Characterization of the key enzymes involved

in anaerobic degradation of phthalate

A dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.)

at the University of Konstanz Faculty of Sciences Department of Biology

Submitted by:

Madan Junghare (M.Sc.) born on 3rd Nov. 1985 in Washim (MH), India

Konstanz, 2016

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-395942

Tag der mündlichen Prüfung: 2. Februar 2017

1. Gutachter: Prof. Dr. rer. nat. Bernhard Schink 2. Gutachter: Prof. Dr. Dieter Spiteller 3. Gutachter: Prof. Dr. Valentin Wittmann

ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere thanks to my supervisor Professor Bernhard Schink for giving me the opportunity to complete my Ph.D dissertation in his lab at the University of Konstanz on such an interesting project. You have provided me valuable guidance, support, and encouragements during my doctoral research, whenever it was needed. Secondly, I would like to thank my co-supervisor Professor Dieter Spiteller for his guidance, nurturing and offering me to use his lab facility for my research. You provided priceless aid for my research project. I wouldn’t have succeeded without your help. I really appreciate everything. Thanks to Professor Valentin Whitmann for acting as a third member of my thesis committee at the Graduate School of Chemical Biology. I am very thankful to Dr. David Schleheck for allowing me to use his proteomics lab facility for doing my research work. Thank you for your helpful discussion, suggestions, and for enriching my scientific knowledge during this period. I would like to thank everyone else in the Schink group for all their kindness, assistance, friendship and providing a great working environment, as well as enjoying coffee and cake time together. It has been a largely very enjoyable time. Vielen Vielen Danke nochmal an Alle! Many thanks to the friendly team at the Welcome Center, University of Konstanz for their support, advice and friendly cooperation in all bureaucratic matter. I like to acknowledge DAAD for providing me a PhD scholarship to begin my doctoral research and giving me an opportunity to study in Germany. A special thanks to my incredible family in India for their endless support, love and for all of the sacrifices that you’ve made on my behalf. You might not have known anything about my research, but you were always there for me. I dedicate this work to my father and mama, I know you would have been very happy and proud of me.

Special thanks to my wife for her support, care and love. And also thank you for reading my thesis, feedback and for spending sleepless nights during the thesis writing.

Lots of love to my sweet daughter Grishma.

I hope my research work has advanced the knowledge in the field of anaerobic phthalate degradation. Enjoy!

Table of contents

Dissertation summary……………………………………………………………………………………….……………………………………………07

Dissertation Zusammenfassung….…………….…………………………………………………………...………………………………………09

Chapter 1. General introduction 1. Aromatic compounds in nature….…………………..………………………………………..………………………………………………...12 1.1 Aromaticity and stability 1.2 Occurrence and sources 2. Microbial degradation of aromatic compounds in nature………………………………………………….………………….…....13 2.1 Aerobic degradation 2.2 Anaerobic degradation 3. Phthalate esters, applications and biodegradation…………….……………………………..…………………………………………19 3.1 Phthalate esters as ‘plasticizers’ 3.2 ‘Man-made’ phthalate as pollutant 4. Microbial degradation of phthalate esters………………………………………………………………..…………………………………22 4.1 Importance of phthalate esters degradation 4.2 Initial steps in phthalate ester degradation 4.3 Aerobic phthalate degradation 4.4 Anaerobic phthalate degradation 5. Aims of the dissertation and objectives………………………………………………………………………………………….…………...28 6. Research co-authored but not included in the dissertation…………………………………….…………………………………..29

Chapter 2. Draft genome sequence of a nitrate-reducing, o-phthalate-degrading bacterium, Azoarcus sp. strain PA01 Abstract…………………………………………………………………..………………………………………………………………………………………31 Introduction…………………………………………………………………………..………………………………………………………………………..31 Organism information……………………………….………………………………..…………………………………………………………………..32 Classification and features Chemotaxonomy Genome sequencing and information……………………………………………………………………………………………………………..36 Genome project history Growth conditions and genomic DNA preparation Genome sequencing and assembly Genome annotation Genome properties………………………………………………………………………………………………………………………………………….38 Insight from the genome sequence……………………………………..……………………………………………………………………...... 41

I

Chapter 3. Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01 Abstract……………………………………………………………………………………………………………………….……………………………….…45 Introduction……………………………………………………………………………………………..……………………………………………………..45 Results………………………………………………………………………………………………………………………………………………..…………..47 Anaerobic growth with o-phthalate and physiological characteristics Differential proteome analysis of o-phthalate-grown cells versus benzoate-grown cells Characterization of the gene cluster involved in anaerobic o-phthalate degradation Enzyme activity measurements with cell-free extracts of Azoarcus sp. strain PA01 Discussion...... …………………………………………………………………….…………………………………………………………….……………59 Experimental procedures…………………………………………………….………………………..………………………………………………..63 Bacterial strain and growth conditions Bacterial growth analysis Preparation of cell-free extracts Differential proteome analysis Protein identification by mass spectrometry Identification of gene clusters and phylogenetic analysis Determination of enzyme activities in cell-free extracts Synthesis of o-phthalyl-CoAs LC-MS/MS analysis of Coenzyme A esters Supporting information……………………………………………………………………………………………………………………………………70

Chapter 4. Cloning and over-expressions of genes involved in anaerobic o-phthalate decarboxylation to benzoate by the Azoarcus sp. strain PA01 Abstract……………………………………………………………………………………..……………………………………………………………………77 Introduction……………………………………………………..……………………………………………………………………………………………..77 Materials and methods…………………………………..……………………………………………………………………………………………….80 Materials and reagents Strains and cultivation conditions Gene amplifications, plasmid construction and sequencing Optimization of protein expression and purifications Molecular weight determination by SDS-PAGE Decarboxylase activity assays and cofactor identification Native protein PAGE analysis Sequence alignment and structure homology modelling Results………………………………………………………………………………………….………………….……………………………………………..86 Gene amplifications, cloning of PhtDa and PhtDb

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Heterologous expression, purification and protein identification Enzyme activity, substrate specificity and cofactor requirement Phylogenetic position of PhtDa and PhtDb Multiple sequence alignment and homology-based structure modelling Native PAGE and protein identification by MS analysis Discussion……………………………………………………………………………………………………………..…………………………………………98

Chapter 5: Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-stain-negative, benzoate-degrading, sulfate-reducing bacterium isolated from a wastewater treatment plant Abstract…………………………………………………………………………………………………………….…………………...... 103 Introduction………………………………………………………………………….………………………………….……………………………………104 Material and methods………………………………………..………………………………..……………………………………………………….104 Results and discussion……………….………………………………………………………….………………………………………………………107 Description of Desulfoprunum gen. nov. Description of Desulfoprunum benzoelyticum gen. nov., sp. nov.

Chapter 6: Enrichment cultures degrading phthalate isomers under nitrate-reducing, sulfate-reducing and fermenting conditions Introduction…………………………………………………………………………………………………………………..………………………………115 Phthalate isomer degradation under denitrifying conditions Phthalate degradation under sulfate-reducing/fermenting conditions Partial characterization of enrichment culture KOPA

Chapter 7: General discussion and concluding remarks General discussion…...…………………………………………………………………………………………………………………………………..121 Activation of o-phthalate by Azoarcus sp. strain PA01 Decarboxylation of activated o-phthalate by a two enzyme component system Concluding remarks and general outlook…………...………………………………………………...... 125

Personal record of achievements……………………………………………..…………………………………………………….126

List of publications……………………………………………………….………………….…………………………...127

Workshops and training courses attended……………………………….………………………………………………….128

References………………………………………………..………………………………..……………………………….129

Abbreviations………………………………………………………………….………………………………….…………146

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Dissertation summary

Anaerobic phthalate degradation was assumed to proceed through initial decarboxylation of phthalate (ortho) to benzoate. However, the intermediates and enzymes involved in anaerobic phthalate decarboxylation were largely unknown. The aim of this dissertation was to investigate the biochemistry of anaerobic phthalate degradation, especially the steps and enzymes involved in the decarboxylation of phthalate to benzoate. In particular, the study focused on the enrichment and isolation of anaerobic phthalate-degrading bacteria under nitrate-reducing, sulfate-reducing and fermenting conditions.

Strain PA01 was isolated and purified from an enrichment culture that degrades phthalate coupled to nitrate reduction. 16S rRNA gene sequencing suggested that strain PA01 is a member of the genus Azoarcus that is known for aromatic compound degradation. Azoarcus sp. strain PA01 could degrade a wide variety of aromatic compounds, including phthalate and benzoate coupled to nitrate reduction. No growth was observed with isophthalate or terephthalate. To gain detailed insights into the biochemistry of phthalate degradation, strain PA01 was genome sequenced. The draft genome of strain PA01 possesses the gene clusters for degradation of aromatic compounds, i.e. for benzoate degradation. Differential two-dimensional protein profiling of phthalate- versus benzoate-grown cells identified the specific proteins induced with phthalate.

The phthalate-induced protein-coding genes were found to constitute a single gene cluster in the genome of Azoarcus sp. strain PA01. Phthalate-induced proteins included a transporter, two CoA- transferases, and UbiX-like/UbiD-like decarboxylases. It was concluded that o-phthalate is first activated to o-phthalyl-CoA by a succinyl-CoA dependent succinyl-CoA:o-phthalate CoA-transferase (PhtSa and PhtSb), and is subsequently decarboxylated to benzoyl-CoA by an o-phthalyl-CoA decarboxylase (PhtDa and PhtDb). In vitro enzyme assays with cell-free extracts of phthalate-grown cells of Azoarcus sp. strain PA01 demonstrated the formation of o-phthalyl-CoA, specifically with o- phthalate and succinyl-CoA as the CoA donor, and established its subsequent decarboxylation to benzoyl-CoA using LC-MS analysis. Neither free CoA nor acetyl-CoA served as the CoA donor. Isophthalyl-CoA and terephthalyl-CoA were not decarboxylated. Phylogenetic analysis of phthalate- induced PhtSa and PhtSb proteins of strain PA01 revealed that they shared high sequence homology to the known enzyme succinyl-CoA:(R)-benzylsuccinate CoA-transferase involved in toluene activation in denitrifying T. aromatica. PhtDa and PhtDb proteins showed high similarity to the recently discovered enzyme family of UbiD-like and UbiX-like decarboxylases that function in ubiquinone synthesis in a wide range of bacteria.

Furthermore, cloning and heterologous expression of the PhtDa and PhtDb proteins in host E. coli verified that these proteins together indeed decarboxylate phthalyl-CoA to benzoyl-CoA. PhtDb is a

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flavin mononucleotide (FMN)-binding protein homologous to known FMN-binding UbiX-like of E. coli, which itself does not possess decarboxylase activity. Instead, it generates a modified-FMN cofactor that is required by PhtDa for decarboxylase activity. Multiple sequence alignments and structure modelling of both proteins suggested that only PhtDb has a binding site for a FMN ligand. This strongly indicates that PhtDb bound FMN plays an essential role in the decarboxylation of o-phthalyl- CoA. Further, it is assumed that FMN functions as a potential electron shuttle between the phthalate ring and the enzyme complex (PhtDa and PhtDb) for facilitating the anaerobic phthalate decarboxylation. Additionally, structural modelling based on known structures of UbiX/UbiD-like enzymes suggested that PhtDb (22 kDa) forms a dodecamer and PhtDa (60 kDa) a homodimer that together build an enzyme complex of about 400 kDa. Native gel analysis of cell-free extract from Azoarcus sp. strain PA01 showed a protein band with an approximate molecular size of 380-400 kDa from which only PhtDa and PhtDb proteins were identified by MS analysis. These results were further supported by native gel analysis of recombinant the PhtDa and PhtDb proteins together which showed a single protein band of molecular size in the same range (380 - 400 kDa).

A mixed culture (KOPA) degrading phthalate was enriched under sulfate-reducing conditions. Isolation and purification of bacteria from the mixed culture resulted in the identification of a novel benzoate-degrading bacterium Desulfoprunum benzoelyticum gen. nov., sp. nov. D. benzoelyticum could not degrade phthalate, but bacterial community analysis of the KOPA culture revealed that, it is a predominant bacterium in the enrichment culture. Other potential phthalate degraders include members of the family Desulfobulbaceae. The enrichment culture could also simultaneously be adapted for benzoate utilization, indicating that phthalate degradation occurs through phthalate decarboxylation to benzoate. Addition of 5 mM molybdate completely inhibited phthalate degradation, suggesting that sulfate reducers are key phthalate degrading bacteria in the enrichment. We assumed that in sulfate-reducing/fermenting bacteria, phthalate is activated to phthalyl-CoA by CoA-transferase possibly with acetyl-CoA as CoA donor, analogous to the denitrifying Azoarcus sp. strain PA01 in which succinyl-CoA acts as CoA donor to form phthalyl-CoA which is then decarboxylated to benzoyl- CoA.

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Dissertation Zusammenfassung

Bisher wurde angenommen, dass der anaerobe Abbau von Phthalat über Benzoat als Intermediat verläuft, wobei Phthalat decarboxyliert wird. Allerdings waren die Intermediate und beteiligten Enzyme dieser ersten Schritte des anaeroben Abbaus von Phthalat bisher unbekannt. Ziel dieser Dissertation war es zu untersuchen, wie Phthalat von Anaerobiern abgebaut wird. Insbesondere die ersten Schritte und die beteiligten Enzyme des Phthalatabbaus sollten identifiziert werden. Dazu wurden Phthalat-abbauende anaerobe Bakterien unter Nitrat-reduzierenden, Sulfat-reduzierenden und fermentativen Bedingungen angereichert bzw. isoliert.

Der Nitrat-reduzierende, Phthalat-abbauende Stamm PA01 konnte isoliert und als Reinkultur erhalten werden. Eine 16S rDNA Analyse ergab, dass dieser Stamm zum Genus Azoarcus gehört, deren Vertreter für den Abbau von aromatischen Verbindungen bekannt sind. Azoarcus sp. Stamm PA01 wuchs mit einer Vielzahl aromatischer Verbindungen, auch mit Phthalat und Benzoat als Kohlenstoffquelle. Mit Isophthalat oder Terephthalat konnte Stamm PA01 allerdings nicht wachsen. Um zu untersuchen, wie Stamm PA01 Phthalate abbaut, wurde das Genom von Stamm PA01 sequenziert. Im Genom von Stamm PA01 konnte ein Gencluster für den Abbau von Benzoat gefunden werden.

Mit Hilfe von differentieller zwei-dimensionaler Proteomanalyse von mit Phthalat angezogenen Stamm PA01-Zellen im Vergleich zu mit Benzoat angezogenen Zellen konnten Proteine identifiziert werden, die spezifisch beim Wachstum mit Phthalat induziert waren: ein Transporter, zwei Coenzym A (CoA)-Transferasen und eine UbiX-artige bzw. eine UbiD-artige Decarboxylase. Die Gene für diese durch Phthalat induzierten Proteine befinden sich in einen Gencluster im Genom von Stamm PA01. Die Ergebnisse legten nahe, dass Phthalat zuerst durch eine Succinyl-CoA-abhängige Succinyl- CoA:o-Phthalat CoA-transferase aktiviert wird. Anschließend wird Benzoyl-CoA durch eine o-Phthalyl- CoA-Decarboxylase erhalten. In vitro Enzymassays mit zellfreien Extrakten von mit Phthalat angezogenen Zellen bildeten o-Phthalyl-CoA in Gegenwart von Phthalat und Succinyl-CoA. Die Decarboxylierung von Phthalyl-CoA zu Benzoyl-CoA wurde durch LC-MS Messungen bestätigt. Weder freies Coenzym-A noch Acetyl-CoA konnten als CoA-Donor fungieren. Isophthalyl-CoA oder Terephthalyl-CoA wurden nicht als Substrate der Decarboxylase akkzeptiert. Eine phylogenetische Analyse von PhtDa und PhtDb zeigte, dass beide Proteine zu den kürzlich gefundenen Enzymfamilien UbiD-artiger und UbiX-artiger Decarboxylasen gehören, die in vielen Bakterien an der Ubichinonbiosynthese beteiligt sind.

Durch Klonierung und heterologe Expression von PhtDa und PhtDb in E. coli konnte belegt werden, dass beide Enzyme eine spezifische und essentielle Rolle bei der Decarboxylierung von Phthalyl-CoA zu Benzoyl-CoA spielen. PhtDb ist ein Flavinmononucleotid (FMN)-bindendes Protein homolog zu UbiX; es zeigt allein keine Decarboxylase Aktivität. Statt dessen bildet PhtDb einen modifizierten FMN-Cofaktor, der von PhtDa (UbiD-artiges Protein) zur Decarboxylase-Aktivität benötigt wird.

