferase -6.1 13.5 CTN_0027 hypothetical protein -4.4 16.0 CTN_0028 glycosyl transferase, group 1 family protein -5.5 16.8 CTN_0029 hypothetical protein -5.7 18.1 CTN_0033 dTDP-glucose 4,6-dehydratase -9.0 17.5 CTN_0039a hypothetical protein -5.1 20.8 CTN_0046a hypothetical protein -4.7 22.2 CTN_0049a lipopolysaccharide biosynthesis protein -5.6 21.1 CTN_0050 HD domain protein -6.9 24.7 CTN_0062 conserved hypothetical protein -2.4 7.5 CTN_0073 hypothetical protein -4.0 20.1 CTN_0105 conserved domain protein -5.2 18.5 CTN_0109 hypothetical protein -5.1 14.9 CTN_0133 putative ATP/GTP-binding protein -6.6 19.5 CTN_0134 conserved hypothetical protein -5.6 33.7 CTN_0158 flagellar export protein FliJ -5.3 15.5 CTN_0192 preprotein translocase, SecG subunit -7.4 8.3 CTN_0236 TRAP transporter, DctQ-like membrane protein -5.9 16.7 CTN_0238 ATP-binding protein of sugar ABC transporter -3.4 12.7 CTN_0239 sugar ABC transporter permease -4.5 21.8 CTN_0241 spermidine/putrescine import ATP-binding protein PotA -4.4 16.8 CTN_0244 ABC transporter, permease protein 16.0 23.2 CTN_0245 ABC transporter periplasmic binding protein -6.7 14.0 CTN_0316 carboxymuconolactone decarboxylase -9.2 15.9 CTN_0318 glycerol-3-phosphate dehydrogenase -6.0 22.2 CTN_0321 conserved protein match to protein family -3.8 9.6 CTN_0323 chorismate synthase -2.4 8.8 CTN_0329a hypothetical protein -2.4 12.9 CTN_0345 probable transporter -2.3 12.0 CTN_0355 α-xylosidase -6.2 17.0 CTN_0357 binding-dependent transport inner membrane component -2.8 17.5 CTN_0358 sugar ABC transporter substrate-binding protein, putative -5.4 13.8 CTN_0360 hypothetical protein -6.7 21.5 CTN_0361 ribose transport ATP-binding protein RbsA -4.5 13.8 CTN_0362 xylose transport system permease protein XylH -3.2 9.6 CTN_0363 branched-chain amino acid transport permease component -5.2 16.6 CTN_0364 putative periplasmic binding protein -5.9 22.9 CTN_0365 oxidoreductase, short chain dehydrogenase/reductase family -5.9 17.3 CTN_0366 ribose transport ATP-binding protein RbsA -8.1 14.6 CTN_0367 ribose transport system permease protein RbsC -6.8 18.1 CTN_0368 hypothetical oxidoreductase YisS -6.3 20.0 CTN_0370 sugar kinase, fggy family -9.2 15.3

325

Table B8 continued CTN_0371 transcriptional regulator, GntR family -7.9 24.7 CTN_0372 succinate-semialdehyde dehydrogenase -13.3 22.7 CTN_0373 glucose-6-phosphate isomerase -8.5 20.7 CTN_0383 putative aldolase 2.1 7.8 CTN_0444 hypothetical protein 2.0 6.9 CTN_0500 sensor histidine kinase protein family HMM PF02518 -2.8 15.2 CTN_0545 putative membrane protein -5.9 24.2 CTN_0555 metallo-β-lactamase family protein 2.3 9.5 CTN_0590 putative membrane protein -5.5 19.8 CTN_0647 hypothetical protein -4.1 17.5 CTN_0660 ABC transporter -14.9 14.1 CTN_0667a oligopeptide ABC transporter, ATP-binding protein -5.8 25.4 CTN_0673 hypothetical protein -4.3 14.3 CTN_0690 conserved hypothetical protein -2.3 11.7 CTN_0710 CRISPR-associated regulatory protein, DevR family -3.3 10.4 CTN_0711 crispr-associated protein Cas5, tneap subtype -8.9 17.8 CTN_0712 CRISPR-associated helicase Cas3 -3.0 12.3 CTN_0713 CRISPR-associated protein Cas4 -2.2 14.4 CTN_0715 crispr-associated protein Cas2 -2.7 13.6 CTN_0778 ABC transporter, permease protein, MalFG family -2.1 11.5 CTN_0779 ABC transporter, permease protein, MalFG family -2.6 10.6 CTN_0781 α-amylase 2.2 9.7 CTN_0784 putative lipoprotein -3.9 16.8 CTN_0823 exonuclease, putative -4.0 15.2 CTN_0859 conserved hypothetical protein -5.3 15.8 CTN_0898 putative ATP-binding protein -7.0 18.6 CTN_0916 putative A-ATPase I-subunit -8.0 18.6 CTN_0917 membrane-associated ATPase C chain -5.8 16.0 CTN_0979 putative membrane protein -4.1 21.9 CTN_0991 translation elongation factor Tu -6.0 25.3 CTN_1016 translation initiation factor IF-1 -7.7 16.5 CTN_1024a conserved hypothetical protein -3.7 13.0 CTN_1056 glycosyl hydrolase, family 57 -6.6 17.5 CTN_1074 sugar nucleotidyltransferase -5.1 18.8 CTN_1080 putative membrane protein -4.2 9.3 CTN_1081 conserved hypothetical protein -4.8 19.6 CTN_1082a conserved hypothetical protein -8.4 7.3 CTN_1083 conserved hypothetical protein -7.8 19.6 CTN_1087 conserved hypothetical protein -7.6 7.8 CTN_1088 HD domain protein -4.6 18.6 CTN_1102a hypothetical protein -6.7 5.8 CTN_1105a hypothetical protein -3.7 8.1 CTN_1160 conserved hypothetical protein -6.8 15.9 CTN_1167 conserved hypothetical protein -7.0 10.4 CTN_1173 conserved hypothetical protein -2.6 17.8 CTN_1174 conserved hypothetical protein -4.4 12.4

326

Table B8 continued CTN_1175 hypothetical protein -5.0 22.0 CTN_1175a hypothetical protein -4.4 17.7 CTN_1183 argininosuccinate lyase -7.1 18.2 CTN_1233 conserved hypothetical protein -5.2 22.3 CTN_1259a hypothetical protein -3.1 15.5 CTN_1262 hypothetical protein -4.8 13.8 CTN_1263 hypothetical protein -7.1 21.2 CTN_1281a conserved hypothetical protein -3.0 18.6 CTN_1285 multidrug resistance protein -6.1 16.9 CTN_1372 putative extracellular solute-binding protein -10.9 23.8 CTN_1373 ABC transporter, permease protein -5.8 15.2 CTN_1374 permease -4.4 21.5 CTN_1387 ggdef domain protein -7.5 18.4 CTN_1388 putative membrane protein -5.5 18.7 CTN_1469 caax amino terminal protease family -3.8 20.8 CTN_1503 ABC transporter, periplasmic binding protein -5.6 14.6 CTN_1543 ABC transporter -4.1 11.0 CTN_1545 altronate hydrolase -7.6 20.7 CTN_1548 hypothetical protein -6.3 24.3 CTN_1549 conserved hypothetical protein -7.1 7.2 CTN_1552 putative lipoprotein -3.6 12.0 CTN_1553 von Willebrand factor type A domain protein -4.9 19.0 CTN_1574 ISTma3, transposase -2.9 16.7 CTN_1622 transketolase -6.1 23.4 CTN_1623 transketolase -7.2 23.9 CTN_1735 conserved hypothetical protein -7.0 22.9 CTN_1784 putative membrane protein -5.1 16.2 CTN_1796 BchE -5.7 19.4 CTN_1821 phosphoglycolate phosphatase, putative -7.5 20.4 CTN_1906 FliY/N -7.0 16.6 CTN_1912 FlgE -6.6 15.4 CTN_1915 hypothetical protein -7.4 8.8 CTN_1916 conserved hypothetical protein -6.7 27.8 CTN_1932 hypothetical protein -5.1 23.2 CTN_1933 general secretion pathway protein D, putative -4.9 19.6 CTN_1934 secretin and TonB N terminus short domain protein -4.4 12.6 CTN_1934a hypothetical protein -4.9 15.4 CTN_1935 hypothetical protein -4.2 8.7 TM_tRNA-Met-2 TM_tRNA-Met-2 2.4 10.6 TM_tRNA-Ser-2 TM_tRNA-Ser-2 2.1 6.8 TM_tRNA-Ser-3 TM_tRNA-Ser-3 2.5 8.1 TM0008 hypothetical protein 2.1 7.6 TM0011 NADP-reducing hydrogenase, subunit B 2.2 9.4 TM0013 hypothetical protein 2.4 8.4 TM0015 pyruvate ferredoxin oxidoreductase, γ subunit 2.1 6.5 TM0019 3-ketoacyl-(acyl-carrier-protein) reductase 3.0 15.1 TM0020 hypothetical protein 3.6 18.7

327

Table B8 continued TM0021 hypothetical protein 2.3 6.3 TM0022 DNA mismatch repair protein 2.5 15.1 TM0028 oligopeptide ABC transporter, ATP-binding protein 2.4 11.9 TM0035 hypothetical protein 3.0 6.3 TM0037 hypothetical protein 3.2 14.5 TM0045 hypothetical protein 2.5 15.3 TM0053 esterase, putative 3.1 13.8 TM0056 ABC transporter, periplasmic binding protein -3.8 24.9 TM0057 oligopeptide ABC transporter, ATP-binding protein -2.8 11.4 TM0061 endo-1,4-β-xylanase A -5.1 25.0 TM0062 hypothetical protein -4.3 23.2 TM0065 transcriptional regulator, IclR family 2.6 15.8 TM0071 ABC transporter, periplasmic binding protein -3.0 20.8 TM0075 oligopeptide ABC transporter, ATP-binding protein -2.3 13.9 TM0077 acetyl xylan esterase -2.4 15.4 TM0082 flagellar hook-associated protein 3 2.3 8.0 TM0094 general secretion pathway protein F, putative 6.0 7.1 TM0095 hypothetical protein 2.2 5.7 TM0096 hypothetical protein 2.1 16.3 TM0108 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 4.1 17.0 TM0120 oxidoreductase, putative 3.2 15.5 TM0129 carboxypeptidase G2, putative 2.8 9.2 TM0134 thioredoxin reductase-related protein 2.3 8.5 TM0144 hypothetical protein 3.4 12.8 TM0149 fatty acid/phospholipid synthesis protein 2.5 20.2 TM0152 hypothetical protein 2.4 12.0 TM0154 hypothetical protein 2.1 12.9 TM0164 hypothetical protein 2.2 12.7 TM0165 Holliday junction DNA helicase 2.1 8.7 TM0167 phosphopentomutase 2.8 9.9 TM0170 hypothetical protein 7.5 23.0 TM0175 acyl carrier protein 2.7 13.4 TM0180 hypothetical protein 4.2 25.9 TM0200 hypothetical protein 2.6 7.5 TM0205 ATP-dependent DNA helicase 2.8 14.1 TM0207 hypothetical protein 5.4 18.9 TM0212 glycine cleavage system H protein 4.6 23.8 TM0220 flagellar motor switch protein G 2.2 10.9 TM0227 Frame Shift 2.5 11.0 TM0240 glucose-1-phosphate adenylyltransferase 2.6 7.2 TM0246 hypothetical protein 2.3 8.2 TM0254 SsrA-binding protein 2.8 11.8 TM0258 DNA topoisomerase 3.6 20.5 TM0261 phosphate permease, putative 4.8 19.0 TM0264 16S pseudouridylate synthase 4.2 16.1 TM0271 hypothetical protein 2.2 7.9 TM0282 aldose 1-epimerase 2.1 18.3

328

Table B8 continued TM0290 citrate synthase 2.3 16.1 TM0292 3-isopropylmalate dehydratase small subunit 2.3 12.3 TM0309 ABC transporter, periplasmic oligopeptide-binding protein -2.3 9.7 TM0310 β-D-galactosidase -2.3 12.4 TM0313 K+ channel, β subunit -2.2 16.4 TM0318 ubiquinone/menaquinone biosynthesis-related protein 2.1 17.9 TM0322 ABC transporter, periplasmic substrate-binding protein -2.3 13.4 TM0329 hypothetical protein 4.6 17.1 TM0331 orotate phosphoribosyltransferase 2.3 11.0 TM0332 orotidine 5'-phosphate decarboxylase, putative 3.4 15.1 TM0338 hypothetical protein 3.0 13.6 TM0340 hypothetical protein 3.5 11.4 TM0349 3-dehydroquinate dehydratase 4.2 11.0 TM0357 hypothetical protein 2.2 8.2 TM0361 hypothetical protein -5.9 14.4 TM0365 putative aminopeptidase 1 2.4 14.5 TM0369 hypothetical protein 3.6 15.3 TM0376 hypothetical protein 2.4 9.1 TM0384 anaerobic ribonucleoside-triphosphate reductase-related protein 2.9 9.8 TM0400 sensor histidine kinase 2.3 14.5 TM0401 thymidine kinase 2.6 11.4 TM0403 nitrogen regulatory protein P-II -2.2 13.1 TM0405 2-oxoglutarate ferredoxin oxidoreductase subunit β 6.0 25.6 TM0407 diacylglycerol kinase, putative 5.7 17.0 TM0409 hypothetical protein 3.0 12.6 TM0410 hypothetical protein 4.3 15.7 TM0415 hypothetical protein 3.5 10.1 TM0420 sugar ABC transporter, permease protein 3.2 14.7 TM0425 oxidoreductase, putative 2.2 18.2 TM0434 α-glucosidase, putative -4.8 22.1 TM0437 exo-poly-α-D-galacturonosidase, putative 2.3 13.6 TM0438 6-phosphogluconate dehydrogenase 2.5 15.3 TM0448 hypothetical protein -3.5 7.6 TM0450 hypothetical protein 2.8 12.2 TM0451 ribosomal protein L33 5.3 20.7 TM0454 ribosomal protein L11 3.4 23.9 TM0457 ribosomal protein L7/L12 4.6 17.8 TM0459 DNA-directed RNA polymerase, β' subunit 2.2 9.3 TM0463 lipoprotein signal peptidase 2.5 12.7 TM0465 hypothetical protein 3.9 16.8 TM0466 hypothetical protein 2.3 9.8 TM0471 hypothetical protein 3.4 21.4 TM0476 hypothetical protein -7.1 19.6 TM0495 phoH-related protein -2.4 15.6 TM0507 hypothetical protein 2.3 10.3 TM0508 recombination factor protein RarA 2.1 14.3 TM0512 hypothetical protein 3.3 12.7