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Aminosäuresequenzvergleich und Proteinstrukturmodellierung beider Proteine unterstützen, dass nur eines eine FMN Bindestelle besitzt. PhtDb gebundenes, modifiziertes FMN spielt eine entscheidende Rolle bei der Decarboxylierung von Phthalyl-CoA und dient als potentieller Elektronenüberträger, um die Decarboxylierung zu erleichtern.

Eine Anreicherungskultur (KOPA) unter Sulfat-reduzierenden Wachstumsbedingungen konnte Phthalate und Benzoate abbauen. Das zeigt, dass unter Sulfat-reduzierenden Bedingungen Phthalate ebenfalls zu Benzoat decarboxyliert werden und dieses weiter verstoffwechselt wird. Mit Hilfe von anoxischen Agar-Verdünnungsreihen konnte das neue Benzoat-abbauende Bakterium Desulfoprunum benzoelyticum gen. nov., sp. nov. erhalten werden,; es konnte jedoch kein Phthalat abbauen. Eine Analyse der bakteriellen Gemeinschaft der KOPA Anreicherungskultur zeigte, dass Desulfoprunum benzoelyticum gen. nov. ein dominierendes Bakterium in dieser Anreicherungskultur ist. Potentiell Phthalat-abbauende Bakterien wären Mitglieder der Familie Desulfobulbaceae. Demnach würde unter Sulfate-reduzierenden Bedingungen Phthalat auch nach initialer Decarboxylierung als Benzoat oder Benzoyl-CoA den Benzoat-Abbauweg durchlaufen. Durch 5 mM Molybdate konnte der Phthalat-Abbau der Sulfat-Reduzierer vollständig gehemmt werden. Dies legt nahe, dass Mitglieder der Familie Desulfobulbaceae die potentiell Phthalat-abbauenden Bakterien in der anreicherungskultur KOPA stellen. Es wird vermutet, dass in Sulfat-reduzierenden Bakterien Phthalat ähnlich wie in den Nitrat-reduzierenden Bakterien aktiviert wird, allerdings verwenden sie vielleicht Acetyl-CoA anstelle von Succinyl-CoA als CoA Donor.

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CHAPTER 1

General Introduction

Introduction

1. Aromatic compounds in nature

1.1 Aromaticity and stability Aromatic compounds are cyclic (ring-shaped), planar (flat) molecules with a delocalised mobile π electron system that builds a ring of resonance bonds exhibiting high stability. E.g. in the benzene molecule six delocalised π electrons are evenly distributed over the six- membered carbon ring. So the position of these electrons cannot be described by a single structural formula, but rather by two resonance structures (Figure 1). The resonance energy provides the stability and also increases the inertness of C-H and C-C bonds, making them resistant to microbial degradation in the environment [Gibson and Subramanian, 1984].

Figure 1. Representation of two different resonance forms of benzene (in square bracket) which combine to produce an average planar structure (right).

1.2 Occurrence and sources Aromatic compounds represent the second most abundant source of organic carbon in nature (after carbohydrates) largely produced by a variety of biological processes [Pérez-Pantoja et al., 2009]. The natural sources of aromatic compounds include tannins, humic compounds, secondary metabolites of plants and the lignin polymer which is one of the most abundant structural polymers in higher plant tissue, as well as the amino acids, e.g. histidine, phenylalanine, tryptophan and tyrosine. Extensive use of natural and synthetic aromatic compounds from the start of the industrial revolution have introduced a wide variety of organic substances into the environment through various processes such as crude oil spillage, fossil fuel combustion, gasoline leakage, as well as natural inputs like forest fire and natural petroleum seepage [Foght, 2008; Weelink et al., 2010].

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Introduction

2. Microbial degradation of aromatic compounds in nature

Animals and plants cannot degrade aromatic compounds and thus, they do not contribute for their mineralization. In contrast, the domain of microorganisms, particularly fungi (which can even degrade lignin), bacteria and some algae can break down most aromatic compounds present in the nature [Heider and Fuchs, 1997]. Because these compounds serve as the important source of carbon and energy for the growth of these diverse microorganisms. Hence, removal of several aromatic and synthetic (man-made) compounds from the environment often relies on microbial activities, i.e., on ‘bioremediation’. Bioremediation include the removal of hazardous pollutants from the environment by the action of microbes which convert them into harmless metabolites, e.g., carbon dioxide and water [Alexander, 1999]. Therefore, microorganisms play a vital role in recycling of global carbon and maintaining the health of the biosphere [Dagley, 1978]. Degradation of aromatic compounds in nature is mainly dominated by aerobic (oxygen-dependent) and anaerobic (oxygen- independent) bacteria including aerobic fungi. In all cases the main challenge for microbial degradation is posed by the inherent stability of aromatic ring and requires a different biochemical strategy for ring destabilization.

2.1 Aerobic degradation The degradation of a wide variety of aromatic compounds by the aerobic bacteria involves several peripheral pathways for aromatic ring opening, in which the stable aromatic ring is first activated by the addition of one or two oxygen atoms by oxygen-dependent mono- or dioxygenases [Gibson and Subramanian, 1984] leading to hydroxy substituted intermediates such as catechol (1,2-dihydroxybenzene) or protocatechuate (3,4-dihydroxybenzoate) [Fuchs et al., 2012; Dagley et al., 1960]. Different peripheral reactions are involved for each aromatic substrate, that lead to the central hydroxy intermediates which are further commonly degraded by ring-cleaving ozygen-dependent dioxygenases [Gibson and Parales, 2000; Lipscomb, 2008]. For instance, aromatic compounds with one hydroxy group, such as phenol or 3-hydroxybenzoate, a monooxygenase adds one oxygen atom to the aromatic ring leading to catechol or protocatechuate, respectively, while the other oxygen atom is reduced to water [Powlowski and Shingler, 1994]. However, aromatic compounds without hydroxy groups, such as benzene or benzoate, a dioxygenase adds two oxygen atoms to the aromatic ring

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Introduction resulting in cis-dihydrodiol formation, which is then finally oxidized to common intermediates such as catechol in case of benzene or protocatechuate in case of benzoate (which can alternatively be metabolized to catechol) as shown in Figure 2 [Dagley et al., 1964].

Figure 2. Pathways for the aerobic degradation of benzene, phenol, benzoate and 3- hydroxybenzoate which are initially activated and hydroxylated through the peripheral reactions to key intermediates, such as catechol or protocatechuate. These intermediates are then further degraded by the ring-cleaving dioxygenase, yielding succinate and acetyl-CoA through β-adipate pathway, which enters into the central metabolism [Figure modified from Fritsche and Hofrichter, 2008; Powlowski and Shingler, 1994; Fuchs et al., 2012].

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Introduction

Although, the aromaticity of these intermediates, e.g., catechol or protocatechuate is still intact. The presence of electron-rich hydroxy group polarizes the ring and weakens the aromatic nature of the ring. As a result, intermediates can be easily cleaved by the action of oxygen-dependent dioxygenases which require another molecule of oxygen [Parales and Resnick, 2006; Gibson and Parales, 2000]. The ring-cleavage may occur in ortho position, between the two hydroxy groups, or in the meta position, adjacent to the introduced hydroxy groups [Vaillancourt et al., 2006]. In the ortho-cleavage pathway of catechol or protochatechuate (Figure 2), a dioxygenase uses two oxygen atoms to cleave the aromatic ring yielding cis,cis-muconate and 3-carboxy muconate, respectively, and finally converted to non-aromatic, unsaturated, open-chain carboxylic acids, e.g., acetyl- and succinyl-CoA derivatives through the well-known β-adipate pathway [Stanier and Ornston, 1973; Harwood and Parales, 1996]. These smaller carboxylic acids are metabolized through central metabolism to CO2. In principle, aerobic bacteria use the reactivity of molecular oxygen with the help of oxygenases to overcome the activation energy needed to destabilize the aromatic ring. Therefore, oxygen is not only acts as the final electron acceptor in aerobic respiration, but also needed as a co-substrate for two key processes, i.e., the hydroxylation and the aromatic ring-cleavage (Figure 2).

2.2 Anaerobic degradation W. C. Evans established that aromatic compounds can be metabolized by bacteria under anoxic conditions [reviewed in Evans, 1977; Evans and Fuchs, 1988; Boll et al., 2002]. In the environment, in fact, many habitats are often anoxic, e.g., aquifers, aquatic sediments, sludge digesters or carbon-rich aerobic sites in which anaerobic degradation is the dominant process [Carmona et al., 2009]. In contrast to aerobic degradation (as described above), the main challenge for anaerobic degradation of aromatic compounds is to activate and destabilize the inert aromatic ring in the absence of molecular oxygen. Therefore, different biochemical strategy is required and anaerobic bacteria destabilize the aromatic ring by reductive manner, which requires energy and strong reducing agents with a highly negative redox potential [Philipp and Schink, 2012].

Different peripheral pathways are involved in the anaerobic breakdown of a wide variety of aromatic compounds in which substrates are partially modified that leading to few central

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Introduction intermediates such as benzoyl-coenzymeA (benzoyl-CoA), resorcinol (1,3- dihydroxybenzene), phloroglucinol (1,3,5-trihydroxybenzene) and hydroxyhydroquinone [Schink et al., 1992; Gibson and Harwood, 2002; Heider and Fuchs, 1997; Harwood et al., 1999]. Except for the benzoyl-CoA, the presence of two or more hydroxy substituents in the central intermediates (as mentioned above) polarizes the ring which decreases the aromaticity and thus facilitates the electron transfer by aromatic ring reductases [Carmona et al., 2009; Philipp and Schink, 2012]. For instance, resorcinol, phloroglucinol and hydroxyhydroquinone possess keto/enol tautomerism that decreases ring-aromaticity and can be easily reduced with biological reductants, such as NAD(P)H in case of phloroglucinol and hydroxyhydroquinone, and ferredoxin in case of resorcinol [Boll, 2005ab].

In anaerobic degradation, benzoyl-CoA is most common intermediate, because a broad variety of aromatic compounds, such as benzene, phenol, hydroxybenzoate and aniline are metabolized through the anaerobic benzoyl-CoA degradation pathway [(Figure 3); Schink et al., 1992; Heider and Fuchs, 1997; Harwood et al., 1999]. In contrast to hydroxy substituted intermediates (as discussed above), the benzene ring in the benzoyl-CoA molecule is still intact and fully aromatic, and thus its reduction becomes difficult. However, Buckel and Keese (1995) described the important role of the CoA thioester linkage for the reduction of the benzene ring in the benzoyl-CoA. The carboxy-thioester group acts as the electron withdrawing substituent [Harwood et al., 1999] and facilitates the transfer of the first electron to the ring by enabling the transient formation of a stable ketyl radical anion to a cyclic, conjugated diene catalyzed by benzoyl-CoA reductase [Boll and Fuchs, 1995; Boll et al., 2000].

In all anaerobic bacteria regardless of the final electron acceptor, formation of benzoyl-CoA involves the ATP-dependent benzoate activation [Harwood et al., 1999]. However, the difference in energy budgets in metabolically diverse anaerobes is drastic: e.g. the highest - energy conservation is reached with nitrate-reducing bacteria (NO3 /N2, E0’= +750 mV), 3+ 2+ followed by iron(III)-reducing bacteria (Fe /Fe , E0’ = -47 mV), and energy conservation is 2- even less with sulfate-reducing bacteria (SO4 /H2S, E0’ = -218 mV) and least with fermentative bacteria [(CO2/CH4, E0’ = -244 mV); Widdel et al., 1993; Evans and Fuchs, 1988; Gibson and Harwood, 2002; Schink et al., 2000; Schink, 1997]. However, depending on the energy budget of the anaerobic bacteria the benzoyl-CoA reduction can be ATP-

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Introduction dependent or ATP-independent. For example, the denitrifying species Thauera aromatica uses an ATP-dependent benzoyl-CoA reductase (Class I) that reduce benzoyl-CoA to 1,5- dienoyl-CoA with the expense of two ATP [Boll and Fuchs, 1995; Unciuleac and Boll, 2001]. Considering strict energy constraints and poor energy balance in obligate anaerobic bacteria (e.g., sulfate-, iron-reducers/fermenters), they cannot afford input of energy (2 ATPs) for ring reduction including ATP-dependent benzoate activation in the first step. Moreover, anaerobic benzoyl-CoA degradation pathway yields three molecules of acetyl-CoA, an equivalent to 3 ATP molecules. Three molecules of ATP are not sufficient for the ATP- dependent benzoate activation and its further reduction. Therefore, they must use an ATP- independent ring reduction in order to balance the energy demand. E.g., the iron(III)-respiring Geobacter metallireducens uses an ATP-independent benzoyl-CoA reductase (Class II) for the ring reduction [Peters et al., 2007; Kung et al., 2009; Wischgoll et al., 2005], allows to partially compensate for the ATP consumed in the activation step [Schöcke and Schink, 1999; Peters et al., 2004; Boll, 2005b; McInerney et al., 2007]. Despite the use of ATP- independent ring reduction in some strict anaerobic bacteria, few of them even adapted for ATP-independent substrate activation relying on CoA-transferase rather than ATP- consuming CoA-ligases. For instance, the fermenting bacterium Sporotomaculum hydroxybenzoicum activates 3-hydroxybenzoate to 3-hydroxybenzoyl-CoA by a CoA- transferase with acetyl-CoA or butyryl-CoA as CoA donor [Müller and Schink, 2000]. Thus, the biochemical strategy used for degradation of aromatic compounds by anaerobic bacteria is largely depends on the energy yields. However, in all cases the key intermediate benzoyl- CoA formed during the degradation of aromatic compounds is then further metabolized to three acetate molecules and carbon dioxide (Figure 3).

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Introduction

Figure 3. General overview of peripheral pathways involved in degradation of different aromatic compounds that lead to benzoyl-CoA as the common intermediate, which is subsequently reduced by I) benzoyl-CoA reductase (both ATP-dependent and ATP- independent pathway) to cyclohex-1,5-diene-1-carbonyl-CoA; II) dienoyl-CoA hydratase to 6-hydroxycyclohex-1-ene-1-carbonyl-CoA; III) hydroxyacyl-CoA dehydrogenase to 6- oxycyclohex-1-ene-carbonyl-CoA; IV) oxoacyl-CoA hydrolase to 3-hydroxy-pimeloyl-CoA and further degradation (arrows) furnishing three molecules of acetyl-CoA and carbon dioxide.

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Introduction

3. Phthalate esters, applications and biodegradation

3.1 Phthalate esters as ‘plasticizers Phthalic acid (benzene dicarboxylic acid) consists of a benzene ring to which two carboxylic groups are attached at different positions that constitute three isomers namely (deprotonated), phthalate (PA), isophthalate (IA), and terephthalate (TA). Characteristics and structures of phthalic acid isomers are shown in Table 1. The esters of phthalic acid isomers, are the dialkyl or alkyl aryl esters (phthalate esters) used mainly as plasticizers, e.g. in polyvinyl chloride products. Plasticizers or softeners are additives used with resin that increase the plasticity or viscosity of a material [Giam et al., 1984; Nilsson, 1994]. Nowadays, more than 60 kinds of phthalate esters are produced for different applications such as in plastic manufacturing, additives in paints, lubricants, adhesives, insecticides, material packaging and cosmetics [reviewed in Vats et al., 2013]. Other applications include building materials, home furnishings, transportation, clothing, and to a limited extent also in food packaging and medical devices.

Table 1. Names, chemical structure and chemical properties of phthalate isomers.

Names Formula Structure Solubility Acidity IUPAC Trivial (g/100 ml) (pKa)

Benzene-1,2- Phthalic acid C8H6O4 0.6 2.89, 5.51 dicarboxylic acid (ortho)

Benzene-1,3- Isophthalic acid C8H6O4 Insoluble in 3.46, 4.46 dicarboxylic acid (meta) water

Benzene-1,4- Terephthalic acid C8H6O4 0.0015 3.51, 4.82 dicarboxylic acid (para)

Note: solubility and acidity values were obtained from wikipedia (https://www.wikipedia.org/). IUPAC, International union of pure and applied chemistry.

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Introduction

3.2 ‘Man-made’ phthalate esters as pollutant Phthalate esters are the synthetic organic compounds were mainly introduced from the early in 1920s. They are globally used for a wide applications in industries and produced in large quantities. The estimated global production is growing rapidly with 1.8 million tons in 1975, reached to 6.2 million tons in 2009 and was more than 8 million tons in 2011 [Peijnenburg and Struijs, 2006; Meng et al., 2014; Net et al., 2015]. The names and structures of most frequently utilized phthalate esters for different applications are shown in Table 2. In order to provide the required flexibility to products, phthalate esters are not bound covalently to the resin [Nilsson, 1994]. Due to the high global utilization phthalate esters, they are released in every environment during production and distribution [Giam et al., 1984] such as air, freshwater, sediments, soil, wastewater, household dust and food products [reviewed in Gao and Wen, 2016] leading to environmental contamination and increasing toxicity.

Phthalate esters are described as man-made priority pollutants due to their high exposure to humans and adverse effects on the health. The experimental exposure of laboratory animals to phthalates showed a variety of effects, and for certain phthalate esters the adverse health effects on the development of the male reproductive system is of great concern, due to their anti-androgenic properties [Latini et al., 2006; Lambrot et al., 2009]. Besides this, they are also considered as a potential carcinogen, teratogens and mutagens [Fushiwaki et al., 2003]. Thus, from the beginning there has been great concern about the release of phthalate esters into the environment [Giam et al., 1978; Mayer et al., 1972]. Furthermore, many phthalate esters have been black listed by the U.S. Environmental Protection Agency and the Chinese Environment Monitoring Center and banned especially for their use in children's toys [Net et al., 2015].

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Introduction

Table 2. Names, sum formulas and structures of the selected phthalate esters that are commonly used for different applications in industry.

Phthalate ester Abbr. Formula Structure Application

Dimethyl DMP C10H10O4 Insect repellent/ectoparasiticide phthalate e.g. mosquitoes and flies.

Diethyl phthalate DEP C12H14O4 Often used in cosmetics, fragrances and other industrial uses include plasticizers, aerosol sprays etc.

Butyl-benzyl BBP C19H20O4 Mostly used as a plasticizer for phthalate PVC. Other uses include traffic cones, food conveyor belts, artificial leather etc.

Diisononyl DINP C26H42O4 Mostly used as a plasticizer. phthalate

Di(2-ethylhexyl) DEHP C24H38O4 Used as plasticizer in medical phthalate devices e.g. intravenous tubing, IV catheters, nasogastric tubes, dialysis bags and tubing, blood bags and transfusion tubing, and air tubes.

Diisodecyl DIDP C28H46O4 Mostly used as a plasticizer. phthalate

Dibutyl phthalate DBP C16H22O4 Used as a plasticizer, additive in adhesives or printing inks.

Abbr. - abbreviation.

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Introduction

4. Microbial degradation of phthalate esters

4.1 Importance of phthalate ester degradation Phthalate esters can be removed from the environment by several methods such as hydrolysis

[Jonsson et al., 2006], UV-light degradation [Lau et al., 2005], TiO2 photocatalysis [Sin et al., 2012] including microbial degradation [Li et al., 2006; Chao et al., 2006]. However, abiotic (non-biological) removal of phthalate esters is very slow and time consuming, e.g., butyl-benzyl phthalate has an aqueous photolysis half-life of >100 days, for dimethyl phthalate is about 3 years and 2000 years for diethyl hexylphthalate [Gledhill et al., 1980; Staples et al., 1997]. Compared to non-biological methods, microbial degradation is rather fast and one of the promising, versatile, cost-effective and environmentally friendly method for removal of phthalate esters from the various environments. In the past studies, removal of phthalate esters from various environments has been reported, including natural water, soils [Carrara et al., 2011], sediments [Chang et al., 2005a], wastewater [Camacho-Munoz et al., 2012], landfills [Boonyaroj et al., 2012].

4.2 Initial steps in phthalate ester degradation Phthalate ester degrading microbes are either aerobic, facultative anaerobic or strict anaerobic bacteria. In all cases, the primary step is the initial hydrolysis of phthalate esters by esterase that release free phthalate and side chain alcohols via mono-esters (Figure 4). Most often the aliphatic or aromatic side chain alcohols are easily utilized by the bacteria and phthalate is accumulated. Because degradation of phthalate requires its decarboxylation and is considered rate-limiting step. Figure 4 shows the general steps involved in hydrolysis of phthalate esters that releases free phthalate. Esterases hydrolyzing the phthalate ester have been characterized from several bacteria. E.g., an esterase purified from Micrococcus sp. strain YGJ1, Bacillus sp. that hydrolyzes medium-chain (C3-C5) mono/di-alkyl phthalate ester to side chain alkyl and phthalate [Maruyama et al., 2005; Niazi et al., 2001]. The complete mineralization of phthalate esters requires a combination of metabolic capacity of diverse microbes that could degrade side chain groups as well as phthalate. Some individual microbes are capable of complete mineralization of phthalate esters alone. On the other hand, in some cases phthalate ester degradation is carried by metabolic cooperation between two bacteria, e.g., di-n-octyl

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Introduction phthalate (DOP) can be completely mineralized by a co-culture of Gordonia sp. strain JDC-2 and Arthrobacter sp. strain JDC-32 [Wu et al., 2010].

Figure 4. Initial steps involved in phthalate ester degradation: de-esterification catalyzed by an esterase ultimately release the free phthalate as intermediate and its further degradation (arrow) is often rate-limiting step (discussed in following section).

4.3 Aerobic phthalate degradation Aerobic degradation of phthalate has been known from about 1970s [Keyser et al., 1976]. The key step in the phthalate degradation is the decarboxylation of phthalate. Similar to degradation pathways of other aromatic compounds (discussed in above section 2.1), phthalate is also first partially metabolized through the peripheral reactions catalyzed by the oxygen-dependent oxygenases leading to intermediate protocatechuate formation. Under aerobic conditions different pathways exist for the removal of the single carboxylic group from the phthalate ring for Gram-positive and Gram-negative bacteria. In both cases, phthalate is converted finally to the hydroxy substituted common key intermediate protocatechuate (Figure 5). For Gram-negative bacteria, phthalate degradation proceeds through the initial oxygenation by dioxygenases which adds two oxygen atoms at carbon positions 4 and 5 that form 4,5-dihydro-4,5-dihydroxyphthalate which is subsequently oxidized and finally decarboxylated yielding protocatechuate via 4,5-dihydroxyphthalate [Chang and Zylstra, 1998; Nomura et al., 1992]. However, for Gram-positive bacteria phthalate is initially oxygenated at carbon 3 and 5 positions by dioxygense leading to 3,4- dihydro-3,4-dihydroxyphthalate which is then subsequently decarboxylated to protocatechuate via 3,4-dihydroxyphthalate [Habe et al., 2003; Eaton and Ribbons, 1982; Eaton, 2001].

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Introduction

Figure 5. Aerobic phthalate decarboxylation pathways in Gram-negative (ortho-cleavage of a carboxyl group) and Gram-positive (meta-cleavage of a carboxyl group) bacteria that convert finally into a common intermediate protocatechuate [Figure modified from Han, 2008] which further degraded to central metabolism.

The currently known pathway for terephthalate degradation also involves oxygenation at carbons 1 and 2 to form 1,2-dihydro-1,2-dihydroxyterephthalate which is subsequently dehydrogenated with concomitant decarboxylation to protocatechuate [Schläfli et al., 1994; Shigematsu et al., 2003]. Besides terephthalate, isophthalate is also known to be metabolized via protocatechuate as the intermediate by aerobic bacteria, e.g., Delftia tsuruhatensis strain T7 [Wang et al., 1995]. The common intermediate of the aerobic degradation pathway, i.e., protocatechuate formed in aerobic phthalate isomer degradation, is further transformed by

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Introduction oxygenolytic ring-cleaving dioxygenases through the ortho- or meta-cleavage pathway leading to acetyl-CoA and/or succinate derivatives (as discussed in the above section 2.1), which enter the tricarboxylic acid cycle and finally metabolized to CO2.

4.4 Anaerobic phthalate degradation Although less understood than aerobic phthalate degradation, Aftring et al., (1981) showed that phthalate isomers are degraded under anoxic conditions by enrichment culture [Aftring et al., 1981]. However, each enrichment culture was specifically for one of the phthalate isomer could able to degrade only single phthalate isomer under anaerobic conditions. Interestingly, all phthalate isomer degrading cultures could simultaneously be adapted for the benzoate- degradation [Kleerebezem et al., 1999ab]. Compared to aerobic degradation, anaerobic phthalate degradation is rather slow and very few anaerobic pure cultures degrading phthalate isomers have been reported so far, including Clostridium sp., Bacillus sp., Pelotomaculum sp., Pseudomonas sp., Thauera sp., [reviewed in Liang et al., 2008; Nozawa and Maruyama, 1988ab; Qiu et al., 2006; Qiu et al., 2004] but were only described briefly. The difficulties in obtaining pure cultures might be because of poor growth of phthalate-degrading anaerobic bacteria. Additionally, the challenge for anaerobic bacteria is the oxygen-independent decarboxylation of phthalate to benzoate. In general the anaerobic degradation of phthalate was reported to occur through the phthalate decarboxylation to benzoate. However, phthalate decarboxylation to benzoate is considered to be the rate-limiting step during anaerobic phthalate degradation [Kleerebezem et al., 1999c].

In the past different hypothetical steps were proposed for anoxic decarboxylation of phthalate to benzoate/benzoyl-CoA. In 1983, Taylor and Ribbons suggested the decarboxylation of phthalic acid through an initial reduction to 1,2-dihydrophthalic acid followed by oxidative decarboxylation to benzoic acid [Taylor and Ribbons, 1983]. But the reduction of the phthalic acid ring with known biological electron donors (e.g., NADH) is not feasible and no experimental evidence for such a reaction has been demonstrated. Similar to benzene ring reduction in benzoyl-CoA by reductase, phthalate would also need to be biologically activated and require electron donor with high negative redox potential. Further, Nozawa and Maruyama observed that a denitrifying Pseudomonas sp. strain P136 accumulated benzoate transiently during growth on phthalate isomers and induced acyl-CoA synthetase activities for phthalate isomers grown conditions [Nozawa and Maruyama, 1988ab]. Therefore, the

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Introduction authors suggested that phthalate isomers are converted to the CoA-ester with Coenzyme A (CoA) as the donor and by acyl-CoA synthetase activity followed by decarboxylation to benzoyl-CoA [Nozawa and Maruyama, 1988a]. In contrast, in few cases phthalate isomers were shown to be first converted to benzoate in the presence of a specific decarboxylase followed by esterification with CoA, i.e., benzoyl-CoA [Kleerebezem et al., 1999ac]. None of these studies presented evidence for the proposed phthalate decarboxylation reaction. However, the current view of anaerobic phthalate degradation was the through the initial decarboxylation to benzoate (Figure 6).

Phthalate degradation under syntrophic condition was proposed to proceed by syntrophic association between phthalate-fermenting (syntroph) bacteria, and methanogens [Kleerebezem et al., 1999a; Qui et al., 2006; Qui et al., 2004;]. Recently, Nobu et al., (2015) proposed two potential hypothetical pathways for the decarboxylation of terephthalate in fermenting bacterium Syntrophorhabdus aromaticivorans. Terephthalate could be anoxically decarboxylated in two steps, in which first terephthalyl-CoA could be formed by an ATP- dependent CoA-ligase that is subsequently decarboxylated to benzoyl-CoA (Figure 6). Alternatively, terephthalate could be directly decarboxylated to benzoate by a specific decarboxylase and benzoate is then degraded further by activation through the anaerobic benzoyl-CoA degradation pathway to acetate and hydrogen in co-culture with methanogens which finally produce methane and carbon dioxide [Nobu et al., 2015].

Under sulfate-reducing conditions, very few reports exist on the phthalate isomers (or phthalate ester) degradation. One such example, dimethyl esters of phthalate isomers could only be transformed to the monomethyl phthalate and/or phthalic acid and could not be mineralized for months [Cheung et al., 2007]. However, it is still unclear if phthalate isomers are degraded through deacarboxylation to benzoate by the sulfate-reducing bacteria. Despite all the studies reported on the anaerobic phthalate degradation, especially of biochemistry phthalate decarboxylation and metabolic intermediates of the initial phthalate degradation still remained unclear.

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Introduction

Figure 6. Hypothetical pathways suggested for anerobic decarboxylation of phthalate (isomers) either to benzoate/benzoyl-CoA by anaerobic bacteria during phthalate degradation: A) phthalate isomers converted to CoA thioesters by acetyl-CoA synthetase (I) following decarboxylation to benzoyl-CoA by decarboxylase (II); B) terephthalate is first converted to terephthalyl-CoA by ATP-dependent CoA ligase (III), which is subsequently transformed to benzoyl-CoA by decarboxylase (II) or terephthalate is directly transformed to benzoate by decarboxylase (IV) and benzoate is then activated to benzoyl-CoA by ATP- dependent benzoyl-CoA ligase (V); and C) phthalic acid is reduced to 1,2-dihydrophthalic acid by reductase (VI) followed by decarboxylation to benzoic acid (benzoate) with the decarboxylase (VII). Generated benzoate or benzoyl-CoA is further degraded through the enzymes of anaerobic benzoyl-CoA degradation pathway as discussed in section 2.2 [Figure modified from Nozawa and Maruyama, 1988a; Nobu et al., 2015 and Taylor and Ribbons, 1983].

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Aims and objectives

5. Aims of the dissertation and objectives

At the beginning of my PhD project, it was still unknown how the challenging reaction of oxygen-independent decarboxylation of phthalate to benzoate is achieved by the anaerobic bacteria. Anoxic decarboxylation of phthalic acid (phthalate) to benzoic acid (benzoate) is considered to be a difficult chemical reaction in organic chemistry. Moreover, phthalate is a synthetic organic compound, which is largely exposed to microorganisms from the last recent decades (mainly from 1920s after the industrial revolution). Therefore, anaerobic bacteria might require to adapt an unusual biochemical strategy for phthalate decarboxylation under anaerobic conditions. Although, several studies proposed different hypothesis for phthalate decarboxylation to benzoate (as discussed in the above section 4.4 and Figure 6). However, none of these studies identified the intermediates, genes or key enzymes involved anaerobic phthalate decarboxylation by anaerobic bacteria. The overall aim of my PhD research project was to identify intermediates, enzymes and genes involved in the anaerobic phthalate decarboxylation in the anaerobic phthalate degrading bacteria using nitrate-reducing Azoarcus sp. strain PA01 and/or sulfate-reducing/methanogenic enrichment cultures. Particularly the following questions were raised:

 How is phthalate anaerobically initially activated by the phthalate-degrading, nitrate- reducing bacterium Azoarcus sp. strain PA01?  How can the difficult step of anaerobic phthalate decarboxylation be accomplished by Azoarcus sp. strain PA01?  Which are the key intermediates in the anoxic phthalate activation and decarboxylation steps?  Which are the specific genes and key enzymes involved in the anaerobic o-phthalate decarboxylation by the Azoarcus sp. strain PA01?  What are the key phthalate-degraders under sulfate-reducing conditions?  Are the phthalate isomers decarboxylation reactions in sulfate-reducing and fermenting bacteria analogous to phthalate decarboxylation by strain PA01?  Are isophthalate and terephthalate decarboxylated analogous to phthalate in other anaerobic bacteria?

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Publications co-authored but not included in the dissertation

6. Publications co-authored but not included in the dissertation

In order to achieve a consistent information for my dissertation on the topic ‘anaerobic degradation of phthalate, the co-authored publications/manuscript from the additional research contributions were not included and as shown below.

1. Patil, Y., Junghare, M., Pester, M., Müller, N., and Schink, B. (2015) Characterization and phylogeny of Anaerobium acetethylicum gen. nov., sp. nov., a strictly anaerobic gluconate-fermenting bacterium isolated from a methanogenic bioreactor. Int J Syst Evol Microbiol 65: 3289-3296.

2. Patil, Y., Junghare, M., and Müller, N. (2016). Fermentation of glycerol by Anaerobium acetethylicum and its potential use in biofuel production. Microbial Biotechnology. (In press).

3. Patil, Y., Müller, N., Schink, B., Whitman, W.B., Huntemann, M., Clum, A., Pillay, M., Palaniappan, K., Varghese, N., Mikhailova, N., Stamatis, D., Reddy, T.B.K., Daum, C., Shapiro, N., Ivanova, N., Kyrpides, N., Woyke, T., Junghare, M. (2016). High-quality-draft genome sequence of the fermenting bacterium Anaerobium acetethylicum type strain GluBS11T (DSM 29698). Standards in Genomic Sciences (Accepted).

4. Patil, Y., Junghare, M., Müller, N. (2016-2017). Fermentation of an oxidized sugar by Anaerobium acetethylicum: evidence for the involvement of a modified Entner- Doudoroff pathway (In preparation).

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CHAPTER 2

Draft genome sequence of a nitrate-reducing, o- phthalate degrading bacterium, Azoarcus sp. strain PA01

Madan Junghare, Yogita Patil and Bernhard Schink

Published in the journal Standards in Genomic Sciences 2015; 10: 90.

Note- Azoarcus sp. PA01 is not a type strain therefore we removed ‘T’ in this thesis as it was published in original publication.

Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Abstract Azoarcus sp. strain PA01 belongs to the genus Azoarcus, of the family Rhodocyclaceae within the class Betaproteobacteria. It is a facultatively anaerobic, mesophilic, non-motile, Gram-stain negative, non-spore-forming, short rod-shaped bacterium that was isolated from a wastewater treatment plant in Constance, Germany. It is of interest because of its ability to degrade o-phthalate and a wide variety of aromatic compounds with nitrate as an electron acceptor. Elucidation of o-phthalate degradation pathway may help to improve the treatment of phthalate-containing wastes in the future. Here, we describe the features of this organism, together with the draft genome sequence information and annotation. The draft genome consists of 4 contigs with 3,908,301 bp and an overall G+C content of 66.08 %. Out of 3,712 total genes predicted, 3,625 genes code for proteins and 87 genes for RNAs. The majority of the protein-encoding genes (83.51 %) were assigned a putative function while those remaining were annotated as hypothetical proteins. Keywords: Azoarcus sp. strain PA01, o-phthalate degradation, Rhodocyclaceae, Betaproteobacteria, anaerobic degradation, wastewater treatment plant, pollutant

Introduction Phthalic acid (PA) consists of a benzene ring to which two carboxylic groups are attached. There are three phthalic acid isomers (o-phthalic acid, m-phthalic acid, p-phthalic acid). Phthalic acid esters (PAEs) are widely used as additives in plastic resins such as polyvinyl resin, cellulosic and polyurethane polymers for the manufacture of building materials, home furnishings, transportation apparatus, clothing, and to a limited extent in food packaging materials and medical products [Vamsee-Krishna et al., 2006; Chen et al., 2007]. Due to the wide-spread use of phthalates there has been great concern about their release into the environment [Staples et al., 2002; Giam et al., 1978]. In addition, phthalates and their metabolic intermediates have been found to be potentially harmful for humans due to their hepatotoxic, teratogenic, and carcinogenic characteristics [Matsumoto et al., 2008; Woodward et al., 1990]. Phthalic acid is also an intermediate in the bacterial degradation of phthalic acid esters [Ribbons et al., 1984] as well as in degradation of certain fused-ring polycyclic aromatic compounds found in fossil fuel [Ribbons and Eaton, 1982], such as

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01 phenanthrene [Kiyohara et al., 1978], fluorene [Grifoll et al., 1994], and fluoranthene [Sepic et al., 1998].

Azoarcus sp. strain PA01 (= KCTC 15483) is a mesophilic, Gram-negative, nitrate-reducing bacterium that was isolated from a wastewater treatment plant in Constance, Germany, for its ability to completely degrade o-phthalate and a wide range of aromatic compounds. Strain PA01 is also able to grow with a variety of organic substrates including short-chain fatty acids, alcohols, selected sugars, and amino acids. These substrates are degraded completely to carbon dioxide coupled to nitrate reduction. The genus Azoarcus is a genus of nitrogen-fixing bacteria [Reinhold et al., 1993] and known for degradation of aromatic compounds. Currently, this genus consists of nine species with validly published names [Parte, 2014]. These species have been isolated from a wide range of environments, including anoxic wastewater sludge and grass root soil [Reinhold et al., 1993]. On the basis of 16S rRNA gene sequence similarity search, the closest relatives of strain PA01 are Azoarcus buckelii DSMZ 14744T [(99 % gene similarity); Mechichi et al., 2002; Val. list no. 87, 2002] and Azoarcus anaerobius [(98 %); Springer et al., 2002]. A. buckelii DSMZ 14744T was also isolated from a sewage treatment plant for its ability to degrade a wide range of aromatic compounds. But the biochemistry and genetics of anaerobic o-phthalate degradation had not been elucidated in detail. Here, we present a summary of the features for Azoarcus sp. strain PA01 and its classification, together with the description of the genomic information and annotation.

Organism information

Classification and features Azoarcus sp. strain PA01 is a member of the family Rhodocyclaceae in the phylum Proteobacteria. It was isolated from an activated sewage sludge sample collected (in 2012) from a wastewater treatment plant in Constance, Germany. Enrichment, isolation, purification and growth experiments were performed in anoxic, bicarbonate-buffered, non-reduced freshwater medium containing (g/l); NaCl, 1.0; MgCl2 x 6 H2O, 0.4; KH2PO4, 0.2; NH4Cl,

0.25; KCl, 0.5; CaCl2 x 2 H2O, 0.15; NaHCO3, 2.5; Na2SO4, 1 mM. The medium was autoclaved at 121 ºC for 25 min and cooled under an oxygen-free mixture of N2/CO2 (80/20) gas phase. Further, 1 ml trace element solution SL-10 [Widdel et al., 1983], 1 ml selenate- tungstate solution [Tschech et al., 1984], and 1 ml seven-vitamin solution [Pfennig, 1978]

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01 were added. The initial pH of the medium was adjusted to 7.3 ± 0.2 with sterile 1 N NaOH or 1 N HCl. Cultivations and transfer of the strain were performed under N2:CO2 (80:20) gas atmosphere. The strain was cultivated in the dark at 30 °C. Enrichment cultures were started by inoculating approximately 2 ml of sludge sample in 50 ml freshwater medium (described above) containing 2 mM neutralized o-phthalic acid as sole carbon source and 10 - 12 mM

NaNO3 as an electron acceptor. Growth was observed after 3 - 4 weeks of incubation. Enrichment cultures were sub-cultured for several passages with o-phthalate as sole carbon source. Pure cultures were obtained in repeated agar (1 %) shake dilutions [Widdel and Bak, 1992]. Single colonies obtained were retrieved by means of finely-drawn sterile Pasteur pipettes and transferred to fresh liquid medium. The strain was routinely examined for purity by light microscopy (Axiophot, Zeiss, Germany) also after growing the culture with 2 mM phthalate plus 1 % (w/v) yeast extract. For genetic and chemotaxonomic analysis, it was cultivated in the described medium containing 8 mM acetate as a carbon source.

Figure 1. General characteristics of Azoarcus sp. strain PA01. A) Phase contrast micrograph of strain PA01T, B) Scanning electron micrograph of strain PA01, C) Agarose gel (1 %) electrophoresis of isolated genomic DNA (gDNA) of strain PA01. Lane 1, 1 kb DNA marker; lane 2, gDNA before RNase treatment; lane 3, high quality gDNA after RNase treatment.

Azoarcus sp. strain PA01 is a mesophilic, non-motile, Gram-negative, short rod-shaped bacterium measuring 0.5 - 0.7 μm (wide), 1.6 - 1.8 μm (length) (Figure 1A and B) and divides by binary fission. Growth was observed from 25 °C to 37 °C with an optimum at 30 °C and optimal pH of 7.3 ± 0.2 (Table 1). Strain PA01 grows anaerobically with nitrate on a

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01 wide variety of substrates, including o-phthalate, benzoate, 3,4-dihydroxy-benzoate, 3- hydroxy-benzoate, 4-hydroxy-benzoate, maltose, fructose, glucose, gluconate, ethanol, 1- butanol, 1-propanol, glycerol, arginine, alanine, malate, pyruvate, succinate, crotonate, propionate, valerate and butyrate. No growth was observed with isophthalate, terephthalate, 4-amino-benzoate, resorcinol, methanol, threonine, choline, betaine, formate, citrate, 2- oxoglutarate and oxaloacetate.

Initial identification and validation of strain PA01 was performed by 16S rRNA gene amplification using a set of universal bacterial primers; 27 F (5′- AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′) as described [Patil et al., 2015]. A phylogenetic tree was constructed from the 16S rRNA gene sequence together with the other representatives of the genus Azoarcus (Figure 2) using the MEGA 4 software package [Tamura et al., 2007]. Phylogenetic analysis indicated that strain PA01 belongs to the genus Azoarcus and is closely related to Azoarcus buckelii (99 %) and Azoarcus anaerobius (98 %). Currently, 30 genome sequences are available for the members of the order Rhodocyclales. The closest neighbors of strain PA01 whose genome sequence is available are Azoarcus sp. strain KH32C [Nishizawa et al., 2012] and Azoarcus sp. strain BH72 [Krause et al., 2006] and Azoarcus toluclasticus ATCC 700605 [Liolios et al., 2008]. The exact phylogenetic position of strain PA01 within the genus Azoarcus is shown in Figure 2 and the 16S rRNA gene sequence of the strain has been deposited to NCBI under accession number KR025921.

Chemotaxonomy Whole-cell fatty acid methyl esters [Kämpfer et al., 1986] were analyzed by the Identification Service of the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany). The cellular fatty acid pattern of Azoarcus sp. strain PA01 is dominated by the presence of an un-saturated branched-chain fatty acid C16:1 ω7c/15 iso-2OH

(49.6 %) and saturated straight-chain fatty acid C16:0 (25.2 %), which have been reported to be common fatty acids also among recently described other species of the genus Azoarcus

[Lee et al., 2014; Chen et al., 2013]. Other fatty acids include C18:1 ω7c (8.8 %), C17:1 cyclo

(0.82 %), C16:1 ω5c (0.68 %), C14:0 (0.73 %), C12:0 (7.19 %), C10:0 3OH (6.27 %), and C10:0 (0.74 %).

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Table 1. Classification and general features of Azoarcus sp. strain PA01 according to the MIGS recommendations [Field et al., 2008]

MIGS ID Property Term Evidence codea

Current classification Domain Bacteria TAS [Woese et al., 1990] Phylum Proteobacteria TAS [Garrity et al., 2005a] TAS [Val. List No. 107, 2006; Class Betaproteobacteria Garrity et al., 2005b] TAS [Val. list No. 107, 2006; Order Rhodocyclales Garrity et al., 2005c] TAS [Val. List No. 107; Garrity et Family Rhodocyclaceae al., 2005d] Genus Azoarcus TAS [Reinhold et al., 1993] TAS [Reinhold et al., 1993; Parte, 2014; Mechichi, 2002; Val. list no. 87, 2002; Springer, et al., Species Azoarcus sp. 1998 ] Strain: PA01 IDA TAS [Reinhold et al., 1993; Val. Gram stain Negative list no. 87, 2002] Cell shape Rod IDA Motility Non-motile IDA Sporulation Not reported IDA Temperature range 25-37 °C IDA Optimum temperature 30 °C IDA pH range 6-8 IDA pH optimum 7±0.3 IDA o-Phthalate, benzoate, 3 hydroxy- benzoate etc. including many organic Carbon source substrates IDA TAS [Reinhold et al., 1993; Val. MIGS-6 Habitat Freshwater, Sewage sludge list no. 87, 2002] MIGS-6.3 Salinity Not reported TAS [Reinhold et al., 1993; Val. MIGS-22 Oxygen requirement Anaerobic/aerotolerant list no. 87, 2002] MIGS-15 Biotic relationship Free-living NAS MIGS-14 Pathogenicity None IDA

MIGS-4 Geographic location Constance, Germany IDA MIGS-5 Sample collection 2012 IDA MIGS-4.1 Latitude 47.67° N IDA MIGS-4.2 Longitude 9.14° E IDA MIGS-4.4 Altitude 397 m IDA a Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [Ashburner et al., 2000]. If the evidence code is IDA, the property was directly observed by one of the authors or an expert mentioned in the acknowledgments.

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Figure 2. Neighbor-joining phylogenetic tree generated using MEGA 4 software package based on 16S rRNA gene sequences. The phylogenetic tree shows the exact position of strain PA01 and the three Azoarcus spp. (in bold) whose genome sequence are published, along with other representatives of the genus Azoarcus. The corresponding 16S rRNA gene accession numbers are given in parenthesis. Bootstrap values are calculated from 1,000 repeats; bar, 0.02 substitutions per nucleotide position.

Genome sequencing and information

Genome project history Strain PA01 was selected for genome sequencing on the basis of its phylogenetic position and its ability to grow on o-phthalate together with numerous aromatic compounds under nitrate- reducing conditions. Genome sequencing was performed at GATC Biotech AG, Konstanz (Germany). High-quality genome draft sequence of Azoarcus sp. strain PA01 is listed in the Genomes Online Database of the Joint Genome Institute under project ID Gp0109270 [Liolios et al., 2008]. The Azoarcus sp. PA01 whole genome shotgun (WGS) project has been deposited at DDBJ/EMBL/GenBank under the project accession LARU00000000. The version described in this paper has the accession number LARU01000000, and consists of

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01 sequences LARU01000001-LARU01000004. The draft genome sequence was released on August 26, 2015. Annotation of the Azoarcus sp. strain PA01 genome, was performed by the DOE Joint Genome Institute using microbial genome annotation pipeline state of the art technology [Mavromatis et al., 2012; Mavromatis et al., 2009]. Table 2 presents the project information and its association with MIGS version 2.0 compliance [Field et al., 2008].

Table 2. Project information

MIGS ID Property Term

MIGS 31 Finishing quality High quality draft MIGS-28 Libraries used 8-12 kb PacBio library MIGS 29 Sequencing platforms PacBio RS MIGS 31.2 Sequencing depth mean coverage 97.42 MIGS 30 Assemblers HGPA3 MIGS 32 Gene calling method Prodigal Locus Tag PA01_ GenBank accession LARU00000000 GenBank Date of Release August 26, 2015 GOLD project ID Gp0109270 IMG taxon ID 2596583641 GenBank BioProject ID PRJNA279928 MIGS 13 Source material identifier KCTC 15483 Project relevance Degradation of aromatic compounds

Growth conditions and DNA preparation For the isolation of genomic DNA, cells were grown in one liter medium with 8 mM acetate plus 10-12 mM nitrate. Cells were harvested in the late stationary phase and cell pellet was stored frozen (-20 °C) until DNA preparation. High-molecular-weight genomic DNA was prepared using modified CTAB DNA extraction protocol [Porebski et al., 1997] with some modifications. Chloroform:isoamyl alcohol (24:1) and phenol:chloroform:isoamyl alcohol (25:24:1) steps were repeated twice and RNase treatment was performed for 2 h. Finally, the DNA was dissolved in RNase and DNase-free molecular grade water. Purity, quality and size of the genomic DNA preparation were analyzed by using nanodrop (639 ng/μl, A260/280

= 1.84, A260/230 = 2.10) and agarose gel electrophoresis (1 % w/v; see Figure 1C).

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Genome sequencing and assembly The genome of Azoarcus sp. strain PA01 was sequenced using a library size of 8-12 kb. Library construction, quantification and sequencing (Pacific Bioscience RS) were performed at GATC Biotech AG (Konstanz, Germany). The final high-quality draft assembly was based on 95,883 reads. The combined libraries provided the 97.42 mean coverage of sequencing depth. Final de novo assembly of the genome from the total reads was performed using the PacBio HGAP3 assembly pipeline with default filter parameters. Minimum read length and polymerase read quality was 500 bp and 0.80, respectively. The minimum seed read length was computed automatically and resulted in 5,181 bp (length cutoff). The final polished assembly of the sequencing reads yielded 4 linear contigs generating a draft genome size of 3.9 Mb.

Genome annotation Annotation was carried out using the DOE-JGI annotation pipeline [Mavromatis et al., 2009] and genes were identified using Prodigal [Hyatt et al., 2010]. The predicted CDSs were translated and used to search the NCBI non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG and InterPro databases. The tRNAScanSE tool [Lowe and Eddy, 1997] was used to find tRNA genes, whereas ribosomal RNA genes were found by searches against models of the ribosomal RNA genes built from SILVA [Pruesse et al., 2007]. Other non-coding RNAs such as the RNA components of the protein secretion complex and the RNase P were identified by searching the genome for the corresponding Rfam profiles using INFERNAL [Nawrocki and Eddy, 2013]. Additional gene prediction analysis and manual functional annotation was performed within the IMG-ER Platform [Markowitz et al., 2009].

Genome properties The draft genome of Azoarcus sp. PA01T is 3,908,301 bp long (with 4 linear contigs, see Figure 3) with an overall GC content of 66.08 % (Table 3). Of a total 3,712 genes predicted, 3,625 were protein-coding genes, and 87 were RNA genes (15 rRNA genes and 59 tRNA genes); 525 genes without function were identified (pseudogenes). The majority of the protein-coding genes (83.51 %) were assigned a putative function while those remaining were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3, the distribution of genes into COGs functional categories is presented

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01 in Table 4. One CRISPR region was found in the genome of strain PA01 which is located in proximity to the CRISPR-associated endonucleases (Cas1 and Cas 2) proteins.

Table 3. Genome statistics

Atribute Value % of Totala

Genome size (bp) 3,908,237 100 %

DNA coding (bp) 3,511,692 89.95 %

DNA G+C (bp) 2,582,614 66.08 %

DNA scaffolds 4

Total genes 3,712 100 %

Protein coding genes 3,625 97.66 %

RNA genes 87 2.43 %

Pseudo genes 13 0.35 %

Genes with function predictions 3,100 83.51 %

Genes without function prediction 525 14.14 %

Genes assigned to COGs 2,579 69.48 %

Genes with Pfam domains 3,178 85.61 %

Genes with signal peptides 311 8.38 %

Genes with transmembrane helices 829 22.33 % CRISPR repeats 1 - a The total is based on either the size of the genome in the base pairs or the total number of protein coding genes in the annotated genome.