329

Table B8 continued TM0523 hypothetical protein 2.7 14.0 TM0531 ABC transporter, periplasmic binding protein -3.2 28.3 TM0540 fumarate hydratase, N-terminal subunit 2.1 6.2 TM0541 fumarate hydratase, C-terminal subunit 4.4 19.3 TM0560 hypothetical protein T -3.7 19.0 TM0567 hypothetical protein -2.1 11.6 TM0569 hypothetical protein 2.1 13.8 TM0571 heat shock serine protease, periplasmic 2.1 10.5 TM0590 penicillin-binding protein 2 2.6 11.9 TM0595 sugar ABC transporter, periplasmic sugar-binding protein 2.9 7.3 TM0598 sugar ABC transporter, permease protein 2.2 17.5 TM0604 single stranded DNA-binding protein, putative 2.7 16.5 TM0616 hypothetical protein -3.5 17.8 TM0623 hypothetical protein 2.1 16.0 TM0626 hypothetical protein 2.5 8.7 TM0640 hypothetical protein 4.1 18.2 TM0643 clostripain-related protein -2.1 8.5 TM0644 hypothetical protein -4.0 8.5 TM0652 hypothetical protein 3.1 14.7 TM0653 hypothetical protein -2.0 7.5 TM0654 spermidine synthase -3.3 7.5 TM0657 rubrerythrin 2.9 13.4 TM0659 rubredoxin 2.5 16.0 TM0665 cysteine synthase -2.1 9.4 TM0667 hypothetical protein 2.4 9.8 TM0669 hypothetical protein 2.8 14.4 TM0680 chemotaxis protein 2.1 8.2 TM0681 dehydrase-related protein 5.1 16.7 TM0683 hypothetical protein 2.0 7.7 TM0690 hypothetical protein 2.7 15.4 TM0698 flagellar biosynthesis protein FliP 2.2 11.0 TM0703 competence-damage inducible protein, putative 3.2 6.8 TM0705 ABC transporter, ATP-binding protein 3.3 21.8 TM0714 hypothetical protein -2.7 11.1 TM0717 propionyl-CoA carboxylase, γ subunit -3.4 17.7 TM0729 (p)ppGpp synthetase 2.6 18.4 TM0730 D-tyrosyl-tRNA deacylase 2.2 7.3 TM0731 hypothetical protein 2.6 8.3 TM0744 hypothetical protein 3.3 17.4 TM0752 α-glucosidase, putative -6.3 31.1 TM0770 hypothetical protein -2.1 10.2 TM0775 translation initiation factor IF-2 2.2 18.3 TM0778 hypothetical protein 3.4 17.4 TM0779 hypothetical protein 5.3 18.8 TM0780 bacterioferritin comigratory protein, ahpC/TSA family 3.1 14.5 TM0785 bacteriocin 2.2 12.1 TM0789 hypothetical protein 2.5 7.0

330

Table B8 continued TM0804 hypothetical protein 3.3 16.8 TM0813 hypothetical protein 2.5 17.1 TM0823 transcriptional regulator, TetR family 2.9 14.8 TM0827 ABC transporter, ATP-binding protein, putative 3.2 11.1 TM0828 sugar kinase, pfkB family 3.8 16.9 TM0829 hypothetical protein 2.5 19.5 TM0831 branched-chain amino acid aminotransferase, putative 3.4 17.8 TM0832 hypothetical protein 3.8 12.0 TM0833 DNA gyrase, subunit B 3.3 10.7 TM0834 hypothetical protein 2.3 10.7 TM0835 cell division protein FtsA, putative 2.9 15.2 TM0842 response regulator 2.5 11.1 TM0853 sensor histidine kinase 3.6 15.9 TM0857 riboflavin kinase/FMN adenylyltransferase -2.0 7.4 TM0858 hypothetical protein 2.2 8.6 TM0865 hypothetical protein 2.3 10.4 TM0872 hypothetical protein 3.2 21.2 TM0876 hypothetical protein 2.2 12.7 TM0877 enolase 2.6 13.1 TM0881 homoserine O-succinyltransferase -2.0 9.5 TM0887 methylated-DNA-protein-cysteine methyltransferase 2.4 11.7 TM0888 hypothetical protein 2.2 12.4 TM0895 glycogen synthase 3.0 15.0 TM0896 galactose-1-phosphate uridylyltransferase, putative 2.0 10.8 TM0904 chemotaxis protein CheC 2.9 16.8 TM0905 hypothetical protein 3.9 20.4 TM0912 basic membrane protein, putative 2.5 9.0 TM0923 hypothetical protein 2.2 16.6 TM0926 chromosomal replication initiator protein 2.0 15.2 TM0932 hypothetical protein -4.7 14.3 TM0937 hypothetical protein 2.2 7.4 TM0942 hypothetical protein -2.0 6.4 TM0944 hypothetical protein -2.3 21.6 TM0947 hypothetical protein 2.1 8.8 TM0953 transketolase, C-terminal subunit -2.2 11.4 TM0956 ribose ABC transporter, ATP-binding protein -3.3 22.3 TM0966 hypothetical protein 2.1 12.1 TM0968 hypothetical protein 2.2 10.8 TM0978 hypothetical protein -2.3 11.7 TM0982 hypothetical protein 2.8 10.3 TM0992 hypothetical protein -2.0 6.2 TM0993 hypothetical protein -2.7 13.7 TM1001 hypothetical protein 2.7 16.3 TM1016 hypothetical protein 2.5 11.0 TM1042 ATP phosphoribosyltransferase 2.7 20.2 TM1044 transposase, IS605-TnpB family 2.3 12.0 TM1047 septum site-determining protein MinC, putative 2.5 7.7

331

Table B8 continued TM1048 endoglucanase 3.5 14.1 TM1049 endoglucanase 2.1 7.6 TM1057 potassium channel, putative 2.2 15.7 TM1068 α-glucosidase, putative -5.8 28.1 TM1069 transcriptional regulator, DeoR family -4.1 18.0 TM1072 rhamnulose-1-phosphate aldolase 2.5 8.3 TM1077 pantoate--β-alanine ligase 2.5 16.0 TM1084 DNA gyrase, subunit A 2.4 20.8 TM1087 hypothetical protein 2.6 13.4 TM1092 hypothetical protein 2.0 5.5 TM1107 hypothetical protein 2.4 12.3 TM1112 hypothetical protein 3.0 12.7 TM1117 general secretion pathway protein D, putative -2.4 8.5 TM1119 hypothetical protein -2.5 13.9 TM1123 flagellar hook-associated protein 2, putative -3.7 8.6 TM1124 hypothetical protein -3.9 16.9 TM1126 hypothetical protein -2.4 13.9 TM1129 5-methylthioadenosine 2.1 13.2 TM1133 hypothetical protein 2.0 12.3 TM1142 hypothetical protein 2.4 10.6 TM1155 glucose-6-phosphate 1-dehydrogenase 2.1 17.2 TM1157 hypothetical protein 2.8 18.5 TM1165 2-oxoacid ferredoxin oxidoreductase subunit β 2.8 18.0 TM1168 frame shift -2.9 15.0 TM1208 hypothetical protein 2.2 15.9 TM1214 NADH dehydrogenase subunit B 4.9 16.7 TM1246 phosphoribosylformylglycinamidine synthase II 2.6 9.8 TM1248 phosphoribosylglycinamide formyltransferase 2.9 18.7 TM1256 ABC transporter, ATP-binding protein 2.1 12.8 TM1260 phosphate transport system regulator PhoU -2.1 13.0 TM1262 phosphate ABC transporter, permease protein -2.2 11.4 TM1269 biotin synthase -2.1 10.7 TM1272 glutamyl tRNA-Gln amidotransferase, subunit A -2.5 8.2 TM1281 6-phospho-β-glucosidase 2.3 11.4 TM1284 oxidase-related protein 2.4 22.2 TM1286 5-methyltetrahydropteroyltriglutamate methyltransferase 7.1 9.1 TM1287 hypothetical protein 2.5 10.9 TM1294 hypothetical protein 3.6 21.8 TM1300 Subtilosin A-like bacteriocin, putative 2.6 16.0 TM1312 HicA toxin 9.0 29.5 TM1320 HicA toxin 4.1 15.9 TM1321 HicB antitoxin 4.0 15.5 TM1332 hypothetical protein -3.1 7.0 TM1343 frame shift 2.4 14.3 TM1379 seryl-tRNA synthetase 2.2 11.7 TM1382 hypothetical protein 4.2 11.5 TM1393 hypothetical protein 3.9 14.3

332

Table B8 continued TM1394 hypothetical protein -2.0 6.9 TM1397 phosphatidate cytidylyltransferase, putative 3.1 12.0 TM1398 hypothetical protein -4.3 19.1 TM1399 ribosome recycling factor 2.5 13.1 TM1421 hydrogenase, putative 3.9 8.3 TM1423 hypothetical protein 2.3 7.7 TM1427 redox-sensing transcriptional repressor Rex 2.8 19.2 TM1430 glycerol kinase -2.7 6.1 TM1431 glycerol uptake operon antiterminator -2.2 14.7 TM1439 hypothetical protein 2.4 12.7 TM1453 ribosomal protein S9 2.3 15.2 TM1454 50S ribosomal protein L13 2.3 15.5 TM1457 hypothetical protein 2.5 5.7 TM1464 hypothetical protein 2.9 21.2 TM1465 cob(I)yrinic acid a,c-diamide adenosyltransferase 3.6 12.6 TM1473 30S ribosomal protein S4 2.8 14.8 TM1474 30S ribosomal protein S11 2.9 16.3 TM1475 30S ribosomal protein S13 3.0 17.3 TM1477 translation initiation factor IF-1 2.6 17.3 TM1479 adenylate kinase 3.2 18.6 TM1483 ribosomal protein S5 2.4 9.8 TM1484 ribosomal protein L18 2.3 11.9 TM1488 50S ribosomal protein L5 2.5 14.2 TM1492 ribosomal protein L29 3.4 10.8 TM1495 ribosomal protein L22 2.4 11.1 TM1496 ribosomal protein S19 2.2 14.8 TM1501 ribosomal protein S10 2.3 11.5 TM1504 30S ribosomal protein S7 2.7 11.9 TM1505 30S ribosomal protein S12 2.5 20.0 TM1508 hypothetical protein 2.2 14.1 TM1509 hypothetical protein 3.2 18.9 TM1522 diaminopimelate epimerase 2.4 12.3 TM1528 1,4-dihydroxy-2-naphthoate octaprenyltransferase, putative 3.0 16.0 TM1531 electron transfer flavoprotein, α subunit -2.1 8.5 TM1532 fixC protein -2.3 13.0 TM1548 lipopolysaccharide biosynthesis protein 2.6 19.1 TM1555 hypothetical protein 2.3 10.1 TM1556 maf protein 3.2 7.2 TM1563 hypothetical protein T 2.4 13.9 TM1570 hypothetical protein 4.3 17.1 TM1572 signal peptidase I, putative 2.6 16.4 TM1576 hemolysin 2.5 7.4 TM1580 transcriptional regulator, putative 2.7 17.1 TM1590 translation initiation factor IF-3 2.1 7.2 TM1591 ribosomal protein L35 3.9 9.7 TM1598 RNA polymerase sigma-E factor 2.0 9.1 TM1605 elongation factor Ts 2.1 11.1

333

Table B8 continued TM1609 ATP synthase F1, subunit epsilon 3.2 19.2 TM1624 β-mannosidase, putative 3.2 15.7 TM1632 hypothetical protein 3.3 12.0 TM1633 ATP-dependent protease LA 2.6 13.5 TM1636 hypothetical protein 3.1 13.6 TM1643 L-aspartate dehydrogenase 3.8 13.3 TM1653 pyrimidine-nucleoside phosphorylase 2.1 10.4 TM1661 polypeptide deformylase 2.3 12.7 TM1663 ABC transporter, ATP-binding protein 2.8 20.4 TM1665 hypothetical protein 2.8 8.4 TM1671 hypothetical protein 2.6 9.5 TM1679 hypothetical protein 2.0 12.8 TM1680 hypothetical protein 2.5 8.2 TM1683 cold shock protein 2.1 14.3 TM1684 ribosomal protein L31 3.5 17.3 TM1688 hypothetical protein 2.0 6.1 TM1697 hypothetical protein 2.6 10.9 TM1706 transcription elongation factor, greA/greB family 2.5 13.7 TM1709 hypothetical protein 2.9 9.2 TM1711 hypothetical protein 2.0 13.8 TM1728 3-methyl-2-oxobutanoate hydroxymethyltransferase 2.4 17.5 TM1731 hypothetical protein -3.1 10.6 TM1739 hypothetical protein 2.4 8.7 TM1742 nagD protein, putative 3.0 21.5 TM1746 ABC transporter, periplasmic binding protein 2.6 17.6 TM1751 endoglucanase 5.3 24.6 TM1756 butyrate kinase -2.2 7.8 TM1761 excinuclease ABC subunit B -2.1 10.7 TM1766 formate--tetrahydrofolate ligase 2.3 13.5 TM1768 exodeoxyribonuclease VII, large subunit 2.5 8.6 TM1772 hypothetical protein 2.3 12.3 TM1773 hypothetical protein 2.3 8.3 TM1774 cofactor-independent phosphoglycerate mutase 3.5 9.4 TM1782 N-acetyl-γ-glutamyl-phosphate reductase 2.4 11.9 TM1790 hypothetical protein 2.3 9.3 TM1792 hypothetical protein 3.3 14.4 TM1793 hypothetical protein 3.0 10.6 TM1794 hypothetical protein 3.2 16.5 TM1796 hypothetical protein 2.4 12.1 TM1799 hypothetical protein 4.3 17.0 TM1801 hypothetical protein 2.3 7.7 TM1802 hypothetical protein 2.7 11.3 TM1804 hypothetical protein 2.2 8.4 TM1805 hypothetical protein 2.2 24.0 TM1806 hypothetical protein 2.9 13.0 TM1809 hypothetical protein 4.0 12.6 TM1816 hypothetical protein 2.8 17.9