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Table 4. Number of genes associated with general COG functional categories

Code Value % age Description

J 201 6.93 Translation, ribosomal structure and biogenesis

A 1 0.03 RNA processing and modification

K 141 4.86 Transcription

L 111 3.83 Replication, recombination and repair

B 1 0.03 Chromatin structure and dynamics

D 35 1.21 Cell cycle control, Cell division, chromosome partitioning

V 55 1.90 Defense mechanisms

T 159 5.48 Signal transduction mechanisms

M 195 6.73 Cell wall/membrane biogenesis

N 87 3.00 Cell motility

U 67 2.31 Intracellular trafficking and secretion

O 154 5.24 Posttranslational modification, protein turnover, chaperones

C 250 8.62 Energy production and conversion

G 111 3.83 Carbohydrate transport and metabolism

E 230 7.93 Amino acid transport and metabolism

F 66 2.28 Nucleotide transport and metabolism

H 165 5.69 Coenzyme transport and metabolism

I 187 6.45 Lipid transport and metabolism

P 166 5.73 Inorganic ion transport and metabolism

Q 79 2.73 Secondary metabolites biosynthesis, transport and catabolism R 210 7.24 General function prediction only S 146 5.04 Function unknown - 1,113 30.52 Not in COGs

The total is based on the total number of protein coding genes predicted in the genome.

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Insight from the genome sequence Azoarcus sp. strain PA01 grows on a wide variety of aromatic compounds (Table 1) linked to nitrate reduction like other bacteria capable of growth via anaerobic degradation of aromatic compounds [Evans and Fuchs, 1988]. In the degradation pathway of most aromatic compounds (including o-phthalate), benzoate is a central intermediate and has also been used routinely as the model compound to study the anaerobic degradation of aromatic compounds via the benzoyl-CoA degradation pathway [Carmona et al., 2009]. Annotation of the genome indicated that strain PA01 has key enzymes for the degradation of aromatic compounds such as benzoate. In the past decade, degradation of benzoate through the benzoyl-CoA pathway has been detailed at the molecular level in facultative anaerobes and the phototrophic strictly anaerobic bacteria, i.e., in the denitrifying bacteria Thauera aromatica and Rhodopseudomonas palustris, respectively [Breese et al., 1998; Harwood et al., 1999].

Unlike other benzoate and/or aromatic compound degrading bacteria, strain PA01 has the genes for benzoate degradation, which involves a one-step reaction that activates benzoate to benzoyl-CoA by an ATP-dependent benzoate-CoA ligase. The genome of PA01 contains in total two copies of the benzoate-CoA ligase, i.e., benzoate-CoA ligase (EC 6.2.1.25) and benzoate-CoA ligase (EC 6.2.1.25) (locus tag PA01_01819, PA01_03223) which are supposed to be involved in the initial activation of benzoate to benzoyl-CoA. They are located in different positions. These two genes show 68.11 % identity to each other and are also found to be present in the genomes of the other bacteria [Nishizawa et al., 2012]. The subsequent enzyme of benzoate degradation, benzoyl-CoA reductase is present in one copy with all its four subunits (locus tags PA01_00623, PA01_00625, PA01_00624, PA01_00626) in the genome of strain PA01. The presence of these gene clusters in the genome of Azoarcus sp. strain PA01 provides evidence for the capacity of strain PA01 to degrade aromatic compounds.

Most of the novel biochemistry of the anaerobic metabolism of aromatic compounds has been discovered with nitrate-reducing bacteria in the past two decades [Heider and Fuchs, 1997; Philipp and Schink, 2012] and little is known about the biochemistry of phthalate degradation in nitrate-reducing and strictly anaerobic (fermenting and sulfate-reducing) bacteria. We are currently exploring the genome of strain PA01 and the enzymes responsible for o-phthalate degradation by using differential proteomics and measuring enzyme activities (unpublished).

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Thus, the draft genome sequence of strain PA01 provides an opportunity to study the biochemistry of o-phthalate degradation into depth.

Conclusions Azoarcus sp. strain PA01 harbors various genes required for degradation of aromatic compounds (which are normally found in the other aromatic degrading bacteria), e.g., benzoate, in the genome of strain PA01. Further, the genome of Azoarcus sp. strain PA01 expands our view to understand the biochemistry of anaerobic degradation of various aromatic compounds including o-phthalate, a priority pollutant. The genome sequence of strain PA01 will provide insight into the putative genes involved in the degradation of all these compounds, especially o-phthalate.

Acknowledgements This work was financially supported by grants from the University of Konstanz, Germany. We are grateful to DAAD (German Academic Exchange Service, Bonn, Germany) for providing a fellowship to MJ during this work. MJ thanks Dr. David Schleheck and Dr. Nicolai Müller, University of Konstanz, for helpful discussion and assistance in genome submission

Authors’ contributions

MJ initiated and BS supervised the study throughout. MJ drafted the manuscript, conducted wet lab work and performed electron microscopy. YP conducted a screening of carbon sources for growth and substrate utilization experiments. MJ, YP and BS discussed, analyzed the data and revised the manuscript. All authors read and approved the final manuscript.

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Draft genome sequence of a nitrate-reducing, o-phthalate degrading bacterium, Azoarcus sp. strain PA01

Figure 3. Graphical representation of different scaffolds of the genome of Azoarcus sp. strain PA01. A) Graphical map of Azoarcus sp. PA01 genome_PA01_unitig_3_quiver.1. From bottom to top: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content (black) and GC skew (purple). B) Graphical map of Azoarcus sp. PA01 genome_PA01_unitig_0_quiver.2. From bottom to top: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content (black) and GC skew (purple). C) Graphical map of Azoarcus sp. PA01T genome_PA01_unitig_2_quiver.3. From bottom to top: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content (black) and GC skew (purple). D) Graphical map of Azoarcus sp. PA01 genome_PA01_unitig_1_quiver.4. From bottom to top: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content (black) and GC skew (purple).

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CHAPTER 3

Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01

Madan Junghare, Dieter Spiteller and Bernhard Schink

Published in the journal Environmental Microbiology 2016, 18: 3175-3188.

Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Abstract The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA- transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalylCoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o- phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl- CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o- phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl- CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygenin sensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.

Introduction Phthalic acids (PAs) are benzoic acids with one additional carboxylic group in either ortho, meta or para position. PA esters are produced massively worldwide and are used in the manufacture of a wide range of plastic products [Vamsee-Krishna et al., 2006; Chen et al., 2007; Liang et al., 2008]. Di-esters of ortho-phthalic acid (o-phthalate) serve mainly as plasticizers, for example, in the production of polyvinyl chloride [Giam et al., 1984].

PA isomers are introduced into the environment from plastics because phthalate esters are non-covalently bound to the plastic polymers in order to maintain the required flexibility of plastic products [Nilsson, 1994]. Additionally, phthalates are released into the environment

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 through liquid and solid waste streams generated during the production of phthalates from the corresponding xylenes [Bemis et al., 1982]. Therefore, high concentrations of PA isomers can be found in soil around chemical factories [Naumov et al., 1996]. PA esters have been detected nearly in every environment, including air [Wensing et al., 2005], soils, sediments, waters [Fatoki and Vernon, 1990] and landfill leachates [Schwarzbauer et al., 2002; Zheng et al., 2007]. PAs are the hydrolysis products of phthalate esters and have also been identified as metabolic intermediates in bacterial degradation of polycyclic aromatic hydrocarbons such as phenanthrene [Kiyohara and Nagao, 1978], fluorene [Grifoll et al., 1994] and fluoranthene [Sepic et al., 1998]. The metabolic derivatives of phthalates are potentially harmful to humans and wildlife due to their hepatotoxic, teratogenic and endocrine disrupting (carcinogenic) characteristics [Woodward, 1988; Matsumoto et al., 2008], and are listed as priority industrial pollutants [Mayer et al., 1972; Giam et al., 1984]. Hence, it is important to remove phthalates from the environment effectively and economically.

Besides slow chemical hydrolysis and photolysis of phthalate esters in the environment, microbial degradation of PAs and their corresponding esters by microorganisms is considered a principal route for removal of phthalates [Liang et al., 2008]. Many studies exist on aerobic degradation of phthalates [Chang and Zylstra, 1998; Wang et al., 2003; Vamsee-Krishna et al., 2006; Li and Gu, 2007] and their corresponding esters [Li et al., 2005a,b; Li et al., 2006; Wang and Gu, 2006a,b]. The pathway of aerobic phthalate degradation has been well characterized in aerobic bacteria, for example, Arthrobacter keyseri 12B and Burkholderia cepacia DBO1, and involves either a 3,4-dihydroxyphthalate decarboxylase [Eaton and Ribbons, 1982] or a 4,5-dihydroxyphthalate decarboxylase for decarboxylation of hydroxylated o-phthalate to dihydroxybenzoate [Pujar and Ribbons, 1985; Chang and Zylstra, 1998]. Since aerobic phthalate degradation uses oxygen as a co-substrate in oxygenase reactions, anaerobic degradation has to proceed differently. Aftring et al., showed that phthalate isomers can be degraded under anoxic conditions [Aftring et al., 1981]. Although less understood than aerobic phthalate degradation, anaerobic degradation of phthalate isomers and phthalate esters was reported for numerous pure and mixed cultures under nitrate- or sulfate-reducing or methanogenic conditions [Battersby and Wilson, 1989; Kleerebezem et al., 1999a; Qiu et al., 2006; Cheung et al., 2007; Liang et al., 2008], but no decisive studies on the degradation pathways have been published so far.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

In 1983, Taylor and Ribbons proposed a hypothetical pathway for anaerobic conversion of o- phthalate to benzoate, which involved two hypothetical enzymatic steps catalyzed by a reductase and a decarboxylase, that is, a reduction and subsequent oxidative decarboxylation [Taylor and Ribbons, 1983]. However, no experimental evidence for this hypothetical pathway was provided. Nozawa and Maruyama (1988) proposed a pathway for anaerobic PA degradation by a nitrate-reducing Pseudomonas sp. that involves initial activation of o- phthalate with coenzyme A (CoA), followed by decarboxylation to benzoyl-CoA [Nozawa and Maruyama, 1988a], however, no precise experimental evidence supported this assumption either. Nonetheless, the current view of anaerobic o-phthalate degradation involves benzoyl-CoA as a key intermediate, and its further degradation would proceed through the well-described anaerobic benzoyl-CoA degradation pathway [Schink et al., 1992; Fuchs et al., 1994; Harwood et al., 1999]. In the present work, we studied the anaerobic o- phthalate metabolism by the recent genome sequenced bacterium Azoarcus sp. strain PA01 [(IMG genome ID: 2596583641, NCBI genome accession: PRJNA279928); Junghare et al., 2015b] that degrades o-phthalate with nitrate as an electron acceptor. We used a combined proteomic and genomic approach in order to identify the catabolic genes and enzymes involved in the initial steps of o-phthalate degradation.

Results Anaerobic growth with o-phthalate and physiological characteristics To compare the growth and substrate utilization by Azoarcus sp. strain PA01 (KCTC 15483), cells were grown with 2 mM o-phthalate or benzoate as sole source of electrons, supplemented with nitrate (10-12 mM) as the final electron acceptor. Irrespective of the substrate, no pronounced difference in growth was observed. Cells showed doubling times of about 10 to 12 h with both substrates (although cells grew slightly faster with benzoate (Supporting Information Figure S1). Nitrite accumulated as an intermediate (0.5-4 mM) in the early log phase. Cells consumed 1.7 mM of o-phthalate and reduced 7 mM of nitrate, whereas benzoate-grown cells consumed 1.8 mM of benzoate and 8.3 mM of nitrate. Thus, the experimentally observed stoichiometry of o-phthalate or benzoate oxidation to nitrate- reduction was approximately 1:4. The biomass produced with benzoate- or o-phthalate was nearly the same that is 34.5 and 36.5 g dry cell mass per mol, respectively. No intermediate organic degradation products were detected in the growth medium.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Fig. S1. Anaerobic growth of Azoarcus sp. strain PA01 with 2 mM o-phthalate or 2 mM benzoate plus nitrate (12 mM) as electron acceptor.

Differential proteome analysis of o-phthalate-grown cells versus benzoate-grown cells The proteome of o-phthalate-grown cells was compared to that of benzoate-grown cells using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Qualitative comparison of the resolved proteomes displayed significant differences in the protein profiles; approximately 15 - 20 protein spots were visible specifically in o-phthalate-grown cells (Figure 1A). Of these, 12 protein spots were selected (labelled in Figure 1A) and identified by mass spectrometry (MS). These protein spots were shown to represent eight different proteins (Table 1). On the other hand, in the proteome of benzoate-grown cells, nine protein spots were selected for MS identification (labelled in Figure 1B), and the identified spots comprised seven different enzymes/proteins (Table 1). The protein spots SBA3, 4, 6 and 9 from the benzoate-grown cells were identified as proteins homologous to the enzymes of the anaerobic benzoyl-CoA degradation pathway of other denitrifying bacteria, for example Thauera aromatica [Breese et al., 1998].

Finally, eight of the identified protein spots (SPA1, 2, 3, 7, 8, 9, 10 and 11) from o-phthalate- grown cells were shown to represent three different proteins and were suspected to be involved in the initial steps of anaerobic o-phthalate degradation (Figure 1; Table 1) and were

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 not observed in benzoate-grown cells. These proteins were identified as a TRAP (tripartite) transporter (SPA7), a benzylsuccinate CoA-transferase (SPA2-3) and a 3-polyprenyl-4- hydroxybenzoate decarboxylase (SPA1, 8-11) and suggested to be involved in o-phthalate metabolism. Furthermore, total proteome analysis of cell-free extracts of o-phthalate-grown cells (Figure 2) and SDS polyacrylamide gel electrophoresis of membrane proteins (Supporting Information Figure S2) strongly supported and complemented the results of the differential 2D-gel protein profiling (Figure 1; Table 1). The identified proteins constituted the most abundant cytosolic proteins (locus tag PA01_00214; PA01_00215; and PA01_00217) that were observed in the 2D-gel proteome of o-phthalate-grown cells (Figure 1; Table 1).

Figure 1. Differential proteome analysis of cytosolic proteins of Azoarcus sp. strain PA01 grown with o-phthalate (A) or benzoate (B): Proteins resolved by two-dimensional differential gel electrophoresis (IEF-SDS PAGE) The induced protein spots are labelled SPA (soluble proteins from o-phthalic acid-grown cells) and SBA (soluble proteins from benzoic acid-grown cells) followed by the corresponding IMG locus tags (green, CoA transferase; red, decarboxylase). For IEF, a strip with a pH gradient from 5 to 8 was used and a prestained molecular protein marker was used for protein size comparison (kDa) on the gel.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

The total proteome results allowed the identification of one extra protein with locus tag PA01_00216 that was identified as the BbsE subunit of benzylsuccinate CoAtransferase (30 % sequence similarity) of T. aromatica and was similar to the previously identified protein spots SPA2-3 (Figure 1; Table 1) with the locus tag PA01_00215 that was also identified as the BbsF subunit of benzylsuccinate CoA-transferase (30 % sequence similarity). The total proteome analysis of benzoate-grown cells revealed the identification of proteins that were homologous to enzymes involved in the anaerobic benzoyl-CoA degradation pathway comprised of: benzoyl-CoA reductase (2-electron) β-subunit (locus tag PA01_03236, SBA3- 4), 6-ketocyclohex-1-ene-1-carbonyl-CoA hydrolase (locus tag PA01_03225, SBA6), benzoate-CoA ligase (locus tag PA01_03196), 6- hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase (locus tag PA01_03226) and 6-hydroxycyclohex-1-ene-1-carbonyl-CoA hydratase (locus tag PA01_03227). As expected, most of these proteins of the anaerobic benzoyl-CoA degradation pathway were also detected in the proteome of o-phthalate-grown cells [Figs 1 and 2; Table 1].

Figure 2. Total proteome analysis of cell-free extracts from the cells of Azoarcus sp. strain PA01 grown with o-phthalate (black bars) and benzoate (grey bars). Proteins were identified by Orbitrap LC-MS analysis showing the o-phthalate specific induced genes with locus tags 00214, 00215, 00216 and 00217 (in bold) during growth on o-phthalate.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Table 1. Identification of selected induced protein spots resolved by two-dimensional difference gel electrophoresis from cell-free extract of Azoarcus sp. strain PA01 cells grown with o-phthalate or benzoate

Spot Gene IMG predicted function Gene IDc Score Massd Sequence IDa locib coverage (%)e 00217 4-hydroxy-3-polyprenylbenzoate decarboxylase phtDa 5060 59 69 SPA1* (UbiD-decarboxylase)

SPA2* 00215 benzylsuccinate CoA-transferase phtSa 7613 44 74 (CoA-transferase family III)

SPA3* 00215 benzylsuccinate CoA-transferase phtSa 7745 44 66 (CoA-transferase family III)

SPA4 01506 ABC-type amide transporter substrate-binding protein livK 7513 44 55 (HAAT family)

SPA5 00796 TRAP-type mannitol/chloroaromatic compound ebA1033 5866 39 79 transport system, periplasmic component

SPA6* 03412 ABC-type Fe3+ transport system substrate-binding ebA4918 13245 37 71 protein

SPA7* 00214 TRAP-type transport system, periplasmic component sO0456 10564 35 76 (TAXI family)

SPA8* 00217 4-hydroxy-3-polyprenylbenzoate decarboxylase phtDa 1102 59 69 (UbiD-decarboxylase)

SPA9* 00217 4-hydroxy-3-polyprenylbenzoate decarboxylase phtDa 1183 59 65 (UbiD-decarboxylase)

SPA10 00217 4-hydroxy-3-polyprenylbenzoate decarboxylase phtDa 1257 59 63 * (UbiD-decarboxylase)

SPA11 00217 4-hydroxy-3-polyprenylbenzoate decarboxylase phtDa 1197 59 61 * (UbiD-decarboxylase)

SPA12 03235 benzoyl-CoA reductase (2-electron) delta subunit oah 11671 28 91

SBA1 00882 chaperonin GroEL (HSP60 family) groL1 1308 58 62

SBA2 03692 isocitrate lyase aceA 916 47 80

SBA3* 03236 benzoyl-CoA reductase (2-electron) beta subunit bcrB 744 50 71

SBA4* 03236 benzoyl-CoA reductase (2-electron) beta subunit bcrB 639 50 55

SBA5 02982 citrate synthase gltA 968 48 79

SBA6* 03225 6-ketocyclohex-1-ene-1-carbonyl-CoA hydrolase oah 388 42 57

SBA7 02989 malate dehydrogenase (NAD) mdh 1092 35 71

SBA8* 03412 ABC-type iron(III) transport system substrate-binding ebA4918 802 37 61 protein SBA9 00613 5-ketocyclohex-1-ene-1-carbonyl-CoA hydratase ebA722 1016 30 76 a SPA1 - 11, protein spots from o-phthalate-grown cells and SBA1 - 9 protein spots from benzoate-grown cells; b IMG gene locus tag PA01_ from the genome of Azoarcus sp. strain PA01; c gene name obtained from IMG annotation; d peptide mass calculated by MS-MS identification; e sequence coverage represents the extent of peptides obtained during MS-MS identification of respective protein and * indicates proteins that were also identified in the total proteome analyzed by Orbitrap LC-MS analysis. Protein spots that were exclusively induced with o-phthalate and likely to be involved in the initial anaerobic degradation of o-phthalate are highlighted in bold.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Characterization of the gene cluster involved in anaerobic o-phthalate degradation Comparative proteome analysis of o-phthalate- versus benzoate-grown cells of Azoarcus sp. strain PA01 was performed using protein sequences obtained from the genome of the Azoarcus sp. strain PA01. The MS identified proteins include the protein-coding genes with locus tags PA01_00214, PA01_00215, PA01_00216 and PA01_00217 that were only induced in o-phthalate-grown cells. To compare and to infer the possible catabolic functions of these genes, similarity searches were performed using the online search programs blastp (www.ncbi.nlm.nih.gov) or UniProtKB (http://www.uniprot.org/blast/). The protein blast searches of o-phthalate induced genes revealed the following similarities: the gene with the locus tag PA01_00214 was similar to TRAP transporters (substrate binding protein), and PA01_00215 and PA01_00216 were similar to CoA-transferases (family III) and PA01_00217 was similar to UbiD-like decarboxylases (UbiD family) respectively.

The genes induced exclusively in o-phthalate-grown cells were localized in a single 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01 (Figure 3A). This indicates that the genes involved in anaerobic o-phthalate decarboxylation to benzoyl-CoA are clustered together, coexpressed and up-regulated in one open reading frame (ORF) during growth with o-phthalate. Based on the proteomic data (Figs 1 and 2; Table 1), the newly identified ophthalate induced gene cluster is predicted to encode four candidate proteins (locus tags from PA01_00214 to PA01_00217) that were proposed to be involved and catalyze the initial steps of anaerobic o-phthalate degradation as shown in Figure 3B. The abbreviation ‘pht’ referring to phthalate was introduced to the IMG (Integrated Microbial Genomes) locus tags from PA01_00215 to PA01_00218, for example, 00215_phtSa.

Phylogenetic analysis of the amino acid sequences of the putative CoA transferases of Azoarcus sp. PA01 revealed that the proteins PhtSa (PA01_00215) and PhtSb (PA01_00216) clustered together in one clade and belong to the CoA-transferase family III (Supporting Information Figure S3A). These two putative CoA-transferases of Azoarcus sp. strain PA01 designated as PhtSa and PhtSb showed 26 % similarity with each other and shared only about 30 % of sequence similarity with the two subunits (BbsEF, AAF89840 and AAF89841) of the previously characterized succinyl-CoA:(R)-benzylsuccinate CoA-transferase (family III) from Thauera aromatica. However, blastp search analysis revealed that these proteins (PhtSa and PhtSb) shared high (>80 %) sequence similarity with uncharacterized CoA-transferases

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 of ‘A. aromaticum EbN1’ (Q5NWH8 and Q5NWH7) and A. toluclasticus (WP_0189914468 and WP_018991467), respectively (Supporting Information Figure S3A).

Figure S3A. Phylogenetic neighbor-joining tree of the putative succinyl-CoA:o-phthalate CoA-transferase of the Azoarcus sp. strain PA01 and representative protein sequences from members of the CoA-transferase families I, II and III from the bacteria. The evolutionary distances were computed using the Poisson correction method [Zuckerkandl and Pauling, 1965] and tree generated using MEGA7 software [Kumar et al., 2016]. The numbers at the corresponding nodes show bootstrap support [(1,000 replicates); Felsenstein, 1981] and accession numbers are given in parenthesis. The bar represents 20 % estimated sequence divergence.

Phylogenetic analysis of the remaining two genes coding for the putative decarboxylases (UbiD- and UbiX-like protein) namely, PhtDa (PA01_00217) and PhtDb (PA01_00218; the latter gene was not detected in the proteome analysis) revealed that they belong to two different clades with the protein sequences of the UbiD- and UbiX-like decarboxylases, respectively (Supporting Information Figure S3B). The gene phtDa_00217 (locus tag PA01_00217) is predicted to code for the putative o-phthalyl-CoA decarboxylase (PhtDa; see discussion) clustered with the protein sequences of the UbiD-like decarboxylases. On the other hand, the putative flavin-binding decarboxylase (UbiX-like protein) designated as

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

PhtDb (locus tag PA01_00218) clustered with the protein sequences of the UbiX-like decarboxylases of other bacteria (Supporting Information Figure S3B). Blastp searches showed that the amino acid sequence of PhtDa (PA01_00217) and PhtDb (PA01_00218) shared >95 % sequence similarity with the uncharacterized UbiD/UbiXlike decarboxylase/carboxy-lyase-like proteins of ‘A. aromaticum EbN1’ (Q5NWH6 and Q5NWG7) and A. toluclasticus (WP_018991466 and WP_040395783), respectively.

Figure S3B. Phylogenetic neighbor-joining tree of the putative o-phthalyl-CoA decarboxylase from Azoarcus sp. strain PA01 and putative amino acid sequences from related bacterial UbiD- or UbiX-like decarboxylases. The evolutionary distances were computed using the Poisson correction method [Zuckerkandl and Pauling, 1965] and tree generated using MEGA 7 software [Kumar et al., 2016]. The numbers at the corresponding nodes show bootstrap support (1000 replicates) [Felsenstein, 1981] and accession numbers are given in parenthesis. The bar represents 20 % estimated sequence divergence.

The gene with the locus tag PA01_00214 (So0456) in the identified o-phthalate gene cluster is predicted to encode a putative TRAP transporter (periplasmic binding protein) which is likely to be involved in o-phthalate transport (substrate transport). Finally, the identified o- phthalate degradation gene cluster comprised a gene with locus tag PA01_00219 (IcIR)

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 coding for a protein that showed high similarity with the transcriptional regulator of the DNA-binding IcIR family protein that could hence act as a regulatory protein for expression/repression of these genes in the o-phthalate gene cluster (Figure 3A). A similar distribution of genes as in the o-phthalate-induced gene cluster in Azoarcus sp. strain PA01 (Figure 3A) was also found to be present in the genomes of other aromatic compound degrading nitrate reducers such as ‘A. aromaticum EbN1’ [Rabus et al., 2005], A. toluclasticus and Thauera chlorobenzoica [Liolios et al., 2008]. In all cases, the putative genes encoding CoA-transferases (00215_phtSa and 00216_phtSb) and decarboxylases (00217_phtDa and 00218_phtDa) were located adjacent to each other in a single gene cluster (Supporting Information Figure S4).

Figure 3. Organization of the genes in the o-phthalate degradation gene cluster and the proposed pathway of anaerobic o-phthalate degradation by Azoarcus sp. strain PA01. A) The gene organization and putative function of individual genes induced with o-phthalate-grown cells are highlighted in bold and different colour. B) Proposed pathway based on proteomics and in vitro enzyme assay results showing reaction steps in the initial degradation of o- phthalate: (1) activation of o-phthalate to o-phthalyl-CoA by the putative succinyl-CoA:o- phthalate CoA-transferase (green, PhtSa; light green, PhtSb); and (2) subsequent decarboxylation of o-phthalyl-CoA to benzoyl-CoA by the putative o-phthalyl-CoA decarboxylase (red, PhtDa). The dotted arrows indicate further degradation of benzoyl-CoA via the anaerobic benzoyl-CoA degradation pathway.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

The genes coding for the enzymes catalyzing the anaerobic degradation of benzoate/benzoyl- CoA to 3-hydroxy-pimeloyl-CoA were identified in the proteome of benzoate-grown cells as well as in the proteome of o-phthalate-grown cells of Azoarcus sp. strain PA01 (Figs 1 and 2; Table 1). These genes coding for the enzymes of the anaerobic benzoate degradation were located in the single gene cluster (17.5 kb) in the genome of Azoarcus sp. strain PA01 (Figure 4A). The enzymatic steps of anaerobic benzoate degradation by Azoarcus sp. strain PA01 were deduced based on the results of our proteome and genome data, and are similar to those of the known anaerobic benzoate degradation pathway [Breese et al., 1998; Carmona et al., 2009; (Figure 4B)].

Figure 4. Scheme of the proposed anaerobic benzoate degradation gene cluster (A), and scheme of the proposed pathway of anaerobic benzoate/benzoyl-CoA degradation (B) by Azoarcus sp. strain PA01: I) ATP-dependent benzoate-CoA ligase (bcIA); II) ATP-dependent benzoyl-CoA reductase-class I (BcrADBC); III) cyclohexa-1,5-diene carbonyl-CoA hydratase (Dch); IV) 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase (Had); V) 6- hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase (Oah) and further degradation (dotted arrows) furnishing three molecules of acetyl-CoA and carbon dioxide. The enzymes which were detected in the proteome analysis are in bold.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Enzyme activity measurements with cell-free extracts of Azoarcus sp. strain PA01 Interpretation of our proteomics data suggested an activation of o-phthalate to o-phthalyl- CoA by a putative succinyl-CoA-dependent CoA-transferase followed by its decarboxylation to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. To establish the assumed pathway, in vitro enzyme activity assays were performed with cell-free extracts of o- phthalate or benzoate-grown cells (the latter as a control) using o-phthalate, succinyl- coenzyme A (Succinyl-CoA, Supporting Information Figure S5; Sigma Aldrich), synthesized o-phthalyl-CoA (rt: 4.9 min, Supporting Information Figure S6) or fluoro-o-phthalyl-CoA (Supporting Information Figure S7) as the substrates.

Succinyl-CoA dependent formation of o-phthalyl-CoA from o-phthalate was observed in cell- free extracts of o-phthalate-grown cells and o-phthalate was converted to o-phthalyl-CoA (Figure 5A and B). No activity was observed when succinyl-CoA was replaced by free CoA (sodium salt of CoA) or acetyl-CoA as possible CoA donor. Furthermore, no activity was detected with either isophthalate and terephthalate or with 3-fluoro-o-phthalate. Cell-free extracts of benzoate-grown cells of Azoarcus sp. strain PA01 did not exhibit any activity with all three isomers of phthalate or 3-fluoro-o-phthalate. In the coupled enzyme assay performed with the addition of o-phthalate and succinyl-CoA in cell-free extract of o-phthalate-grown cells, formation of benzoyl-CoA was also observed, that is, enzymatically formed o-phthalyl- CoA was subsequently decarboxylated to benzoyl-CoA (Figure 5C and D). Further, the decarboxylase activity was tested individually in cell-free extracts by the addition of synthesized o-phthalyl-CoA (Supporting Information Figure S6), isophthalyl-CoA (Supporting Information Figure S8), terephthalyl-CoA (Supporting Information Figure S9) and fluoro-o-phthalyl-CoA (Supporting Information Figure S7). Cell-free extracts of o- phthalate-grown cells converted synthesized o-phthalyl-CoA to benzoyl-CoA (rt: 11.4 min, Supporting Information Figure S10) and similarly fluoro-o-phthalyl-CoA was also efficiently decarboxylated to fluoro-benzoyl-CoA (rt: 11.5 min, Supporting Information Figure S11). No decarboxylase activity was observed with either isophthalyl-CoA or terephthalyl-CoA.

Benzoate-grown cell-free extract or heat-inactivated cellfree extract of o-phthalate-grown cells did not form o-phthalyl-CoA or benzoyl-CoA. Exposure of cell-free extract to air did not affect the activities of o-phthalyl-CoA formation or decarboxylation of the o-phthalyl- CoA to benzoyl-CoA (Supporting Information Figure S12). Storage of cell-free extract for 1-

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

2 day at 4 ºC did not result in a loss of enzyme activity. Synthesized o-phthalyl-CoA standing overnight at room temperature was not decarboxylated and was found to be stable for at least 1-2 days.

Figure 5. LC-MS/MS ion traces following the product formation in a coupled enzyme assay performed with cell-free extracts of o-phthalate-grown cells of Azoarcus sp. strain PA01. A. Time course of the formation of o-phthalyl-CoA from o-phthalate in the presence of succinyl- CoA (succinyl-CoA:o-phthalate CoA-transferase) monitoring the specific ion traces m/z 409 of the MS/MS fragmentation of o-phthalyl-CoA (quasimolecular ion m/z 916). B. ESI- MS/MS of the quasimolecular ion m/z 916 of o-phthalyl-CoA. C. Time course of enzymatic benzoyl-CoA formation due to subsequent decarboxylation of formed o-phthalyl-CoA by the putative o-phthalyl-CoA decarboxylase monitoring the specific ion trace m/z 365 of the MS/MS of benzoyl-CoA (quasimolecular ion m/z 872). D. ESI-MS/MS of the quasimolecular ion m/z 916 of benzoyl-CoA [Park et al., 2007].

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Discussion In the present study, we elucidated the pathway of anaerobic o-phthalate degradation in Azoarcus sp. strain PA01 using differential proteomics and in vitro enzyme assays. Based on the draft genome sequence of Azoarcus sp. strain PA01 (IMG genome ID: 2596583641, NCBI genome accession: PRJNA279928), we identified the genes coding for the enzymes involved in the anaerobic conversion of o-phthalate to benzoyl-CoA. The combined proteome and genome analysis of Azoarcus sp. strain PA01 identified an o-phthalate-induced gene cluster coding for the enzymes likely involved in the initial steps of anaerobic degradation of o-phthalate to benzoyl-CoA (Figure 3; Table 1). They comprised enzymes homologous to a dicarboxylic acid transporter (TRAP transporter, PA01_00214), the CoAtransferases (putative succinyl-CoA:o-phthalate CoAtransferase) encoded by two adjacent genes phtSa and phtSb (locus tag PA01_00215 and PA01_00216) and a putative o-phthalyl-CoA decarboxylase (UbiD-like decarboxylase) encoded by phtDa (locus tag PA01_00217), respectively.