334

Table B8 continued TM1827 riboflavin synthase subunit γ 4.5 20.3 TM1831 transposase, putative 2.9 11.2 TM1832 transposase 3.6 11.7 TM1844 hypothetical protein TM1844 2.2 13.1 TM1855 sugar ABC transporter, periplasmic sugar-binding protein -8.5 25.9 TM1858 recX protein, putative 2.9 7.3 TM1860 hypothetical protein 4.2 14.0 TM1863 hypothetical protein 3.0 16.1 TM1866 membrane bound protein LytR, putative 2.4 13.0 TM1869 ATP-dependent protease LA, putative 2.3 11.1 TM1871 hypothetical protein 4.1 16.0 TM1873 ornithine decarboxylase 2.5 15.8 TM1876 hypothetical protein 2.9 13.1

335

Table B9. ORFs differentially transcribed in the T. zoo vs. T.petrophila in glucose culture. Positive values indicate up-regulation in the zoo, negative values indicate up-regulation in the pure culture Fold P- Probe ID Annotation Change value TM_rnpB TM_rnpB -2.7 13.6 TM_tRNA-Arg-3 TM_tRNA-Arg-3 -3.2 13.0 TM_tRNA-Arg-4 TM_tRNA-Arg-4 -3.1 9.5 TM_tRNA-Glu-2 TM_tRNA-Glu-2 -2.1 10.0 TM_tRNA-Gly-2 TM_tRNA-Gly-2 -2.1 8.2 TM_tRNA-Ile-1 TM_tRNA-Ile-1 -2.9 12.5 TM_tRNA-Leu-3 TM_tRNA-Leu-3 2.2 6.7 TM_tRNA-Leu-4 TM_tRNA-Leu-4 2.5 10.0 TM_tRNA-Lys-2 TM_tRNA-Lys-2 -3.3 11.5 TM_tRNA-Met-2 TM_tRNA-Met-2 5.9 17.3 TM_tRNA-Ser-2 TM_tRNA-Ser-2 4.2 11.8 TM_tRNA-Ser-3 TM_tRNA-Ser-3 6.4 14.0 TM_tRNA-Thr-1 TM_tRNA-Thr-1 -2.2 11.2 TM_tRNA-Thr-3 TM_tRNA-Thr-3 -3.9 20.7 TM0008 hypothetical protein 4.4 13.1 TM0011 NADP-reducing hydrogenase, subunit B 5.0 15.8 TM0012 NADP-reducing hydrogenase, subunit A 3.6 15.2 TM0013 hypothetical protein 5.8 14.5 TM0014 methyl-accepting chemotaxis protein, putative -3.5 12.9 TM0015 pyruvate ferredoxin oxidoreductase, γ subunit 4.3 11.6 TM0017 pyruvate ferredoxin oxidoreductase, α subunit 2.2 11.8 TM0019 3-ketoacyl-(acyl-carrier-protein) reductase 8.8 22.5 TM0020 hypothetical protein 13.2 26.3 TM0021 hypothetical protein 5.3 11.2 TM0022 DNA mismatch repair protein 6.2 22.6 TM0024 laminarinase 2.0 6.7 TM0026 hypothetical protein 3.6 20.7 TM0028 oligopeptide ABC transporter, ATP-binding protein 5.5 18.8 TM0033 hypothetical protein -2.8 7.9 TM0034 iron-sulfur cluster-binding protein -2.2 6.3 TM0035 hypothetical protein 8.8 13.7 TM0037 hypothetical protein 10.2 21.8 TM0038 6-pyruvoyl tetrahydrobiopterin synthase, putative 3.6 15.0 TM0039 hypothetical protein -3.1 14.5 TM0043 ABC transporter, ATP-binding protein -3.3 6.3 TM0045 hypothetical protein 6.0 22.8 TM0046 hypothetical protein 3.3 13.2 TM0052 hypothetical protein 3.3 25.0 TM0053 esterase, putative 9.4 21.1 TM0054 hypothetical protein 2.3 7.8

336

Table B9 continued TM0055 α-glucuronidase -3.0 16.0 TM0056 ABC transporter, periplasmic binding protein -14.7 33.2 TM0057 oligopeptide ABC transporter, ATP-binding protein -7.8 18.3 TM0058 oligopeptide ABC transporter, ATP-binding protein -4.8 8.0 TM0059 oligopeptide ABC transporter, permease protein -3.2 10.4 TM0060 oligopeptide ABC transporter, permease protein -5.7 9.0 TM0061 endo-1,4-β-xylanase A -26.3 33.2 TM0062 hypothetical protein -19.4 31.5 TM0065 transcriptional regulator, IclR family 6.9 23.3 TM0067 2-keto-3-deoxygluconate kinase 2.1 6.4 TM0070 endo-1,4-β-xylanase B -2.5 10.7 TM0071 ABC transporter, periplasmic binding protein -9.3 28.8 TM0073 oligopeptide ABC transporter, permease protein -2.6 5.8 TM0074 oligopeptide ABC transporter, ATP-binding protein -4.4 8.3 TM0075 oligopeptide ABC transporter, ATP-binding protein -5.4 21.3 TM0076 xylosidase -3.9 16.9 TM0077 acetyl xylan esterase -5.8 23.1 TM0078 iron(III) ABC transporter, ATP-binding protein 2.1 9.2 TM0082 flagellar hook-associated protein 3 5.2 13.8 TM0083 flagellar hook-associated protein 1 2.2 9.9 TM0094 general secretion pathway protein F, putative 34.6 12.5 TM0095 hypothetical protein 4.9 10.4 TM0096 hypothetical protein 4.2 23.9 TM0098 GTPase ObgE 2.3 8.3 TM0104 sugar ABC transporter, permease protein 2.5 8.0 TM0107 hypothetical protein -2.5 9.1 TM0108 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 17.0 24.6 TM0109 pyruvate formate lyase activating enzyme, putative 2.8 22.7 TM0111 alcohol dehydrogenase, iron-containing 2.3 11.1 TM0112 sugar ABC transporter, permease protein 2.4 12.3 TM0114 sugar ABC transporter, periplasmic sugar-binding protein -2.6 10.3 TM0119 acetamidase, putative 2.5 9.9 TM0120 oxidoreductase, putative 10.2 22.9 TM0122 ferric uptake regulation protein -2.0 9.5 TM0126 response regulator 3.3 16.7 TM0128 oxaloacetate decarboxylase 2.5 15.1 TM0129 carboxypeptidase G2, putative 8.0 15.4 TM0130 hypothetical protein 3.0 16.4 TM0133 isochorismatase-related protein -2.5 10.8 TM0134 thioredoxin reductase-related protein 5.4 14.4 TM0138 tryptophan synthase subunit β -3.7 15.9

337

Table B9 continued TM0140 indole-3-glycerol phosphate synthase 3.6 13.6 TM0141 anthranilate synthase component II -3.8 17.1 TM0144 hypothetical protein 11.5 19.8 TM0145 secreted metalloendopeptidase Gcp, putative 3.9 14.5 TM0146 ATP-dependent protease ATP-binding subunit 3.8 16.9 TM0147 hypothetical protein 2.8 8.5 TM0148 glucosamine--fructose-6-P aminotransferase 3.8 23.2 TM0149 fatty acid/phospholipid synthesis protein 6.2 28.2 TM0152 hypothetical protein 5.6 18.9 TM0153 hypothetical protein 3.4 12.4 TM0154 hypothetical protein 4.5 20.1 TM0159 ham1 protein 3.8 20.8 TM0160 hypothetical protein 6.0 9.7 TM0163 hypothetical protein 2.3 7.9 TM0164 hypothetical protein 4.8 19.7 TM0165 Holliday junction DNA helicase 4.2 14.7 TM0167 phosphopentomutase 7.5 16.3 TM0169 redox-sensing transcriptional repressor Rex -3.3 14.0 TM0170 hypothetical protein 55.9 31.1 TM0171 hypothetical protein 2.3 6.4 TM0175 acyl carrier protein 7.1 20.6 TM0176 hypothetical protein -2.5 12.0 TM0178 primosomal protein N' 3.5 18.3 TM0180 hypothetical protein 17.3 33.6 TM0181 hypothetical protein 3.1 18.3 TM0182 hypothetical protein 2.7 17.7 TM0183 hypothetical protein 2.7 6.3 TM0184 phosphoglucomutase/phosphomannomutase family protein 2.3 8.2 TM0188 hypothetical protein 2.3 9.6 TM0193 hypothetical protein 2.6 15.0 TM0194 ABC transporter, ATP-binding protein -2.6 9.5 TM0196 hypothetical protein -2.7 18.8 TM0197 hypothetical protein -2.4 7.8 TM0200 hypothetical protein 6.5 13.1 TM0201 NADP-reducing hydrogenase, subunit D, putative 3.7 18.3 TM0203 ABC transporter, permease protein, cysTW family 2.3 15.0 TM0205 ATP-dependent DNA helicase 8.0 21.4 TM0207 hypothetical protein 29.5 26.8 TM0208 pyruvate kinase 2.9 11.5 TM0209 6-phosphofructokinase 2.3 10.1 TM0211 aminomethyltransferase -2.7 11.6 TM0212 glycine cleavage system H protein 21.2 32.0

338

Table B9 continued TM0213 glycine dehydrogenase subunit 1 3.6 9.0 TM0217 glycyl-tRNA synthetase, β subunit -2.4 5.8 TM0219 flagellar export/assembly protein 3.8 15.1 TM0220 flagellar motor switch protein G 4.6 17.5 TM0226 hypothetical protein -2.3 7.2 TM0227 frame shift 6.5 17.8 TM0229 hypothetical protein -3.8 16.4 TM0234 UDP-N-acetylmuramoylalanine--D-glutamate ligase 2.6 18.1 TM0235 phospho-N-acetylmuramoyl-pentapeptide-transferase 2.6 11.4 TM0237 ligase -2.5 5.7 TM0238 hypothetical protein 2.9 17.6 TM0239 glucose-1-phosphate adenylyltransferase 2.1 5.9 TM0240 glucose-1-phosphate adenylyltransferase 6.7 12.7 TM0241 hypothetical protein -3.1 10.0 TM0246 hypothetical protein 5.1 14.0 TM0250 DNA processing chain A 2.4 8.5 TM0252 glutamyl tRNA-Gln amidotransferase, subunit C 3.8 7.4 TM0254 SsrA-binding protein 7.6 18.7 TM0256 hypothetical protein 3.2 19.7 TM0257 frame shift -3.2 19.9 TM0258 DNA topoisomerase 12.9 28.4 TM0260 hypothetical protein -2.1 10.9 TM0261 phosphate permease, putative 23.0 26.8 TM0264 16S pseudouridylate synthase 17.2 23.7 TM0268 5-methyltetrahydrofolate S-homocysteine methyltransferase -3.1 16.7 TM0271 hypothetical protein 4.6 13.6 TM0275 transcriptional regulator, GntR family 2.1 8.1 TM0277 frame shift -2.2 12.1 TM0280 hypothetical protein -2.1 9.4 TM0282 aldose 1-epimerase 4.6 26.1 TM0283 L-ribulose-5-phosphate 4-epimerase 3.6 14.8 TM0284 sugar kinase, FGGY family 2.3 9.2 TM0285 araM protein, putative 3.2 9.4 TM0289 6-phosphofructokinase, pyrophosphate-dependent -2.2 12.3 TM0290 citrate synthase 5.1 23.6 TM0291 3-isopropylmalate dehydratase large subunit -2.1 12.1 TM0292 3-isopropylmalate dehydratase small subunit 5.1 19.2 TM0293 γ-glutamyl phosphate reductase -2.1 12.0 TM0297 oxidoreductase, short chain dehydrogenase/reductase family 3.5 11.3 TM0302 oligopeptide ABC transporter, permease protein -3.3 14.2 TM0308 α-xylosidase YicI 2.7 5.5 TM0309 ABC transporter, periplasmic binding protein -5.4 16.1