Although the putative succinyl-CoA:o-phthalate CoA-transferase of Azoarcus sp. strain PA01 was only distantly related (30 % similarity) to the succinyl-CoA:(R)-benzylsuccinate CoA- transferase of T. aromatica which belongs to the CoA-transferase family III [Leutwein and Heider, 2001; Leutwein and Heider, 1999], both enzyme showed similar function, that is, transfer of CoA to a free acids. The succinyl-CoA:(R)-benzylsuccinate CoA-transferase of T. aromatica consists of two subunits BbsEF (28 % identical) of similar sizes (44 and 45 kDa) constituting an α2β2 tetramer [Heider, 2001; Leutwein and Heider, 2001]. Similarly, Azoarcus sp. strain PA01 grown with o-phthalate induced the expression of the putative CoA- transferases (PhtSa, PA01_00215 and PhtSb, PA01_00216) of similar sizes (44 kDa and 41 kDa). Thus, the two o-phthalate-induced CoAtransferases, PhtSa and PhtSb (26 % similarity) might work together as a single enzyme entity, similar to the BbsEF subunits of the succinyl- CoA:(R)-benzylsuccinate CoA-transferase of T. aromatica [Heider, 2001; Leutwein and Heider, 2001].

Phylogenetic analysis supported our view that the putative succinyl-CoA:o-phthalate CoA- transferase of Azoarcus sp. strain PA01 belongs to the enzymes of CoA-transferase family III. No significant sequence similarity was observed with the proteins of CoA-transferase families I and II. The currently characterized CoA-transferases of family III are known to

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 transfer CoA to free acids in anaerobic degradation pathways and activate organic acids for subsequent metabolization [Heider, 2001]. Therefore, we suspected that the putative succinyl-CoA:o-phthalate CoA-transferase of Azoarcus sp. strain PA01 activates o-phthalate to o-phthalyl-CoA with succinyl-CoA as a CoA donor, followed by decarboxylation of o- phthalyl-CoA to benzoyl-CoA (Figure 3B).

Phylogenetic analysis supported our view that the putative succinyl-CoA:o-phthalate CoA- transferase of Azoarcus sp. strain PA01 belongs to the enzymes of CoA-transferase family III. No significant sequence similarity was observed with the proteins of CoA-transferase families I and II. The currently characterized CoA-transferases of family III are known to transfer CoA to free acids in anaerobic degradation pathways and activate organic acids for subsequent metabolization [Heider, 2001]. Therefore, we suspected that the putative succinyl-CoA:o-phthalate CoA-transferase of Azoarcus sp. strain PA01 activates o-phthalate to o-phthalyl-CoA with succinyl-CoA as a CoA donor, followed by decarboxylation of o- phthalyl-CoA to benzoylCoA (Figure 3B).

Our conclusion was experimentally proven by observing the conversion of o-phthalate to o- phthalyl-CoA and its subsequent decarboxylation to benzoyl-CoA in cell-free extracts of o- phthalate-grown cells in the presence of succinyl-CoA (Figure 5). This is in contrast to an earlier report on the anaerobic phthalate degradation by Pseudomonas sp. strain P136, in which formation of phthalyl-CoA and its subsequent decarboxylation to benzoyl-CoA was reported in the presence of free CoA plus ATP (adenine triphosphate) by an acyl-CoA synthetase [Nozawa and Maruyama, 1988a]. However, our enzyme assay results did not show o-phthalyl-CoA formation when succinyl-CoA was replaced with free CoA or acetyl- CoA as a possible CoA donor. Moreover, earlier enzyme assays with ATP and o-phthalate supplied with free CoA did not show the formation of o-phthalyl-CoA [Junghare and Schink, 2014; unpublished results]. Also in the proteome analysis of o-phthalate-grown cells of Azoarcus sp. strain PA01, no induced acyl-CoA synthetase was found (Figs 1 and 2; Table 1). Thus, we conclude that in Azoarcus sp. strain PA01 o-phthalate is activated by a CoA- transferase rather than an ATP-dependent CoA-ligase or synthetase.

Isophthalate or terephthalate were not converted to the corresponding phthalyl-CoAs by the putative succinyl-CoA:o-phthalate CoA-transferase. Moreover, Azoarcus sp. strain PA01 was unable to grow with isophthalate or terephthalate [Junghare et al., 2015b]. The observed high

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 substrate specificity of the putative CoA-transferase is in line with the observation that the enzymes of CoA-transferase family III catalyse the transfer of the CoA moiety in a highly substrate- and stereospecific manner [Heider, 2001]. The activity of the CoA-transferase was insensitive to air exposure, similar to the previously studied CoA-transferase BbsEF [Leutwein and Heider, 2001]. However, Azoarcus sp. strain PA01 was unable to grow aerobically with o-phthalate or benzoate.

The o-phthalyl-CoA formed in the first step of o-phthalate degradation is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase (Figure 5C and D). In the o-phthalate-induced gene cluster (Figure 3A), there are two alternative genes predicted to code for putative decarboxylases (phtDa, PA01_00217 and phtDb, PA01_00218). Of these, only the PhtDa was found to be highly induced exclusively in o- phthalate-grown cells of Azoarcus sp. strain PA01 and thus was suspected to catalyze the decarboxylation of o-phthalyl-CoA to benzoyl-CoA as shown in Figure 3B. In contrast, the gene with locus tag PA01_00218 (phtDb) is predicted to encode an UbiX-like decarboxylase (flavoprotein) of about 22 kDa size that was never detected in our proteome analyses (Figs 1 and 2 and Supporting Information Figure S2). Therefore, the role of gene phtDb (PA01_00218; UbiX-like protein) in o-phthalate metabolism is still unclear. However, recently White et al. (2015) reported that the UbiX-like protein in bacterial ubiquinone biosynthesis acts as a flavin mononucleotide (FMN)-binding protein with no decarboxylase activity. The FMN-binding protein (UbiX-like protein) is a metal-independent flavin prenyltransferase involved in the formation of a novel flavin-derived cofactor that is required for the decarboxylase activity of the UbiD-like protein [Lin et al., 2015; White et al., 2015].

Phylogenetic analysis of the putative o-phthalyl-CoA decarboxylase revealed that PhtDa (PA01_00217) was affiliated with UbiD-like decarboxylases [Bhuiya et al., 2015] that are known to decarboxylate, for example 3-polyprenyl-4-hydroxybenzoate in Escherichia coli [Baba et al., 2006]. UbiD-like decarboxylases are commonly found in bacteria [Jacewicz et al., 2013] and are involved in ubiquinone biosynthesis [Zhang and Javor, 2000]. The protein designated as PhtDb (00218) is affiliated to flavin prenyltransferases of the UbiX-like protein family (Supporting Information Figure S3B). It is possible that PhtDb might play a similar role in the decarboxylation of o-phthalyl-CoA.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Enzyme assays with synthesized o-phthalyl-CoA resulted in the formation of benzoyl-CoA (Supporting Information Figure S10) or conversion of the synthesized fluorinated o-phthalyl- CoA analogue fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA (Supporting Information Figure S11). No decarboxylase activity was observed with isophthalyl-CoA and terephthalyl-CoA or with extracts of benzoate-grown cells, indicating that the putative o-phthalyl-CoA decarboxylase (00217_PhtDa) is rather selective for its substrate.

The newly identified o-phthalate-induced gene cluster comprised also a gene with the locus tag PA01_00214 (So0458) encoding a protein homologous to a subunit of TRAP transporters (TRAP: transporter-tripartite ATP-independent periplasmic) that are specialised in the transport of dicarboxylic acids, for example, malate or succinate [Forward et al., 1997]. Thus, the TRAP transporter subunit that was highly induced in o-phthalate-grown cells is most likely involved in the transport of o-phthalate. This conclusion is further backed by the observation that this protein was not induced in benzoate-grown cells (Figs 1 and 2; Table 1). Finally, the o-phthalate gene cluster contains a gene with the locus tag 00214_IcIR that is highly similar to the proteins of the IclR family. Members of this family include transcriptional regulators, which act as both activators and repressors [Molina-Henares et al., 2006]. Therefore, this protein might be involved in the regulation of genes of the o-phthalate gene cluster. Future experiments are needed to address mechanistic details of the enzymes involved in o-phthalate degradation by Azoarcus sp. strain PA01.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Experimental procedures

Bacterial strain and growth conditions Azoarcus sp. strain PA01 (KCTC 15483) was isolated from the wastewater treatment plant in Constance, Germany. It is a mesophilic, non-motile, Gram-negative, short rod-shaped bacterium that grows optimally at 30 ºC and pH 7.3 ± 0.2 [Junghare et al., 2015b]. Cells were cultured anaerobically without shaking in 50 or 100 ml infusion bottles in non-reduced, bicarbonate-buffered mineral medium under a N2/CO2 (80:20) atmosphere. Benzoate or o- phthalate was added anoxically from sterile anoxic stock solutions to final concentration of 2 mM as growth substrate plus nitrate (10 - 12 mM) as terminal electron acceptor. Cultures were inoculated from pre-cultures adapted over >20 generations on the respective substrates (o-phthalate or benzoate) in 100 ml serum bottles containing 75 ml of culture medium.

Bacterial growth analysis

Cell growth was monitored by measuring the optical density at 600 nm wavelength (OD600) in a spectrophotometer (Uvikon 860, Zurich, Switzerland). At different time intervals, samples were removed for growth measurement and substrate utilization analysis. Samples were centrifuged (8000 x g for 10 min) and the supernatant was applied to HPLC analysis for quantification of benzoate, o-phthalate, nitrate and nitrite (data not shown). Fifty microliter of sample supernatant was injected into an HPLC (LC prominence Shimadzu, Japan) equipped with a GromTM Sil 120 ODS-5 ST, column (4 mm x 150 mm, 5 µm) using an UV detector (SPD-M20A prominence) at 230 nm for signal detection. Eluent A (acetonitrile) and eluent B (20 mM H3PO4) were used at a flow rate of 0.8 ml/min at 30 ºC. A gradient of eluent B was applied for 30 min: 5 min isocratic of 92 %; 3 min gradient to 89 %; 10 min gradient to 80 %; 5 min isocratic of 80 %; 2 min gradient to 95 %; 3 min gradient to 92 %; 2 min isocratic 92 %.

Preparation of cell-free extracts Cultivation of bacterial cell mass for proteomics or enzyme activity measurements was performed in 1 l infusion bottles inoculated with adapted pre-cultures grown with benzoate or o-phthalate. Parallel cultures were grown on 2 mM of benzoate or o-phthalate and harvested in identical physiological states. All steps for preparation of cell-free extracts were performed

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 under anoxic conditions. Cells of Azoarcus sp. strain PA01 were harvested in the late exponential growth phase (OD600 = 0.2 - 0.3) by centrifugation (7 000 x g for 20 min at 4 ºC, Dupont Sorvall). Cell pellets from 1 l growth medium were washed with about 300 ml of anoxic Tris-HCl buffer (0.1 M pH 7.6) and re-suspended in 4 - 5 ml of the same buffer by adding 0.5 mg of DNase and RNase (Sigma-Aldrich). Prior to cell disruption, approximately 2 mg of protease inhibitor (Complete Mini tablets, Roche Diagnostics, Germany) was added. Cells were broken anoxically by two to three passages through a cooled MiniCell French pressure cell (SLM Aminco, Cat. No. FA003) operated at 137 MPa. Cell debris was removed by centrifugation at 27,000 x g for 30 min at 4 ºC to obtain the crude extract. The soluble protein fraction was obtained by high speed centrifugation (60,000 x g for 60 min) of the crude extract and insoluble membrane proteins were pelleted. The supernatant containing the soluble cytosolic proteins was filtered through a Sephadex NAP-25 column (GE Healthcare, Germany) for proteomic analysis. For enzyme assays, 3 - 4 ml of cell-free extract was processed further using vivaspin centrifugal concentrators (GE Healthcare, Life Sciences) with a 10,000 molecular weight cut-off (MWCO) filter to remove smaller molecules. The cytosolic soluble protein fraction was washed at least 3 times with 3 - 4 ml of Tris-HCl buffer (0.1 M, pH 7.6). The protein concentration was determined with the Bradford assay using bovine serum albumin as standard [Bradford, 1976].

Differential proteome analysis Two-dimensional (2D) gel electrophoresis (isoelectric focusing (IEF) and SDS-PAGE of cytosolic soluble proteins was carried out with cells grown with o-phthalate or benzoate. IEF was performed using 17 cm long immobilized pH gradient (IPG) strips (BioRad ReadyStripTM) of pH gradient range 5 - 8 (results of pH range of 3 - 10 and 5.7 - 6.7 strips are not shown). Approximately 1 mg of total protein from the soluble fraction was precipitated by the addition of at least 5 volumes of ice-cold acetone and incubated overnight at -20 ºC. The protein precipitate was collected by centrifugation (13,000 x g 10 min, 4 ºC), the protein pellet was air-dried at room temperature and solubilized in 600 µl of rehydration buffer (8 M urea, 2 M thiourea and 0.2 % CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate). Prior to use, 60 mM of dithiothreitol (DTT) and 0.2 % ampholyte (Sigma- Aldrich) were added to rehydration buffer. The IPG strips (pH 5 - 8) were rehydrated using 300 µl of rehydration buffer (protein load 500 mg) overnight in a rehydration tray covered

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 with mineral oil. IEF was performed using a maximal current of 50 mA per strip at 20 ºC. It was started for 1 h at a maximal voltage of 500 V (desalting), followed by a voltage ramp (rapid) to a maximal voltage of 10,000 V within 3 h and additional focusing at 10,000 V until a total of 40,000 Volt-hours (Vh) were reached [Schmidt et al., 2013]. The second dimension, that is, SDS-PAGE was performed using the BioRad Protean II system (17 x 20 cm). After focusing, the strips were equilibrated in SDS equilibration buffers I (6 M urea; 0.375 M Tris- HCl, pH 8.8; SDS 2 %; glycerol 20 %, and DTT 2 % w/v) and II (6 M urea, 0.375 M Tris- HCl pH 8.8, SDS 2 % glycerol 20 %, and iodoacetamide 2.5 % w/v) for 10 min, respectively. Strips were rinsed with 1 x Tris-glycine-SDS buffer (0.025 M Tris-HCl, pH 8.6; 0.192 M glycine and 0.1 % SDS). IPG Strips were mounted onto 12 % acrylamide gel and kept firm using an overlay of agarose (0.5 % w/v) solution. Electrophoresis was performed at 40 mA for 15 - 16 h under cooling conditions (8 - 10 ºC). One-dimensional (1D) SDS-PAGE was performed for analysis of membrane proteins (see above). The membranes were washed with Tris-HCl (50 mM, pH 7.6) and solubilised in 0.5 ml of Tris-HCl (20 mM pH 8.0) containing 0.5 % dodecyl β-D-maltoside, and incubated on ice for 2 h. The suspension was centrifuged at 60,000 x g for 30 min to remove remaining insoluble debris. The supernatant was mixed with 2 volumes of SDS-gel loading buffer containing 5 % β-mercapto ethanol and incubated at 100 ºC for 5 min. Approximately 50 mg of protein (20 ml) per sample was loaded onto the SDS-gel (8 x 6 cm x 1.0 mm BioRad Protean Mini cell, 12 % resolving and 4 % stacking gel). Electrophoresis was performed for about 2 h at 120 V. 1D and 2D gels were stained with a colloidal solution of Coomassie Brilliant Blue R-250 [Neuhoff et al., 1988] and analyzed for specific protein spots. All protein gel electrophoresis experiments were repeated at least three times.

Protein identification by mass spectrometry Coomassie-stained gels were scanned (HP Scanjet G4050) and the gel images were printed on transparency film (Ink Jet). Specifically induced protein spots of o-phthalate-grown and benzoate-grown cells were identified by manual overlapping of the printed gel images. Protein spots of interest were excised and submitted for MS analysis to the Proteomics Center of the University of Konstanz. Protein spots were destained, reduced with DTT, alkylated with iodo acetamide and digested with trypsin. Trypsinized peptides were analyzed by liquid

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Chromatography-tandem mass spectrometry (LC-MS/MS) using a Bruker Esquire 30001 with an Agilent 1100 HPLC or a Bruker amaZon Speed ETD with a Bruker Advance nanoHPLC. Total proteome analysis of the cells grown with ophthalate or benzoate was performed using a LTQ Orbitrap Discovery with an Eksigent 2D-nano HPLC (Thermo Fisher Scientific). Short peptide sequences obtained by MS (peptide spectral data) were matched using the Mascot search engine [(v2.2.2 from Matrix Science); Perkins et al., 1999] with the locally established protein database of the IMG annotated genome of Azoarcus sp. strain PA01 [Junghare et al., 2015b] for the identification of protein spots. Search results were validated on the basis of top hits and scores obtained in the Mascot search engine. MS identification of protein spots and Mascot search were performed at least twice from independent protein spots analysis.