339

Table B9 continued TM0310 β-D-galactosidase -5.3 19.7 TM0312 hypothetical protein -2.1 11.2 TM0313 K+ channel, β subunit -4.9 23.9 TM0314 hypothetical protein -2.5 16.3 TM0316 hypothetical protein 2.5 14.5 TM0318 ubiquinone/menaquinone biosynthesis-related protein 4.3 25.4 TM0319 hypothetical protein -2.2 14.1 TM0321 hypothetical protein 3.9 21.7 TM0322 ABC transporter, periplasmic substrate-binding protein -5.3 20.6 TM0324 hypothetical protein -2.5 10.9 TM0328 m4C-methyltransferase -3.0 14.5 TM0329 hypothetical protein 20.9 24.8 TM0330 hypothetical protein -2.8 17.2 TM0331 orotate phosphoribosyltransferase 5.3 17.6 TM0332 orotidine 5'-phosphate decarboxylase, putative 11.4 22.3 TM0334 dihydroorotate dehydrogenase electron transfer protein -3.4 17.2 TM0335 dihydroorotase -2.9 10.6 TM0337 hypothetical protein -3.4 13.5 TM0338 hypothetical protein 9.0 20.4 TM0340 hypothetical protein 11.9 18.1 TM0342 permease, putative -3.3 9.2 TM0343 3-deoxy-7-phosphoheptulonate synthase -3.7 6.8 TM0344 prephenate dehydrogenase 2.9 9.1 TM0345 3-phosphoshikimate 1-carboxyvinyltransferase -3.6 14.0 TM0349 3-dehydroquinate dehydratase 17.8 17.7 TM0350 hypothetical protein 2.1 10.3 TM0351 hypothetical protein 2.4 8.1 TM0354 hypothetical protein 2.9 13.8 TM0357 hypothetical protein 4.9 14.2 TM0358 hypothetical protein 3.7 11.5 TM0361 hypothetical protein -34.2 21.7 TM0365 putative aminopeptidase 1 5.7 21.8 TM0366 endonuclease III 2.5 11.9 TM0369 hypothetical protein 12.8 22.8 TM0376 hypothetical protein 5.6 15.3 TM0384 anaerobic ribonucleoside-triphosphate reductase-related protein 8.1 16.1 TM0388 hypothetical protein 2.3 9.0 TM0399 response regulator 2.6 12.4 TM0400 sensor histidine kinase 5.4 21.8 TM0401 thymidine kinase 6.5 18.2 TM0403 nitrogen regulatory protein P-II -4.7 20.2 TM0405 2-oxoglutarate ferredoxin oxidoreductase subunit β 36.5 33.8

340

Table B9 continued TM0407 diacylglycerol kinase, putative 32.7 24.6 TM0408 chemotaxis-specific methylesterase 2.7 6.3 TM0409 hypothetical protein 8.7 19.6 TM0410 hypothetical protein 17.9 23.0 TM0411 transcriptional regulator, XylR-related -2.3 7.8 TM0412 alcohol dehydrogenase, zinc-containing -2.4 17.6 TM0413 creatinine amidohydrolase, putative -3.2 8.2 TM0415 hypothetical protein 11.9 16.5 TM0418 sugar ABC transporter, periplasmic sugar-binding protein -3.0 16.4 TM0419 sugar ABC transporter, permease protein -2.9 15.5 TM0420 sugar ABC transporter, permease protein 10.1 22.0 TM0421 sugar ABC transporter, ATP-binding protein -3.9 15.7 TM0422 hypothetical protein -2.3 13.1 TM0425 oxidoreductase, putative 5.0 25.6 TM0426 PHT4-related protein 3.6 22.5 TM0428 oxidoreductase, putative 3.1 10.5 TM0430 sugar ABC transporter, permease protein -2.9 7.3 TM0431 sugar ABC transporter, permease protein -2.3 10.6 TM0432 sugar ABC transporter, periplasmic sugar-binding protein -2.3 9.9 TM0433 pectate lyase 3.4 17.7 TM0434 α-glucosidase, putative -22.5 30.1 TM0435 acetyl xylan esterase-related protein -2.0 6.0 TM0436 alcohol dehydrogenase, zinc-containing 2.9 8.5 TM0437 exo-poly-α-D-galacturonosidase, putative 5.2 20.9 TM0438 6-phosphogluconate dehydrogenase 6.3 22.7 TM0447 phosphoribosylaminoimidazole carboxylase ATPase subunit -2.2 9.1 TM0448 hypothetical protein -11.8 13.1 TM0450 hypothetical protein 7.8 19.1 TM0451 ribosomal protein L33 27.9 28.6 TM0453 N utilization substance protein G 4.0 17.0 TM0454 ribosomal protein L11 11.3 32.0 TM0457 ribosomal protein L7/L12 21.2 25.5 TM0458 DNA-directed RNA polymerase, β subunit 3.6 23.0 TM0459 DNA-directed RNA polymerase, β' subunit 4.7 15.5 TM0463 lipoprotein signal peptidase 6.2 19.8 TM0465 hypothetical protein 15.4 24.4 TM0466 hypothetical protein 5.2 16.0 TM0469 hypothetical protein -2.0 5.8 TM0471 hypothetical protein 11.8 29.4 TM0472 amidotransferase, putative 3.6 18.8 TM0476 hypothetical protein -50.9 27.6 TM0479 hypothetical protein 7.3 8.9

341

Table B9 continued TM0480 excinuclease ABC, subunit A -2.3 5.8 TM0481 hypothetical protein -2.3 8.2 TM0484 pyrimidine precursor biosynthesis enzyme, putative 2.8 14.4 TM0485 ABC transporter, permease protein, cysTW family 2.0 7.9 TM0486 hypothetical protein 3.4 20.0 TM0489 hypothetical protein 3.8 13.2 TM0490 regulatory protein, SIR2 family 2.8 10.9 TM0493 hypothetical protein -2.2 12.2 TM0495 phoH-related protein -5.5 22.6 TM0499 hypothetical protein 2.8 9.6 TM0500 oligopeptide ABC transporter, ATP-binding protein 3.8 12.8 TM0501 oligopeptide ABC transporter, ATP-binding protein 3.6 14.9 TM0502 oligopeptide ABC transporter, permease protein 3.1 16.1 TM0503 oligopeptide ABC transporter, permease protein 2.0 6.4 TM0507 hypothetical protein 5.4 20.2 TM0508 recombination factor protein RarA 4.4 21.5 TM0512 hypothetical protein 11.0 19.7 TM0516 clostripain-related protein -2.5 13.1 TM0519 hypothetical protein -3.2 11.9 TM0523 hypothetical protein 7.2 21.3 TM0525 tRNA delta-2-isopentenylpyrophosphate transferase -3.0 9.8 TM0526 host factor I 2.5 7.2 TM0529 heavy metal binding protein 4.1 8.1 TM0531 ABC transporter, periplasmic binding protein -10.4 36.6 TM0539 tryptophan synthase subunit β -2.7 8.2 TM0540 fumarate hydratase, N-terminal subunit 4.4 11.7 TM0541 fumarate hydratase, C-terminal subunit 19.7 27.2 TM0542 malate oxidoreductase 2.3 15.9 TM0544 ABC transporter, ATP-binding protein -3.0 11.5 TM0547 aspartokinase II 2.8 15.7 TM0549 acetolactate synthase, small subunit 2.0 10.2 TM0550 ketol-acid reductoisomerase -2.8 8.7 TM0560 hypothetical protein -13.3 26.8 TM0561 divalent cation transport-related protein -3.3 14.1 TM0562 hypothetical protein -2.3 8.7 TM0564 hypothetical protein 3.3 19.2 TM0565 sugar fermentation stimulation protein, putative -2.9 8.7 TM0566 hypothetical protein 2.9 13.9 TM0567 hypothetical protein -4.2 18.3 TM0568 hypothetical protein 2.3 17.4 TM0569 hypothetical protein 4.4 21.0 TM0571 heat shock serine protease, periplasmic 4.5 17.0

342

Table B9 continued TM0577 hypothetical protein 2.8 15.7 TM0579 hypothetical protein -2.9 10.6 TM0581 hypothetical protein -3.1 6.0 TM0590 penicillin-binding protein 2 6.6 18.8 TM0591 amino acid ABC transporter, ATP-binding protein -2.3 9.7 TM0592 amino acid ABC transporter, permease protein 3.6 11.4 TM0593 ABC transporter, periplasmic binding protein -3.9 18.3 TM0595 sugar ABC transporter, periplasmic sugar-binding protein 8.5 13.0 TM0598 sugar ABC transporter, permease protein 4.8 25.2 TM0600 hypothetical protein 2.4 8.5 TM0602 iron-dependent transcriptional repressor, putative -2.6 7.8 TM0603 ribosomal protein S6 -2.7 7.8 TM0604 single stranded DNA-binding protein, putative 7.3 24.1 TM0606 hypothetical protein 2.5 13.1 TM0607 hypothetical protein -2.9 12.4 TM0612 hypothetical protein 2.3 7.8 TM0616 hypothetical protein -12.4 25.5 TM0621 frame shit 2.2 11.3 TM0623 hypothetical protein 4.4 23.5 TM0624 N-acetylglucosaminyl-phosphatidylinositol biosynthesis -2.9 8.3 TM0626 hypothetical protein 6.0 14.8 TM0633 flagellar-related protein -3.1 13.9 TM0636 hypothetical protein 2.9 14.3 TM0639 hypothetical protein -2.5 5.5 TM0640 hypothetical protein 17.0 25.9 TM0641 hypothetical protein -2.2 5.8 TM0643 clostripain-related protein -4.3 14.3 TM0644 hypothetical protein -15.6 14.4 TM0648 hypothetical protein -2.4 7.3 TM0649 hypothetical protein 3.3 12.4 TM0650 hypothetical protein -2.5 11.2 TM0652 hypothetical protein 9.9 22.1 TM0653 hypothetical protein -4.0 13.1 TM0654 spermidine synthase -10.6 13.0 TM0657 rubrerythrin 8.6 20.6 TM0659 rubredoxin 6.4 23.6 TM0663 hypothetical protein -3.2 10.4 TM0665 cysteine synthase -4.4 15.9 TM0667 hypothetical protein 5.6 16.1 TM0668 pleiotropic regulatory protein 2.9 11.3 TM0669 hypothetical protein 7.9 21.7 TM0672 hypothetical protein 2.2 6.8

343

Table B9 continued TM0674 flagellar protein, putative -2.7 8.7 TM0676 motility protein B -2.1 11.1 TM0680 chemotaxis protein 4.2 13.9 TM0681 dehydrase-related protein 25.5 24.3 TM0683 hypothetical protein 4.1 13.2 TM0686 DNA polymerase III, γ and ηsubunit 2.0 20.0 TM0690 hypothetical protein 7.5 22.8 TM0691 hypothetical protein -3.3 13.2 TM0694 trigger factor, putative -3.1 13.5 TM0695 ATP-dependent Clp protease, proteolytic subunit 2.1 5.7 TM0696 ray-related protein 2.6 6.7 TM0697 flagellar biosynthesis protein FliQ -3.4 13.1 TM0698 flagellar biosynthesis protein FliP 4.8 17.6 TM0700 chemotaxis response regulator CheY 3.2 10.1 TM0703 competence-damage inducible protein, putative 9.9 12.0 TM0705 ABC transporter, ATP-binding protein 10.9 29.9 TM0707 glucose-inhibited division protein B 2.5 10.2 TM0708 hypothetical protein 3.1 9.7 TM0709 hypothetical protein -3.5 14.8 TM0710 transcriptional regulator, MarR family 3.9 15.4 TM0714 hypothetical protein -7.1 17.7 TM0715 tRNA nucleotidyl transferase-related protein 2.7 11.6 TM0717 propionyl-CoA carboxylase,γ subunit -11.6 25.4 TM0719 cysteinyl-tRNA synthetase -2.7 10.7 TM0721 uracil phosphoribosyltransferase 2.5 7.0 TM0725 hypothetical protein 3.2 15.5 TM0727 pmbA-related protein 3.4 12.7 TM0728 hypothetical protein 2.6 11.3 TM0729 (p)ppGpp synthetase 6.9 26.2 TM0730 D-tyrosyl-tRNA deacylase 4.8 13.2 TM0731 hypothetical protein 6.5 14.2 TM0736 mannose-6-phosphate isomerase 3.4 18.9 TM0737 hypothetical protein 2.0 12.1 TM0738 hypothetical protein -2.2 9.9 TM0739 hypothetical protein 2.4 12.0 TM0743 hypothetical protein 2.7 14.8 TM0744 hypothetical protein 11.0 25.1 TM0746 hypothetical protein -2.2 12.7 TM0749 hypothetical protein 3.1 10.1 TM0752 α-glucosidase, putative -40.0 39.4 TM0757 hypothetical protein -2.2 10.3 TM0759 acyltransferase, putative 3.0 9.7

344

Table B9 continued TM0760 lipopolysaccharide biosynthesis protein, putative 3.1 15.9 TM0764 hypothetical protein -2.5 9.6 TM0768 hypothetical protein -3.3 14.2 TM0770 hypothetical protein -4.2 16.7 TM0772 hypothetical protein 3.2 10.9 TM0773 hypothetical protein 7.1 9.3 TM0774 hypothetical protein 3.6 15.4 TM0775 translation initiation factor IF-2 4.7 26.1 TM0778 hypothetical protein 11.2 25.1 TM0779 hypothetical protein 28.2 26.7 TM0780 bacterioferritin comigratory protein, ahpC/TSA family 9.7 21.9 TM0781 hypothetical protein 2.5 11.8 TM0785 bacteriocin 4.8 19.0 TM0787 putative thiazole biosynthetic enzyme -2.9 10.3 TM0788 thiamine biosynthesis protein ThiC -3.1 11.5 TM0789 hypothetical protein 6.8 12.9 TM0792 hypothetical protein 2.8 10.0 TM0794 hypothetical protein -2.2 7.1 TM0797 2-phosphosulfolactate phosphatase 2.9 12.2 TM0803 CTP synthetase -2.5 6.6 TM0804 hypothetical protein 11.0 24.4 TM0805 lipophilic protein, putative 2.3 7.9 TM0813 hypothetical protein 6.3 24.7 TM0814 N-acetylglucosamine-6-phosphate deacetylase 2.7 10.5 TM0816 transcriptional regulator, putative, Mar family -2.7 10.9 TM0823 transcriptional regulator, TetR family 8.4 22.1 TM0825 astB/chuR-related protein 2.3 12.6 TM0826 hypothetical protein 2.7 12.5 TM0827 ABC transporter, ATP-binding protein, putative 10.7 18.0 TM0828 sugar kinase, pfkB family 14.1 24.5 TM0829 hypothetical protein 6.3 27.5 TM0830 hypothetical protein 3.5 12.2 TM0831 branched-chain amino acid aminotransferase, putative 11.5 25.6 TM0832 hypothetical protein 14.2 19.0 TM0833 DNA gyrase, subunit B 10.5 17.4 TM0834 hypothetical protein 5.0 17.2 TM0835 cell division protein FtsA, putative 8.4 22.6 TM0838 hypothetical protein 3.7 15.9 TM0840 hypothetical protein 2.9 8.6 TM0841 S-layer-like array protein -3.5 15.0 TM0842 response regulator 6.3 17.7 TM0843 formiminotransferase-cyclodeaminase -2.7 8.3

345

Table B9 continued TM0848 hypothetical protein 3.2 19.3 TM0849 dnaJ protein -3.0 8.6 TM0851 heat shock operon repressor HrcA -2.7 11.7 TM0853 sensor histidine kinase 13.0 23.4 TM0857 riboflavin kinase/FMN adenylyltransferase -4.0 12.8 TM0858 hypothetical protein 4.6 14.6 TM0860 protein-export membrane protein SecD, putative 2.3 9.1 TM0863 ribosomal protein L9 -3.3 18.2 TM0864 hypothetical protein -3.4 17.5 TM0865 hypothetical protein 5.0 16.9 TM0868 glutaredoxin-related protein -3.6 14.9 TM0870 penicillin-binding protein 2 3.5 16.7 TM0872 hypothetical protein 9.9 29.2 TM0874 hypothetical protein 2.0 6.5 TM0876 hypothetical protein 4.8 19.8 TM0877 enolase 6.7 20.3 TM0879 ferredoxin -3.1 18.6 TM0880 oxaloacetate decarboxylase, β subunit -2.8 6.7 TM0881 homoserine O-succinyltransferase -4.1 15.8 TM0887 methylated-DNA-protein-cysteine methyltransferase 5.8 18.5 TM0888 hypothetical protein 4.8 19.5 TM0891 gcpE protein -3.1 9.5 TM0892 hypothetical protein 2.6 8.3 TM0895 glycogen synthase 9.1 22.5 TM0896 galactose-1-phosphate uridylyltransferase, putative 4.0 17.4 TM0899 hypothetical protein 3.7 17.8 TM0901 hypothetical protein -2.5 10.6 TM0902 RNA polymerase sigma-28 factor, putative 4.4 8.3 TM0903 chemotaxis methylation protein 2.9 11.1 TM0904 chemotaxis protein CheC 8.1 24.2 TM0905 hypothetical protein 15.4 28.2 TM0906 hypothetical protein 2.8 14.9 TM0911 translation initiation factor, aIF-2B α subunit-related 2.8 13.0 TM0912 basic membrane protein, putative 6.4 15.1 TM0915 ribonuclease HII 2.2 12.6 TM0916 hypothetical protein 3.2 9.2 TM0923 hypothetical protein 4.7 24.2 TM0924 hypothetical protein -2.9 11.5 TM0926 chromosomal replication initiator protein 4.1 22.3 TM0927 ferredoxin 3.0 13.6 TM0932 hypothetical protein -21.6 21.6 TM0933 hypothetical protein -3.0 5.6

346

Table B9 continued TM0935 hypothetical protein -3.9 20.0 TM0937 hypothetical protein 4.8 13.0 TM0938 hypothetical protein -2.6 8.1 TM0941 hypothetical protein -3.8 20.2 TM0942 hypothetical protein -3.9 11.4 TM0943 glutamine synthetase -3.7 12.3 TM0944 hypothetical protein -5.5 29.6 TM0945 hypothetical protein -2.9 8.9 TM0947 hypothetical protein 4.2 14.9 TM0950 hypothetical protein -3.1 11.1 TM0951 hypothetical protein -6.7 9.4 TM0952 glycerol kinase -3.7 11.4 TM0953 transketolase, C-terminal subunit -4.8 18.1 TM0955 ribose ABC transporter, permease protein 2.3 10.5 TM0956 ribose ABC transporter, ATP-binding protein -10.7 30.3 TM0962 hypothetical protein -3.0 7.5 TM0963 oligoendopeptidase, putative -3.4 12.8 TM0964 hypothetical protein -2.9 14.0 TM0966 hypothetical protein 4.4 19.0 TM0968 hypothetical protein 5.0 17.4 TM0972 conserved hypothetical protein, GGDEF domain -2.8 8.6 TM0973 methyl-accepting chemoreceptor-related protein -2.2 8.2 TM0974 hypothetical protein 2.2 12.2 TM0977 hypothetical protein -3.6 8.2 TM0978 hypothetical protein -5.2 18.5 TM0979 hypothetical protein 2.6 6.6 TM0982 hypothetical protein 7.6 16.7 TM0984 hypothetical protein -3.4 10.1 TM0985 hypothetical protein 3.6 20.1 TM0986 hypothetical protein -3.4 10.5 TM0988 hypothetical protein -3.1 15.2 TM0989 hypothetical protein -2.4 10.2 TM0992 hypothetical protein -3.9 11.0 TM0993 hypothetical protein -7.0 20.9 TM0998 heavy metal resistance transcriptional regulator -3.0 14.4 TM1001 hypothetical protein 7.1 23.4 TM1002 hypothetical protein 2.4 10.7 TM1003 transposase-related protein 2.9 18.7 TM1005 transcriptional regulator, putative -2.5 10.5 TM1015 glutamate dehydrogenase -2.9 8.6 TM1016 hypothetical protein 6.0 17.8 TM1017 hypothetical protein -2.5 11.9

347

Table B9 continued TM1018 hypothetical protein -2.4 9.8 TM1027 hypothetical protein -2.2 8.6 TM1028 ABC transporter, ATP-binding protein 4.0 15.9 TM1033 mannose-1-phosphate guanylyltransferase 2.7 7.8 TM1035 phosphoribosyl-AMP cyclohydrolase -3.1 23.6 TM1042 ATP phosphoribosyltransferase 7.1 28.2 TM1043 histidyl-tRNA synthetase-related protein 5.8 6.4 TM1044 transposase, IS605-TnpB family 5.5 18.7 TM1046 hypothetical protein 2.4 9.9 TM1047 septum site-determining protein MinC, putative 5.9 13.4 TM1048 endoglucanase 12.3 21.4 TM1049 endoglucanase 4.3 13.2 TM1057 potassium channel, putative 4.9 23.3 TM1058 glutamate synthase-related protein -2.8 16.0 TM1064 ABC transporter, ATP-binding protein -2.5 7.8 TM1066 ABC transporter, permease protein -2.3 9.9 TM1067 ABC transporter, periplasmic binding protein -2.3 6.4 TM1068 α-glucosidase, putative -33.6 36.4 TM1069 transcriptional regulator, DeoR family -17.2 25.8 TM1072 rhamnulose-1-phosphate aldolase 6.1 14.2 TM1073 sugar kinase 5.2 9.6 TM1076 hypothetical protein -2.3 7.0 TM1077 pantoate--β-alanine ligase 6.1 23.5 TM1080 sugar-phosphate isomerase 2.0 8.0 TM1082 lexA repressor 2.9 13.4 TM1084 DNA gyrase, subunit A 6.0 28.8 TM1085 methionyl-tRNA synthetase 2.9 7.6 TM1087 hypothetical protein 6.6 20.6 TM1091 hypothetical protein -2.4 14.8 TM1092 hypothetical protein 4.0 10.1 TM1098 hypothetical protein -3.1 7.5 TM1102 ribonuclease III -2.9 8.0 TM1107 hypothetical protein 5.8 18.8 TM1111 hypothetical protein -3.0 6.5 TM1112 hypothetical protein 9.0 19.7 TM1114 hypothetical protein -2.3 5.5 TM1115 hypothetical protein -2.5 12.4 TM1117 general secretion pathway protein D, putative -5.6 14.4 TM1119 hypothetical protein -6.2 21.2 TM1120 G-3-P ABC transporter, periplasmic G-3-P-binding protein 2.2 12.4 TM1123 flagellar hook-associated protein 2, putative -13.3 14.5 TM1124 hypothetical protein -15.3 24.5

348

Table B9 continued TM1125 hypothetical protein 2.6 12.8 TM1126 hypothetical protein -5.9 21.1 TM1129 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase 4.5 20.4 TM1133 hypothetical protein 4.1 19.4 TM1135 ABC transporter, periplasmic binding protein 2.8 15.2 TM1138 ABC transporter, ATP-binding protein -2.6 6.1 TM1142 hypothetical protein 5.9 17.2 TM1145 hypothetical protein -2.2 7.3 TM1148 isocitrate dehydrogenase -3.3 11.8 TM1154 oxidoreductase, sol/devB family 3.0 15.9 TM1155 glucose-6-phosphate 1-dehydrogenase 4.3 24.7 TM1157 hypothetical protein 8.0 26.3 TM1161 Mg2+ transporter MgtE, putative 2.5 5.6 TM1163 conserved hypothetical protein, GGDEF domain 3.6 7.7 TM1164 2-oxoacid ferredoxin oxidoreductase, α subunit 2.9 9.7 TM1165 2-oxoacid ferredoxin oxidoreductase subunit β 7.6 25.7 TM1166 oxygen-independent coproporphyrinogen III oxidase, putative -4.0 6.9 TM1168 frame shift -8.6 22.6 TM1169 3-oxoacyl-(acyl carrier protein) reductase -3.4 16.4 TM1171 transcriptional regulator, crp family -2.8 11.0 TM1176 transcriptional regulator, metal-sensing -3.4 14.3 TM1177 hypothetical protein -3.6 14.7 TM1180 hypothetical protein 3.8 18.7 TM1183 oxidoreductase, aldo/keto reductase family 4.0 15.6 TM1185 methylglyoxal synthase 3.0 12.1 TM1186 hypothetical protein 3.5 11.7 TM1190 galactokinase -2.8 12.0 TM1192 α-galactosidase 3.1 13.8 TM1194 oligopeptide ABC transporter, ATP-binding protein 3.3 20.3 TM1200 transcriptional regulator, LacI family 3.3 16.2 TM1202 maltose ABC transporter, permease protein 3.5 15.9 TM1203 maltose ABC transporter, permease protein 2.2 6.7 TM1204 maltose ABC transporter, periplasmic maltose-binding protein 2.6 14.2 TM1206 putative monovalent cation/H+ antiporter subunit F -2.7 7.9 TM1207 putative monovalent cation/H+ antiporter subunit G -3.0 11.8 TM1208 hypothetical protein 4.8 23.4 TM1211 NADH dehydrogenase, putative -2.1 9.1 TM1214 NADH dehydrogenase subunit B 24.0 23.9 TM1215 NADH dehydrogenase, 30 kDa subunit, putative 3.8 19.3 TM1222 oligopeptide ABC transporter, permease protein -2.4 7.9 TM1224 transcriptional regulator, XylR-related 3.8 20.3 TM1227 endo-1,4-β-mannosidase -2.8 9.9

349

Table B9 continued TM1228 transcriptional regulator, RpiR family 3.3 15.7 TM1230 hypothetical protein 2.3 9.2 TM1231 α-mannosidase-related protein -2.6 13.2 TM1232 sugar ABC transporter, ATP-binding protein -3.3 15.2 TM1236 hypothetical protein 2.5 10.7 TM1239 hypothetical protein 2.6 6.4 TM1241 hypothetical protein -2.7 13.2 TM1245 phosphoribosylformylglycinamidine synthase I 17.1 8.8 TM1246 phosphoribosylformylglycinamidine synthase II 6.8 16.2 TM1247 amidophosphoribosyltransferase -2.8 13.9 TM1248 phosphoribosylglycinamide formyltransferase 8.4 26.5 TM1250 phosphoribosylamine--glycine ligase 2.3 15.4 TM1251 phosphoribosylaminoimidazole synthetase 2.8 17.7 TM1252 hypothetical protein -3.7 15.0 TM1255 aspartate aminotransferase -3.2 12.3 TM1256 ABC transporter, ATP-binding protein 4.4 19.7 TM1259 phosphate regulon transcriptional regulatory protein PhoB -2.5 12.2 TM1260 phosphate transport system regulator PhoU -4.3 20.2 TM1261 phosphate ABC transporter, ATP-binding protein -3.3 14.2 TM1262 phosphate ABC transporter, permease protein -4.9 18.3 TM1264 phosphate ABC transporter, periplasmic binding protein -2.3 11.7 TM1265 hypothetical protein -4.0 18.4 TM1269 biotin synthase -4.4 17.2 TM1272 glutamyl tRNA-Gln amidotransferase, subunit A -5.9 14.0 TM1273 aspartyl/glutamyl-tRNA amidotransferase subunit B -2.7 12.3 TM1276 sugar ABC transporter, ATP-binding protein -2.9 14.2 TM1281 6-phospho-β-glucosidase 5.2 18.2 TM1283 hypothetical protein 3.9 17.0 TM1284 oxidase-related protein 5.8 30.3 TM1286 methyltransferase 52.4 15.5 TM1287 hypothetical protein 6.5 17.6 TM1290 hypothetical protein -2.9 13.5 TM1293 hypothetical protein -2.9 8.3 TM1294 hypothetical protein 13.2 29.3 TM1295 hypothetical protein 2.5 12.0 TM1299 hypothetical protein -2.4 9.7 TM1300 hypothetical protein 6.9 23.5 TM1301 astB/chuR-related protein -2.8 10.8 TM1302 ABC transporter, ATP-binding protein -3.1 14.4 TM1310 ABC transporter, ATP-binding protein -3.0 7.9 TM1311 hypothetical protein 2.1 7.2 TM1312 hypothetical protein 81.1 37.9

350

Table B9 continued TM1313 hypothetical protein -2.7 15.7 TM1320 frame shift 16.6 23.4 TM1321 hypothetical protein 15.8 22.9 TM1323 hypothetical protein 2.0 6.8 TM1329 hypothetical protein -2.8 27.9 TM1332 hypothetical protein -9.1 12.4 TM1333 hypothetical protein 2.6 12.6 TM1337 hypothetical protein -2.3 13.1 TM1343 frame shift 5.7 21.6 TM1350 lipase, putative 2.4 13.1 TM1351 glutamyl-tRNA synthetase 2.9 14.6 TM1354 inosine-5-monophosphate dehydrogenase-related protein 2.9 11.5 TM1356 hypothetical protein 2.7 8.0 TM1359 sensor histidine kinase -2.8 9.4 TM1360 response regulator 3.5 12.4 TM1365 flagellar basal-body rod protein FlgC 3.3 10.9 TM1368 ABC transporter, ATP-binding protein -2.8 12.7 TM1369 hypothetical protein 2.6 6.2 TM1371 aminotransferase, class V 3.3 14.8 TM1373 hypothetical protein 2.4 12.7 TM1375 ABC transporter, periplasmic binding protein 2.2 10.6 TM1379 seryl-tRNA synthetase 5.0 18.5 TM1381 hypothetical protein 2.6 7.0 TM1382 hypothetical protein 17.1 18.2 TM1385 glucose-6-phosphate isomerase 3.3 8.5 TM1386 hypothetical protein -2.0 7.8 TM1388 hypothetical protein -2.7 10.6 TM1392 hypothetical protein 3.6 12.5 TM1393 hypothetical protein 15.7 21.7 TM1394 hypothetical protein -4.0 12.2 TM1397 phosphatidate cytidylyltransferase, putative 9.8 18.6 TM1398 hypothetical protein -18.6 26.9 TM1399 ribosome recycling factor 6.4 20.2 TM1402 hypothetical protein -2.6 12.2 TM1404 antibiotic ABC transporter, transmembrane protein, putative -2.2 7.6 TM1405 lipopolysaccharide biosynthesis protein-related protein -3.0 14.2 TM1406 hypothetical protein -3.1 13.7 TM1410 hypothetical protein 3.4 14.3 TM1417 ABC transporter, ATP-binding protein 2.2 17.0 TM1421 hydrogenase, putative 15.0 14.4 TM1422 rnfB-related protein 3.0 12.7 TM1423 hypothetical protein 5.2 13.4

351

Table B9 continued TM1424 Fe-hydrogenase, subunit γ -2.0 10.4 TM1425 Fe-hydrogenase, subunit β 2.0 6.6 TM1427 redox-sensing transcriptional repressor Rex 7.7 27.1 TM1429 glycerol uptake facilitator protein -2.7 11.2 TM1430 glycerol kinase -6.8 10.9 TM1431 glycerol uptake operon antiterminator -4.8 21.7 TM1433 oxidoreductase, putative -3.0 13.8 TM1434 hypothetical protein 2.8 15.6 TM1439 hypothetical protein 5.9 19.8 TM1441 aspartyl-tRNA synthetase 2.6 8.5 TM1445 ribosomal protein S1 2.7 9.5 TM1446 hypothetical protein 3.2 18.3 TM1447 hypothetical protein 3.3 19.0 TM1450 transcription-repair coupling factor, putative 2.9 10.6 TM1452 DNA primase 4.2 8.8 TM1453 ribosomal protein S9 5.3 22.6 TM1454 50S ribosomal protein L13 5.2 22.6 TM1455 hypothetical protein 3.0 14.9 TM1456 50S ribosomal protein L27 2.5 11.0 TM1457 hypothetical protein 6.2 10.3 TM1459 hypothetical protein 2.2 11.0 TM1460 jag protein, putative 3.1 20.0 TM1463 ribonuclease P protein component 2.2 7.4 TM1464 hypothetical protein 8.4 29.2 TM1465 cob(I)yrinic acid a,c-diamide adenosyltransferase 13.1 19.7 TM1466 hypothetical protein 2.2 6.9 TM1468 hypothetical protein 3.3 12.1 TM1469 glucokinase -2.8 9.6 TM1472 DNA-directed RNA polymerase subunit α -3.2 12.5 TM1473 30S ribosomal protein S4 7.8 22.2 TM1474 30S ribosomal protein S11 8.4 23.9 TM1475 30S ribosomal protein S13 8.8 24.9 TM1476 50S ribosomal protein L36 -2.3 7.1 TM1477 translation initiation factor IF-1 7.0 25.0 TM1479 adenylate kinase 10.1 25.9 TM1481 ribosomal protein L15 2.6 18.3 TM1483 ribosomal protein S5 5.9 16.4 TM1484 ribosomal protein L18 5.0 18.7 TM1486 ribosomal protein S8 -2.7 11.8 TM1487 ribosomal protein S14 2.0 7.8 TM1488 50S ribosomal protein L5 6.3 21.5 TM1489 50S ribosomal protein L24 2.2 9.5

352

Table B9 continued TM1491 30S ribosomal protein S17 2.4 7.4 TM1492 ribosomal protein L29 11.2 17.4 TM1493 50S ribosomal protein L16 -2.4 18.6 TM1495 ribosomal protein L22 5.8 18.1 TM1496 ribosomal protein S19 5.0 22.2 TM1498 ribosomal protein L23 2.1 11.6 TM1500 ribosomal protein L3 2.3 12.7 TM1501 ribosomal protein S10 5.5 18.3 TM1503 translation elongation factor G 2.0 14.3 TM1504 30S ribosomal protein S7 7.3 18.8 TM1505 30S ribosomal protein S12 6.2 27.9 TM1506 hypothetical protein 2.6 18.1 TM1507 phoH-related protein 2.5 14.5 TM1508 hypothetical protein 4.8 21.3 TM1509 hypothetical protein 10.6 26.8 TM1510 hypothetical protein 2.4 7.9 TM1514 hypothetical protein 2.4 14.0 TM1516 hydrolase, ama/hipO/hyuC family -2.1 7.8 TM1519 2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase -3.1 13.5 TM1522 diaminopimelate epimerase 5.8 19.3 TM1525 endoglucanase -3.0 10.7 TM1528 1,4-dihydroxy-2-naphthoate octaprenyltransferase, putative 8.8 23.5 TM1529 hypothetical protein -2.5 6.3 TM1531 electron transfer flavoprotein, α subunit -4.7 14.7 TM1532 fixC protein -5.2 20.2 TM1534 hypothetical protein 2.2 10.6 TM1536 hypothetical protein -2.7 12.2 TM1544 rod shape-determining protein MreB 2.1 10.9 TM1548 lipopolysaccharide biosynthesis protein 6.6 26.8 TM1549 methicillin resistance protein 2.3 6.6 TM1550 hypothetical protein -3.5 14.0 TM1555 hypothetical protein 5.1 16.6 TM1556 maf protein 10.0 12.8 TM1557 DNA repair protein 4.5 6.6 TM1559 deoxyribose-phosphate aldolase 2.0 9.3 TM1563 hypothetical protein 5.6 21.1 TM1567 hypothetical protein 3.7 20.0 TM1568 16S rRNA processing protein, putative 3.8 24.9 TM1569 tRNA guanine-N1 methyltransferase 3.2 12.5 TM1570 hypothetical protein 18.7 24.3 TM1571 50S ribosomal protein L19 2.4 10.4 TM1572 signal peptidase I, putative 6.7 24.0

353

Table B9 continued TM1573 hypothetical protein -2.1 11.0 TM1574 pseudouridylate synthase I -2.7 14.6 TM1576 hemolysin 6.2 13.4 TM1578 preprotein translocase subunit SecA 2.7 9.6 TM1579 peptide chain release factor 2 2.6 13.8 TM1580 transcriptional regulator, putative 7.1 24.8 TM1582 hypothetical protein 3.8 13.1 TM1584 comFC protein, putative 2.7 11.4 TM1586 hypothetical protein 3.6 20.0 TM1587 hypothetical protein 2.6 14.0 TM1588 conserved hypothetical protein, GGDEF domain 2.4 9.7 TM1589 clostripain-related protein 3.7 17.3 TM1590 translation initiation factor IF-3 4.7 13.1 TM1591 ribosomal protein L35 14.9 16.0 TM1594 conserved hypothetical protein, GGDEF domain 3.3 17.4 TM1596 purine nucleoside phosphorylase -2.7 13.7 TM1597 hypothetical protein -2.7 14.3 TM1598 RNA polymerase sigma-E factor 4.1 15.3 TM1600 hypothetical protein -3.1 15.2 TM1602 transcriptional regulator, biotin repressor family -2.1 8.0 TM1603 permease, putative 3.3 7.5 TM1605 elongation factor Ts 4.2 17.6 TM1606 cytoplasmic axial filament protein, putative 3.4 5.9 TM1608 hypothetical protein -3.4 12.6 TM1609 ATP synthase F1, subunit ε 10.3 27.1 TM1613 ATP synthase F1, subunit δ -2.5 9.5 TM1618 cheX protein -3.7 14.6 TM1621 hypothetical protein 2.2 16.2 TM1624 β-mannosidase, putative 9.9 23.1 TM1627 general stress protein Ctc 2.8 9.8 TM1629 UDP-N-acetylglucosamine pyrophosphorylase -3.4 13.7 TM1632 hypothetical protein 10.5 18.9 TM1633 ATP-dependent protease LA 6.8 20.7 TM1636 hypothetical protein 9.7 20.8 TM1637 hypothetical protein -2.7 9.5 TM1639 ferredoxin--NADP(+) reductase subunit α 3.3 12.0 TM1643 L-aspartate dehydrogenase 14.4 20.6 TM1647 hypothetical protein -2.7 10.4 TM1648 hypothetical protein -3.1 7.1 TM1650 α-amylase, putative -2.1 5.5 TM1651 translation elongation factor G 3.9 16.0 TM1653 pyrimidine-nucleoside phosphorylase 4.4 16.7

354

Table B9 continued TM1654 sensor histidine kinase HpkA 3.5 16.2 TM1659 hypothetical protein -2.3 6.7 TM1660 hypothetical protein 3.2 16.5 TM1661 polypeptide deformylase 5.1 19.9 TM1662 stationary phase survival protein 2.5 12.4 TM1663 ABC transporter, ATP-binding protein 7.9 28.4 TM1665 hypothetical protein 7.5 14.2 TM1666 succinyl-diaminopimelate desuccinylase 3.6 18.8 TM1671 hypothetical protein 6.5 15.8 TM1674 hypothetical protein -3.4 17.5 TM1677 transposase, putative 3.5 12.3 TM1679 hypothetical protein 4.1 19.8 TM1680 hypothetical protein 6.3 14.1 TM1681 hypothetical protein 2.5 13.9 TM1682 hypothetical protein 2.7 14.2 TM1683 cold shock protein 4.5 21.6 TM1684 ribosomal protein L31 12.3 25.0 TM1687 DNA/pantothenate metabolism flavoprotein 2.5 12.9 TM1688 hypothetical protein 4.0 11.0 TM1690 hypothetical protein 2.4 9.1 TM1691 hypothetical protein 3.9 25.6 TM1696 type IV prepilin peptidase -2.3 7.1 TM1697 hypothetical protein 7.0 17.6 TM1700 hypothetical protein -2.9 12.1 TM1701 hypothetical protein 2.5 12.6 TM1702 hypothetical protein 3.4 8.5 TM1703 hypothetical protein -3.5 16.1 TM1704 hypothetical protein 2.7 12.5 TM1705 lysyl-tRNA synthetase -2.8 11.1 TM1706 transcription elongation factor, greA/greB family 6.2 20.9 TM1708 hypothetical protein 2.9 14.3 TM1709 hypothetical protein 8.0 15.3 TM1711 hypothetical protein 4.1 20.6 TM1712 hypothetical protein 3.6 13.8 TM1713 proline dipeptidase, putative -2.6 9.5 TM1715 hypothetical protein -3.5 15.0 TM1717 hypothetical protein 2.3 14.9 TM1722 hypothetical protein -2.4 6.3 TM1724 3-oxoacyl-(acyl carrier protein) reductase 2.2 13.7 TM1728 3-methyl-2-oxobutanoate hydroxymethyltransferase 5.9 25.2 TM1729 outer membrane protein 2.1 8.4 TM1730 Holliday junction DNA helicase B 2.4 10.7

355

Table B9 continued TM1731 hypothetical protein -9.7 17.2 TM1733 hypothetical protein 2.8 16.5 TM1739 hypothetical protein 5.6 14.7 TM1740 hypothetical protein 2.6 12.8 TM1742 nagD protein, putative 8.7 29.3 TM1746 ABC transporter, periplasmic binding protein 6.5 25.4 TM1747 oligopeptide ABC transporter, permease protein -2.5 10.3 TM1749 oligopeptide ABC transporter, ATP-binding protein 3.1 15.7 TM1750 oligopeptide ABC transporter, ATP-binding protein 2.8 10.8 TM1751 endoglucanase 28.4 32.8 TM1753 excinuclease ABC, subunit B-related protein 2.5 13.5 TM1754 butyrate kinase -3.7 15.2 TM1755 phosphate butyryltransferase -3.1 6.9 TM1756 butyrate kinase -4.7 13.7 TM1759 2-ketoisovalerate ferredoxin reductase -2.8 11.4 TM1761 excinuclease ABC subunit B -4.3 17.5 TM1762 transketolase -3.9 22.3 TM1766 formate--tetrahydrofolate ligase 5.2 20.6 TM1768 exodeoxyribonuclease VII, large subunit 6.1 14.6 TM1771 hypothetical protein 3.3 10.5 TM1772 hypothetical protein 5.1 19.4 TM1773 hypothetical protein 5.2 14.2 TM1774 cofactor-independent phosphoglycerate mutase 12.1 15.6 TM1779 hypothetical protein -2.7 9.2 TM1782 N-acetyl-γ-glutamyl-phosphate reductase 6.0 18.8 TM1783 bifunctional ornithine acetyltransferase -2.5 7.7 TM1784 acetylglutamate kinase -3.6 17.3 TM1785 acetylornithine aminotransferase 2.2 5.4 TM1788 conserved hypothetical protein, GGDEF domain -2.8 10.7 TM1790 hypothetical protein 5.2 15.5 TM1791 hypothetical protein 3.9 19.9 TM1792 hypothetical protein 10.8 21.7 TM1793 hypothetical protein 9.5 17.5 TM1794 hypothetical protein 10.3 24.2 TM1796 hypothetical protein 5.8 19.1 TM1799 hypothetical protein 18.5 24.5 TM1801 hypothetical protein 5.6 13.8 TM1802 hypothetical protein 7.3 18.3 TM1804 hypothetical protein 4.7 14.3 TM1805 hypothetical protein 5.0 32.1 TM1806 hypothetical protein 8.3 20.1 TM1808 hypothetical protein 2.2 5.8

356

Table B9 continued TM1809 hypothetical protein 15.4 19.5 TM1812 hypothetical protein 3.5 15.4 TM1814 hypothetical protein -2.6 9.8 TM1815 ferredoxin 3.1 28.1 TM1816 hypothetical protein 7.7 25.6 TM1822 ftsH protease activity modulator HflK -2.5 5.6 TM1826 bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase -3.2 8.6 TM1827 riboflavin synthase subunit α 20.0 28.3 TM1828 riboflavin-specific deaminase -2.1 8.2 TM1829 hypothetical protein -3.9 20.7 TM1831 transposase, putative 8.0 17.9 TM1832 transposase 12.6 18.6 TM1836 maltose ABC transporter, permease protein 3.2 10.7 TM1838 hypothetical protein -3.4 8.9 TM1841 hypothetical protein 3.1 19.6 TM1844 hypothetical protein 4.7 20.3 TM1848 cellobiose-phosphorylase -2.7 10.8 TM1849 hypothetical protein -2.5 12.1 TM1851 α-mannosidase, putative -2.8 11.3 TM1852 hypothetical protein -3.4 17.6 TM1855 sugar ABC transporter, periplasmic sugar-binding protein -72.6 34.1 TM1856 transcriptional regulator, LacI family -2.9 12.7 TM1858 recX protein, putative 8.0 12.6 TM1860 hypothetical protein 17.2 21.3 TM1861 CDP-diacylglycerol--glycerol-3-P3-phosphatidyltransferase -4.0 12.7 TM1862 hypothetical protein 4.5 8.4 TM1863 hypothetical protein 9.2 23.6 TM1866 membrane bound protein LytR, putative 5.5 20.0 TM1869 ATP-dependent protease LA, putative 5.3 17.7 TM1871 hypothetical protein 16.6 23.5 TM1872 hypothetical protein 2.8 14.4 TM1873 ornithine decarboxylase 6.2 23.3 TM1875 glutamyl-tRNA synthetase 2.7 11.4 TM1876 hypothetical protein 8.4 20.2 TM1878 bifunctional UDP-sugar hydrolase 4.5 8.6 TMrrnaA16 TMrrnaA16 -2.6 17.7 TMrrnaA23 TMrrnaA23 -2.0 12.9 Tpet_0213 hypothetical protein 6.0 15.0 Tpet_0485 extracellular solute-binding protein, family 5 2.3 10.5 Tpet_0636 extracellular solute-binding protein, family 1 2.9 10.3 Tpet_0944 hypothetical protein 2.2 10.2 Tpet_1751 phosphoadenosine phosphosulfate reductase -2.5 6.6

357

Table B9 continued Tpet_1753 hypothetical protein -2.8 12.8 Tpet_1765 metal dependent phosphohydrolase 3.2 14.7 Tpet_1768 hypothetical protein 24.5 33.2 Tpet_1774 hypothetical protein -2.8 6.2 Tpet_1776 ATPase (AAA+ superfamily)-like protein -3.3 14.5 Tpet_1785 DNA mismatch repair protein MutS domain protein 2.8 18.3

358

Table B10. ORFs differentially transcribed in the T. zoo vs. T. sp RQ2 in glucose culture. Positive values indicate up-regulation in the zoo, negative values up-regulation in the pure culture Fold P- Probe ID Annotation Change value TRQ2_0153 hypothetical protein, 2A7 TRQ2_0154 PSP1 domain -2.0 13.0 2C2 TRQ2_1109 metallophosphoesterase -2.2 13.3 2F2 TRQ2_0970 extracellular solute-binding protein family 1 -2.1 12.5 2H5 TRQ2_0512 extracellular solute-binding protein family 1 -2.5 13.5 A8 TRQ2_0662 glycoside hydrolase family 43 2.1 12.4 B5 TRQ2_1838 hypothetical protein -2.0 9.2 TRQ2_0317 hypothetical protein, E2 TRQ2_0318 hypothetical protein 2.2 12.5 TRQ2_0303 glucose-1-phosphate thymidylyltransferase, E9 TRQ2_0304 dTDP-4-dehydrorhamnose 3,5 epimerase 2.1 8.0 GAPDH TRQ2_0241 glyceraldehyde-3-phosphate dehydrogenase, -2.6 18.1 TRQ2_1548 type IV pilin-related protein, KJ3B6 TRQ2_1549 endo-1,4-β-xylanase -4.3 13.1 KJB3 TRQ2_0658 α-N-arabinofuranosidase 2.2 11.6 NTPE TRQ2_1103 H+ transporting two-sector ATPase E subunit -2.2 8.4 TAA12 TRQ2_0305 dTDP-glucose 4,6-dehydratase 2.6 13.3 TAA49 TRQ2_1833 hypothetical protein -2.1 8.4 TAA57 TRQ2_0563 major facilitator superfamily MFS_1 -2.3 10.7 TRQ2_1548 type IV pilin-related protein, TAA59 TRQ2_1549 endo-1,4-β-xylanase -3.0 11.6 TAA96 TRQ2_0503 hypothetical -2.1 9.0 TRQ2_0522 iron-containing alcohol dehydrogenase, TAB17 TRQ2_0524 hypothetical protein -2.2 9.2 TAC01 TRQ2_0657 hypothetical protein 2.0 7.8 TAC08 TRQ2_0661 extracellular solute-binding protein family 1 2.4 9.3 TAC47 TRQ2_1656 hypothetical protein -3.4 14.3 TRQ2_0665 hypothetical protein, TAC83 TRQ2_0666 hypothetical protein 2.1 7.5 TAD02 TRQ2_1840 pseudouridine synthase, RluA family -2.3 12.4 TRQ2_0086 PEGA domain protein, TAD39 TRQ2_0087 hypothetical protein 3.1 14.2 TAD66 TRQ2_0973 LacI transcriptional regulator -2.3 11.7 TAD72 TRQ2_0284 lipopolysaccharide biosynthesis protein -2.0 9.8 TAE01 TRQ2_1645 metal dependent phosphohydrolase -2.5 13.2 TAE55 TRQ2_0611 TRAP dicarboxylate transporter, DctP subunit -2.2 9.3 TAE56 TRQ2_0283 NAD+ synthetase -2.3 13.5 TAE82 TRQ2_0658 α-N-arabinofuranosidase -2.0 12.5 TM_tRNA-Arg-3 TM_tRNA-Arg-3 -2.2 10.9 TM_tRNA-Arg-4 TM_tRNA-Arg-4 -2.0 6.6 TM_tRNA-Gly-2 TM_tRNA-Gly-2 -2.0 9.6 TM_tRNA-Met-2 TM_tRNA-Met-2 2.2 9.5

359

Table B10 continued TM_tRNA-Ser-1 TM_tRNA-Ser-1 2.0 6.2 TM_tRNA-Ser-2 TM_tRNA-Ser-2 2.1 7.1 TM_tRNA-Ser-3 TM_tRNA-Ser-3 2.1 6.1 TM0012 NADP-reducing hydrogenase, subunit A 2.2 11.3 TM0019 3-ketoacyl-(acyl-carrier-protein) reductase 2.8 14.2 TM0020 hypothetical protein 2.7 15.1 TM0023 methyl-accepting chemotaxis protein 2.6 6.1 TM0024 laminarinase 2.4 10.8 TM0025 β-glucosidase 2.1 7.6 TM0030 oligopeptide ABC transporter, permease protein 2.0 8.5 TM0033 hypothetical protein -2.3 7.2 TM0037 hypothetical protein 3.1 14.0 TM0045 hypothetical protein 2.3 14.3 TM0065 transcriptional regulator, IclR family 2.2 13.1 TM0071 ABC transporter, periplasmic binding protein 2.3 16.3 TM0073 oligopeptide ABC transporter, permease protein 2.1 5.6 TM0082 flagellar hook-associated protein 3 2.1 6.6 TM0096 hypothetical protein 2.2 17.2 TM0108 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 3.1 13.7 TM0113 xylU-related protein 2.3 12.5 TM0114 sugar ABC transporter, periplasmic sugar-binding protein 2.1 9.3 TM0120 oxidoreductase, putative 2.0 9.3 TM0129 carboxypeptidase G2, putative 2.4 7.3 TM0134 thioredoxin reductase-related protein 2.4 8.8 TM0138 tryptophan synthase subunit β -2.6 13.8 TM0141 anthranilate synthase component II -2.7 15.7 TM0144 hypothetical protein 2.5 9.3 TM0148 glucosamine--fructose-6-P aminotransferase, isomerizing 2.1 17.3 TM0149 fatty acid/phospholipid synthesis protein 2.2 18.4 TM0160 hypothetical protein 2.9 6.8 TM0164 hypothetical protein 2.3 13.5 TM0167 phosphopentomutase 2.4 8.2 TM0169 redox-sensing transcriptional repressor Rex -2.0 9.9 TM0170 hypothetical protein 2.0 8.7 TM0175 acyl carrier protein 2.8 14.1 TM0176 hypothetical protein -2.0 11.3 TM0178 primosomal protein N' 2.0 13.0 TM0180 hypothetical protein 2.3 16.2 TM0194 ABC transporter, ATP-binding protein -2.1 8.8 TM0201 NADP-reducing hydrogenase, subunit D, putative 2.3 14.9 TM0205 ATP-dependent DNA helicase 2.2 10.4 TM0207 hypothetical protein 3.0 12.9 TM0211 aminomethyltransferase -2.1 10.2 TM0212 glycine cleavage system H protein 2.3 14.8 TM0216 glycyl-tRNA synthetase subunit α -2.5 14.9 TM0217 glycyl-tRNA synthetase, βsubunit -2.0 5.5

360

Table B10 continued TM0219 flagellar export/assembly protein 2.2 10.7 TM0229 hypothetical protein -2.2 11.9 TM0254 SsrA-binding protein 3.0 12.7 TM0258 DNA topoisomerase 2.3 14.0 TM0261 phosphate permease, putative 2.9 13.3 TM0264 16S pseudouridylate synthase 2.8 11.6 TM0268 5-methyltetrahydrofolate S-homocysteine methyltransferase -2.6 17.1 TM0302 ABC transporter, permease protein -2.0 10.1 TM0309 ABC transporter, periplasmic binding protein 2.2 9.0 TM0328 m4C-methyltransferase -2.1 12.1 TM0329 hypothetical protein 3.2 13.4 TM0332 orotidine 5'-phosphate decarboxylase, putative 2.4 10.9 TM0335 dihydroorotase -2.0 8.2 TM0338 hypothetical protein 2.5 10.2 TM0340 hypothetical protein 2.7 9.7 TM0345 3-phosphoshikimate 1-carboxyvinyltransferase -2.4 11.0 TM0349 3-dehydroquinate dehydratase 3.1 8.4 TM0361 hypothetical protein -4.4 12.0 TM0365 putative aminopeptidase 1 2.0 11.6 TM0376 hypothetical protein 2.3 8.6 TM0384 anaerobic ribonucleoside-triphosphate reductase-related protein 2.3 7.5 TM0392 hypothetical protein -2.1 12.9 TM0400 sensor histidine kinase 2.1 12.8 TM0401 thymidine kinase 2.5 11.0 TM0405 2-oxoglutarate ferredoxin oxidoreductase subunit β 2.8 16.9 TM0407 diacylglycerol kinase, putative 3.8 13.3 TM0409 hypothetical protein 2.1 8.3 TM0410 hypothetical protein 2.6 11.4 TM0415 hypothetical protein 2.9 8.4 TM0420 sugar ABC transporter, permease protein 2.4 11.0 TM0430 sugar ABC transporter, permease protein -2.2 6.4 TM0434 α-glucosidase, putative -2.4 13.4 TM0438 6-phosphogluconate dehydrogenase 2.2 12.8 TM0450 hypothetical protein 2.4 10.4 TM0451 ribosomal protein L33 3.1 14.9 TM0453 N utilization substance protein G 2.0 10.7 TM0454 ribosomal protein L11 2.1 15.8 TM0457 ribosomal protein L7/L12 3.8 15.8 TM0463 lipoprotein signal peptidase 2.3 11.3 TM0465 hypothetical protein 2.5 11.6 TM0471 hypothetical protein 2.6 17.3 TM0472 amidotransferase, putative 2.2 15.2 TM0489 hypothetical protein 2.3 10.1 TM0495 phoH-related protein -2.2 14.5 TM0512 hypothetical protein 2.5 9.2 TM0519 hypothetical protein -2.3 10.2 TM0523 hypothetical protein 2.1 10.7

361

Table B10 continued TM0525 tRNA δ-2-isopentenylpyrophosphate transferase -2.1 7.7 TM0539 tryptophan synthase subunit β -2.1 7.5 TM0541 fumarate hydratase, C-terminal subunit 3.4 16.3 TM0544 ABC transporter, ATP-binding protein -2.1 8.9 TM0550 ketol-acid reductoisomerase -2.6 10.0 TM0560 hypothetical protein -2.4 13.0 TM0561 divalent cation transport-related protein -2.1 10.9 TM0566 hypothetical protein 2.7 15.6 TM0569 hypothetical protein 2.1 13.3 TM0570 cell division protein FtsY -2.1 5.5 TM0571 heat shock serine protease, periplasmic 4.0 19.0 TM0579 hypothetical protein -2.2 9.2 TM0604 single stranded DNA-binding protein, putative 2.2 13.1 TM0607 hypothetical protein -2.2 11.3 TM0626 hypothetical protein 2.5 9.0 TM0640 hypothetical protein 2.6 12.7 TM0643 clostripain-related protein -2.5 10.8 TM0650 hypothetical protein -2.2 11.5 TM0653 hypothetical protein -2.4 9.9 TM0663 hypothetical protein -2.2 8.5 TM0669 hypothetical protein 2.3 11.5 TM0674 flagellar protein, putative -2.1 7.5 TM0681 dehydrase-related protein 3.4 12.8 TM0690 hypothetical protein 2.4 13.4 TM0691 hypothetical protein -2.3 10.8 TM0694 trigger factor, putative -2.2 11.5 TM0697 flagellar biosynthesis protein FliQ -2.1 9.2 TM0709 hypothetical protein -2.2 10.9 TM0710 transcriptional regulator, MarR family 2.0 9.7 TM0714 hypothetical protein -2.3 8.9 TM0719 cysteinyl-tRNA synthetase -2.1 9.1 TM0727 pmbA-related protein 2.2 9.4 TM0744 hypothetical protein 2.8 15.0 TM0745 hypothetical protein -2.1 6.7 TM0768 hypothetical protein -2.3 11.7 TM0770 hypothetical protein -2.3 11.9 TM0775 translation initiation factor IF-2 2.1 17.4 TM0778 hypothetical protein 2.1 10.8 TM0779 hypothetical protein 2.7 11.6 TM0780 bacterioferritin comigratory protein, ahpC/TSA family 2.5 11.7 TM0787 putative thiazole biosynthetic enzyme -2.2 9.1 TM0788 thiamine biosynthesis protein ThiC -2.7 12.6 TM0804 hypothetical protein 2.8 14.6 TM0813 hypothetical protein 2.2 15.0 TM0828 sugar kinase, pfkB family 2.5 11.8 TM0830 hypothetical protein 2.1 8.2 TM0832 hypothetical protein 2.4 7.2

362

Table B10 continued TM0833 DNA gyrase, subunit B 2.3 6.9 TM0834 hypothetical protein 2.1 9.3 TM0835 cell division protein FtsA, putative 2.5 13.1 TM0841 S-layer-like array protein -2.6 13.9 TM0842 response regulator 3.1 13.6 TM0843 formiminotransferase-cyclodeaminase -2.0 6.6 TM0848 hypothetical protein 2.1 15.7 TM0849 dnaJ protein -2.1 6.7 TM0853 sensor histidine kinase 2.4 10.7 TM0857 riboflavin kinase/FMN adenylyltransferase -2.5 10.3 TM0868 glutaredoxin-related protein -2.3 11.7 TM0872 hypothetical protein 2.3 16.7 TM0876 hypothetical protein 3.0 17.2 TM0877 enolase 2.4 12.2 TM0881 homoserine O-succinyltransferase -2.5 12.9 TM0891 gcpE protein -2.3 8.3 TM0895 glycogen synthase 2.2 10.8 TM0904 chemotaxis protein CheC 2.3 13.8 TM0912 basic membrane protein, putative 2.4 8.2 TM0923 hypothetical protein 2.3 17.9 TM0924 hypothetical protein -2.1 9.7 TM0932 hypothetical protein -2.2 6.7 TM0935 hypothetical protein -2.0 13.3 TM0941 hypothetical protein -2.5 17.8 TM0942 hypothetical protein -2.6 9.7 TM0943 glutamine synthetase -2.4 9.6 TM0950 hypothetical protein -2.3 9.9 TM0956 ribose ABC transporter, ATP-binding protein -2.5 18.3 TM0963 oligoendopeptidase, putative -2.2 10.1 TM0972 conserved hypothetical protein, GGDEF domain -2.1 7.4 TM0982 hypothetical protein 2.0 6.4 TM0984 hypothetical protein -2.4 8.7 TM0986 hypothetical protein -2.2 8.2 TM0988 hypothetical protein -2.3 14.2 TM1001 hypothetical protein 2.1 11.2 TM1015 glutamate dehydrogenase -2.0 6.3 TM1016 hypothetical protein 2.4 10.5 TM1028 ABC transporter, ATP-binding protein 2.0 9.8 TM1035 phosphoribosyl-AMP cyclohydrolase -2.2 19.5 TM1047 septum site-determining protein MinC, putative 2.1 6.0 TM1058 glutamate synthase-related protein -2.2 14.7 TM1084 DNA gyrase, subunit A 2.2 18.8 TM1091 hypothetical protein -2.2 15.8 TM1107 hypothetical protein 2.1 9.5 TM1119 hypothetical protein -2.0 10.4 TM1142 hypothetical protein 2.2 9.2 TM1148 isocitrate dehydrogenase -2.4 10.6

363

Table B10 continued TM1171 transcriptional regulator, crp family -2.1 9.2 TM1176 transcriptional regulator, metal-sensing -2.0 9.9 TM1177 hypothetical protein -2.5 12.6 TM1186 hypothetical protein 2.1 8.3 TM1192 α-galactosidase 2.0 10.3 TM1200 transcriptional regulator, LacI family 2.4 14.9 TM1202 maltose ABC transporter, permease protein 2.1 11.4 TM1206 putative monovalent cation/H+ antiporter subunit F -2.5 8.5 TM1214 NADH dehydrogenase subunit B 3.1 11.1 TM1224 transcriptional regulator, XylR-related 2.7 18.6 TM1231 α-mannosidase-related protein -2.0 11.9 TM1246 phosphoribosylformylglycinamidine synthase II 2.9 11.0 TM1247 amidophosphoribosyltransferase -2.1 12.6 TM1252 hypothetical protein -2.4 12.1 TM1255 aspartate aminotransferase -2.3 10.3 TM1261 phosphate ABC transporter, ATP-binding protein -2.2 11.0 TM1262 phosphate ABC transporter, permease protein -2.6 13.7 TM1265 hypothetical protein -2.1 12.2 TM1272 glutamyl tRNA-Gln amidotransferase, subunit A -2.0 6.1 TM1276 sugar ABC transporter, ATP-binding protein -2.4 14.4 TM1281 6-phospho-β-glucosidase 2.9 14.7 TM1299 hypothetical protein -2.0 9.5 TM1300 Subtilosin A-like bacteriocin, putative 2.6 15.6 TM1320 HicA toxin, putative 2.8 11.5 TM1321 HicB antitoxin, putative 3.5 14.2 TM1336 permease, putative -2.0 7.6 TM1343 frame shift 2.2 12.7 TM1359 sensor histidine kinase -2.1 8.0 TM1382 hypothetical protein 3.0 8.5 TM1388 hypothetical protein -2.3 10.7 TM1394 hypothetical protein -2.0 7.0 TM1397 phosphatidate cytidylyltransferase, putative 2.6 9.7 TM1398 hypothetical protein -2.4 11.9 TM1399 ribosome recycling factor 3.0 15.4 TM1402 hypothetical protein -2.5 14.0 TM1405 lipopolysaccharide biosynthesis protein-related protein -2.1 11.5 TM1406 hypothetical protein -2.1 10.6 TM1429 glycerol uptake facilitator protein -2.1 10.1 TM1433 oxidoreductase, putative -2.4 13.2 TM1453 ribosomal protein S9 2.2 14.0 TM1460 jag protein, putative 2.2 17.8 TM1464 hypothetical protein 2.2 16.9 TM1465 cob(I)yrinic acid a,c-diamide adenosyltransferase 2.2 7.1 TM1469 glucokinase -2.3 9.6 TM1472 DNA-directed RNA polymerase subunit α -2.2 10.3 TM1473 30S ribosomal protein S4 2.7 14.1 TM1474 30S ribosomal protein S11 2.9 16.5

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Table B10 continued TM1475 30S ribosomal protein S13 2.9 16.7 TM1477 translation initiation factor IF-1 2.5 16.2 TM1479 adenylate kinase 2.6 15.7 TM1484 ribosomal protein L18 2.1 11.0 TM1488 50S ribosomal protein L5 2.3 12.8 TM1492 ribosomal protein L29 2.2 6.4 TM1495 ribosomal protein L22 2.2 10.0 TM1496 ribosomal protein S19 2.1 13.9 TM1500 ribosomal protein L3 2.4 15.6 TM1501 ribosomal protein S10 2.6 13.2 TM1504 30S ribosomal protein S7 2.2 9.3 TM1505 30S ribosomal protein S12 2.7 21.1 TM1508 hypothetical protein 2.0 12.7 TM1509 hypothetical protein 2.3 13.6 TM1522 diaminopimelate epimerase 2.7 13.8 TM1528 1,4-dihydroxy-2-naphthoate octaprenyltransferase, putative 2.1 10.6 TM1533 ferredoxin 2.1 6.5 TM1536 hypothetical protein -2.1 10.6 TM1550 hypothetical protein -2.1 9.8 TM1556 maf protein 2.7 5.9 TM1568 16S rRNA processing protein, putative 2.2 19.5 TM1570 hypothetical protein 3.2 15.0 TM1572 signal peptidase I, putative 2.1 12.7 TM1578 preprotein translocase subunit SecA 2.1 8.5 TM1597 hypothetical protein -2.2 13.7 TM1598 RNA polymerase sigma-E factor 2.7 13.0 TM1600 hypothetical protein -2.2 13.4 TM1608 hypothetical protein -2.2 9.7 TM1609 ATP synthase F1, subunit epsilon 2.7 16.7 TM1618 cheX protein -2.4 11.7 TM1624 β-mannosidase, putative 2.7 13.6 TM1629 UDP-N-acetylglucosamine pyrophosphorylase -2.3 11.3 TM1632 hypothetical protein 2.5 9.1 TM1633 ATP-dependent protease LA 2.5 13.0 TM1636 hypothetical protein 2.3 9.8 TM1637 hypothetical protein -2.4 10.2 TM1647 hypothetical protein -2.1 9.4 TM1663 ABC transporter, ATP-binding protein 2.2 16.0 TM1665 hypothetical protein 2.1 5.7 TM1671 hypothetical protein 2.7 10.0 TM1674 hypothetical protein -2.1 13.6 TM1677 transposase, putative 2.1 8.2 TM1680 hypothetical protein 2.3 7.3 TM1684 ribosomal protein L31 3.0 15.4 TM1691 hypothetical protein 2.0 18.5 TM1703 hypothetical protein -2.7 15.4 TM1705 lysyl-tRNA synthetase -2.2 9.8

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Table B10 continued TM1706 transcription elongation factor, greA/greB family 2.3 12.2 TM1708 hypothetical protein 2.0 11.4 TM1709 hypothetical protein 2.7 8.6 TM1712 hypothetical protein 2.3 11.1 TM1715 hypothetical protein -2.3 12.4 TM1728 3-methyl-2-oxobutanoate hydroxymethyltransferase 2.2 15.9 TM1731 hypothetical protein -2.1 6.5 TM1739 hypothetical protein 2.1 7.2 TM1742 nagD protein, putative 2.2 16.8 TM1751 endoglucanase 2.0 12.0 TM1756 butyrate kinase -2.1 7.6 TM1761 excinuclease ABC subunit B -2.4 13.3 TM1766 formate--tetrahydrofolate ligase 2.2 13.0 TM1772 hypothetical protein 2.6 14.3 TM1773 hypothetical protein 2.2 7.8 TM1774 cofactor-independent phosphoglycerate mutase 3.1 8.4 TM1783 bifunctional ornithine acetyltransferase -2.1 7.5 TM1792 hypothetical protein 2.7 11.8 TM1794 hypothetical protein 2.3 11.7 TM1796 hypothetical protein 2.2 10.4 TM1799 hypothetical protein 2.8 11.6 TM1806 hypothetical protein 2.7 11.9 TM1809 hypothetical protein 2.7 8.7 TM1816 hypothetical protein 2.1 13.2 TM1826 bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase -3.0 9.9 TM1827 riboflavin synthase subunit α 3.0 15.5 TM1829 hypothetical protein -2.0 14.0 TM1831 transposase, putative 2.7 10.4 TM1832 transposase 2.6 8.5 TM1844 hypothetical protein 2.5 15.4 TM1848 cellobiose-phosphorylase -2.2 10.0 TM1858 recX protein, putative 2.6 6.3 TM1860 hypothetical protein 2.7 9.5 TM1861 CDP-diacylglycerol--glycerol-3-P 3-phosphatidyltransferase -2.3 8.9 TM1863 hypothetical protein 2.5 13.6 TM1869 ATP-dependent protease LA, putative 2.0 9.0 TM1871 hypothetical protein 3.0 12.8 TM1872 hypothetical protein 2.0 12.1 TM1873 ornithine decarboxylase 2.1 12.9 TM1878 bifunctional UDP-sugar hydrolase 2.8 6.8

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