Identification of gene clusters and phylogenetic analysis To identify the o-phthalate gene cluster, we analyzed the genome of Azoarcus sp. strain PA01 and selected protein sequences were BLASTp-searched using the NCBI (https://www.ncbi.nlm.nih.gov/) or UniProt (http://www.uniprot.org/blast/) online search tool. The relative positions of the genes within the genome and putative transcription direction of the genes were determined using online tools provided by the IMG, JGI [http://genome.jgi.doe.gov/, Markowitz et al., 2009]. Phylogenetic analysis of predicted amino acid sequences of the o-phthalate induced genes with the IMG gene loci PA01_00215 to PA01_00218 were obtained from the Azoarcus sp. strain PA01 genome were aligned using ClustalW from the MEGA7 software [Kumar et al., 2016] with closely related and characterized CoA-transferases or decarboxylases from other bacteria, repectively. Protein sequences for the alignments were obtained from GenBank and UniProtKB/SwissProt or from the IMG database. Phylogenetic analysis was performed using the neighbor-joining algorithm [Saitou and Nei, 1987], and Poisson correction method [Zuckerkandl and Pauling, 1965]. The phylogenetic tree was constructed using MEGA 7 [Kumar et al., 2016]. Bootstrap values were calculated as a percentage of 1,000 replicates [Felsenstein, 1981].

Determination of enzyme activities in cell-free extracts Cell-free extracts (~2 - 3 g wet weight grown either with o-phthalate or benzoate) were prepared anoxically as described. Enzyme activities were measured anoxically (unless

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01 mentioned otherwise) in 5 ml serum vials closed with butyl rubber septa. Vials were flushed with nitrogen and all additions and samples were taken with gas-tight Unimetrix microliter syringes (Macherey-Nagel, Duren, Germany). All enzyme assays were run at least in triplicates at 28 - 30 ºC.

(i) Succinyl-CoA:o-phthalate CoA-transferase assay: CoA-transferase activity catalyzing the conversion of o-phthalate to o-phthalyl-CoA with succinyl-CoA as CoA donor was measured discontinuously by LC-MS/MS screening for the formation of o-phthalyl-CoA and benzoyl- CoA. The standard assay mixture (500 µl) contained: triethanolamine buffer (0.1 M pH 7.6), 0.2 - 0.3 mg protein, succinyl-CoA (2 mM, Sigma-Aldrich) and o-phthalate (6 mM, Fluka Chemie). After initial incubation for about 5 min, the enzyme reactions were started by the addition of the CoA acceptor (o-phthalate) or CoA donor (succinyl-CoA, free CoA or acetyl- CoA). Occasionally 0.1 - 0.2 mM of N, N-dicyclohexylcarbodiimide was added to inhibit ATPase activity to minimize succinyl-CoA hydrolysis. For determination of the substrate specificity, free acids; such as isophthalate, terephthalate (Fluka Chemie) or 3-fluoro-o- phthalate (Sigma) were used, or succinyl-CoA was replaced by free CoA or acetyl-CoA (Sigma-Aldrich) as a possible alternative CoA donor.

(ii) o-Phthalyl-CoA decarboxylase assay: The standard enzyme assay mixture (500 µl) contained; triethanolamine buffer (0.1 M, pH 7.6) and 0.2 - 0.3 mg protein extract. After initial incubation of cell-free extract for about 5 min in buffer, the enzyme reaction was started by the addition of 50 µl synthesized o-phthalyl-CoA (approximately 20 - 30 mM). Alternatively, isophthalyl-CoA, terephthalyl-CoA or fluoro-o-phthalyl-CoA were used in enzyme assays in order to investigate the substrate specificity of the putative decarboxylase. Formation of benzoyl-CoA/fluoro-benzoyl-CoA was measured by LC-MS/MS analysis. Control assays were performed either after heat inactivation of the cell-free extract (90 ºC x 10 min) or by excluding one key substrate in the enzyme assay, for example, succinyl-CoA or o-phthalate or o-phthalyl-CoA. The effect of oxygen exposure on the enzyme activities was studied by incubating the enzyme assays exposed to air. For analysis of the enzyme products, 70 µl of the samples was withdrawn at different time points, 0 min (before the start of the reaction), 3, 5, 10 or 20 min and the reaction was stopped by addition of an equal volume of methanol and centrifuged (13,000 x g for 5 min). The supernatant was transferred into 200 µl glass vial inserts and analyzed for the respective enzyme product formation by LC-MS/MS.

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Synthesis of o-phthalyl-CoAs Phthalic anhydride (3 mg, 20 µmol; Sigma-Aldrich) was dissolved in 100 µl of acetone and mixed with 200 µl of NaOH (0.3 N) containing coenzyme A trilithium salt (10 mg, 13 µmol) for 3 min. Acetone was removed by a gentle stream of nitrogen and the formation of o- phthalyl-CoA was analyzed by LCMS/MS (Supporting Information Figure S4). Isophthalyl- CoA and fluoro-o-phthalyl-CoA were generated using 16 µmol of each phthalate dissolved in 100 µl of dry tetrahydrofuran in a dried 4 ml glass vial fitted with a stirring bar under nitrogen. To this solution, ethyl chloroformate (2 µl, 21 µmol) and trimethylamine (2 µl, 15 µmol) were added. The reaction mixture was stirred for 1 min and coenzyme A trilithium salt (10 mg, 13 µmol) dissolved in 200 ml of NaOH (0.3 N) was added and mixed for 3 min. Tetrahydrofuran was removed by blowing it off in a gentle nitrogen stream. Terephthalyl- CoA was synthesized analogously, but terephthalate (16 µmol) was dissolved in dimethylformate (DMF, 500 µl) and activated with ethyl chloroformate (2 µl, 21 µmol) and trimethylamine (2 µl, 15 µmol) to the anhydride that was reacted with free CoA in 500 µl of NaOH (0.3 N). DMF was removed by freeze drying the sample. The formation of phthalyl- CoAs was checked by LC-MS/MS (Supporting Information Figure S5). Phthalyl-CoAs were obtained in about 70 - 90 % yield as determined by the peak area ratio of free-CoA/CoA- ester, and were used directly for the enzyme assays.

LC-MS/MS analysis of Coenzyme A esters LC-MS/MS analysis of coenzyme A esters (CoAs) was performed with an Agilent 1100 HPLC system fitted with a Phenomenex Synergi polar-RP (250 x 2 mm, 4 µm) column. The mobile phase consisted of solvent A, 30 mM ammonium acetate buffer (pH 7.4), and solvent B, acetonitrile with 0.1 % acetate. The CoAs were separated by programmed elution: 2 min isocratic 2 % B, in 20 min gradient elution to 100 % B at a flow rate of 0.25 ml/min. The assay samples of 30 - 90 µl was injected. For detection of the respective CoAs, the HPLC was connected to a Finnigan LCQ ion trap mass spectrometer fitted with an electrospray ion (ESI) source and operated in the MS/MS mode to specifically detect CoA esters [Park et al., 2007]. In addition, reference compounds were measured for all CoA esters studied except for fluoro-benzoyl-CoA.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Acknowledgments MJ is very grateful to the German Academic Exchange Service (DAAD) Bonn, Germany, for providing a PhD scholarship (A/11/75732). The authors thank Dr. David Schleheck, University of Konstanz, for providing space in his proteomics laboratory to conduct 2D- PAGE experiments. We are indebted to Andreas Marquardt, University of Konstanz, for the MS protein identification service.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Supporting information

Fig. S2. SDS-PAGE analysis of solubilized membrane proteins treated with dodecyl β- maltoside. Lane 1,-prestained protein ladder; lane 2, benzoate-grown and lane 3, o-phthalate- grown cells. The table shows the LC-MS/MS identification of the respective protein spots and their predicted functions are shown.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Figure S4. Comparison of the genes from the phthalate gene cluster from the genome of Azoarcus sp. strain PA01 and distribution of genes with similar functions in the genomes of the other nitrate-reducing bacteria ‘A. aromaticum EbN1’, Azoarcus toluclasticus ATCC 700605 and Thauera chlorobenzoica. The neighborhoods of the genes from the genome with the same top COG hit (Clusters of Orthologous Groups of protein) are compared. Genes having significant similar IMG predicted function are shown in the same color (top COG hit). Grey, 4-hydroxy-3-polyprenylbenzoate decarboxylase (UbiX-like decarboxylase/flavin binding protein); red, 4-hydroxy-3-polyprenylbenzoate decarboxylase (UbiD-like decarboxylase); green, benzylsuccinate CoA-transferase (CoA-transferase family III); purple, TRAP-type uncharacterized transport system periplasmic component; blue, TRAP-type uncharacterized transport system, fused permease components and black, hypothetical protein respectively.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Figure S5. ESI MS/MS of the [M+H]+ m/z 868 of succinyl-CoA (Sigma-Aldrich).

Figure S6. ESI-MS/MS of the [M+H]+ m/z 916 of synthesized o-phthalyl-CoA.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Figure S7. ESI-MS/MS of the [M+H]+ m/z 934 of synthesized fluoro-o-phthalyl-CoA.

Figure S8. ESI-MS/MS of the [M+H]+ m/z 916 of isophthalyl-CoA.

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Enzymes involved in the anaerobic degradation of ortho-phthalate by Azoarcus sp. strain PA01

Figure S9. ESI-MS/MS of the [M+H]+ m/z 916 of terephthalyl-CoA.

Figure S10. The time course of benzoyl-CoA formation (rt: 11.4 min) by the enzymatic decarboxylation of the synthesized o-phthalyl-CoA in the enzyme assay performed with cell- free extract of o-phthalate-grown cells, monitoring the specific ion trace m/z 365 of the MS/MS fragmentation of benzoyl-CoA (quasimolecular ion m/z 872).

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Figure S11. The time course of fluoro-benzoyl-CoA formation (rt: 11.5 min) by the enzymatic decarboxylation of the synthesized fluoro-o-phthalyl-CoA in the enzyme assay performed with cell-free extract of o-phthalate-grown cells, monitoring the specific ion trace m/z 383 of the MS/MS fragmentation of fluoro-benzoyl-CoA (quasimolecular ion m/z 890).

Figure S12. The time course of benzoyl-CoA formation (rt: 11.4 min) in a coupled enzyme assay with o-phthalate and succinyl-CoA by the cell-free extract of o-phthalate-grown cells monitoring the specific ion trace m/z 365 of the MS/MS fragmentation of benzoyl-CoA (quasimolecular ion m/z 872) under aerobic incubation conditions.

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CHAPTER 4

Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase from Azoarcus sp. strain PA01

Madan Junghare, Dieter Spiteller, Bernhard Schink

Manuscript to be communicated soon

Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase

Abstract o-Phthalyl-CoA decarboxylase (PhtDa and PhtDb) encoded by the two genes PA01_00217 and PA01_00218 which catalyses the decarboxylation of activated o-phthalate to benzoyl- CoA, an essential step in anaerobic o-phthalate degradation by Azoarcus sp. strain PA01. Both genes were originally annotated as an UbiD-like/UbiX-like protein. In our previous study, we suspected that these two genes are involved in the decarboxylation of activated phthalate, but their specific role remained unknown. Cloning and overexpression of both genes was performed using the pET100/D-TOPO expression vector in E. coli. The functional gene PA01_00217 is 1,584 bp long coding for protein PhtDa (60 kDa), whereas PA01_00218 is 600 bp long codes for protein PhtDb (22 kDa). Here, we demonstrate that both genes are essential for decarboxylation, and each has a different role. PhtDb is a flavin mononucleotide (FMN)-binding protein which does not function as a decarboxylase alone. Rather, PhtDb is assumed to generate a modified FMN-containing cofactor that is required by the PhtDa for decarboxylase activity. Alone, PhtDa does not function as a decarboxylase either. Recombinantely expressed PhtDa and PhtDb together showed activity for decarboxylation of o-phthalyl-CoA to benzoyl-CoA, only if PhtDb was previously incubated with FMN and dimethylallyl monophosphate. Phylogenetically, the proteins PhtDa and PhtDb are closely related to UbiD-like/UbiX-like enzymes that catalyses the decarboxylation of 4-hydroxy-3- octaprenylbenzoic acid to 2-octaprenylphenol, an intermediate step in ubiquinone biosynthesis. Furthermore, multiple sequence alignment and structural modelling of both proteins suggested that only PthDb possesses the binding site for FMN that forms a dodecameric structure, while the PhtDa protein forms a homodimer. These results strongly indicate that the flavin-containing cofactor is essential for decarboxylation of o-phthalyl-CoA to benzoyl-CoA during anaerobic o-phthalate degradation by Azoarcus sp. strain PA01.

Introduction o-Phthalic acid (1,2-dicarboxybenzene) is a synthetic compound most commonly used in the manufacturing of phthalate esters. Phthalate esters are globally produced in huge quantities each year for a wide range of applications, e.g., as plasticizers [Liang et al., 2008]. They are considered as priority pollutants due to their toxicity, and adverse effect on human health and animals. Thus, their environmental removal is of great concern. Degradation of phthalate

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase esters by bacteria involves the initial de-esterification step which releases readily degradable side chain alcohols and often phthalate accumulates which requires decarboxylation for further breakdown. However, decarboxylation of phthalate is a challenging reaction in microbial degradation and limits its degradation, especially for anaerobic bacteria [Kleerebezem et al. 1999c].

In the aerobic breakdown of phthalate, decarboxylation is facilitated by oxygenase-dependent oxygenation reactions, forming a 3,4-dihydrodiol (in Gram-positive bacteria) or 4,5- dihydrodiol (in Gram-negative bacteria) which is further converted to a dihydroxyphthalate derivative by dehydrogenation. Dihydroxyphthalate is then decarboxylated by corresponding dihydroxyphthalate decarboxylases to the common intermediate 3,4-dihydroxybenzoate [Eaton and Ribbons, 1982; Batie et al., 1987; Pujar and Ribbons, 1985; Chang and Zylstra, 1998]. Similarly, decarboxylation of m-phthalate (isophthalate) and p-phthalate (terephthalate) also follow the pathway analogous to phthalate decarboxylation leading to 3,4-dihydroxybenzoate as the intermediate [Schläfli et al., 1994; Fukuhara et al., 2008; Fukuhara et al., 2010]. In essence, aerobic phthalate-degrading bacteria introduce molecular oxygen into the phthalate ring that partially polarizes the ring, facilitating the difficult step of phthalate decarboxylation.

Due to absence of molecular oxygen, anaerobic phthalate-degrading bacteria cannot facilitate phthalate decarboxylation by introducing oxygen atoms into the phthalate molecule. Thus, anaerobic decarboxylation of phthalate to benzoate is considered to be challenging and regarded as the rate-limiting step in anaerobic phthalate degradation [Kleerebezem et al., 1999c]. Although, anaerobic degradation of man-made phthalates (which have been introduced only after the 1920s) has been known for many years, but still the anaerobic phthalate decarboxylation reaction was poorly understood. In the past, different hypotheses were proposed for anaerobic decarboxylation of phthalate to benzoate. E.g., Taylor and Ribbon (1983) suggested that phthalic acid is reduced by two electrons leading to 3,5- cyclohexadiene-1,2-dicarboxylic acid before its decarboxylation to benzoic acid [Taylor and Ribbons, 1983]. However, reduction of phthalic acid with NADH is not a feasible reaction and could never be demonstrated. Further, it was assumed that decarboxylation of phthalate isomers to benzoate involves formation of a phthalate ester with coenzyme A (CoA), which is subsequently decarboxylated to benzoyl-CoA [Nozawa and Maruyama, 1988ab]. But such a

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase reaction lacked experimental proof until now. In 2016, first experimental evidence was provided that anaerobic phthalate decarboxylation by denitrifying bacteria such as Azoarcus sp. strain PA01 or Thauera chlorobenzoica is a two-step reaction proceeding through an initial activation of o-phthalate to o-phthalyl-CoA catalysed by a succinyl-CoA-dependent CoA-transferase, which is subsequently decarboxylated to benzoyl-CoA [Junghare et al., 2016; Ebenau-Jehle et al., 2016]. We confirmed the formation of o-phthalyl-CoA from o- phthalate and succinyl-CoA, and observed its decarboxylation to benzoyl-CoA in cell-free extract of Azoarcus sp. strain PA01 [Junghare et al., 2016]. Benzoyl-CoA is further metabolized by the enzymes of the established anaerobic benzoyl-CoA degradation pathway [Fuchs et al., 2012; Breese et al., 1998] and the genome of Azoarcus sp. strain PA01 possess the required genes for anaerobic benzoate-degradation pathway [Junghare et al., 2015B].

Azoarcus sp. strain PA01 cells grown with o-phthalate induced a set of proteins that were identified by differential protein profiling using benzoate-grown cells as the control. Interestingly, phthalate-induced proteins were encoded by genes that are placed together in a single gene cluster coding for the protein homologous to a solute transporter (locus tag PA01_00214), two CoA-transferases (PA01_00215 and PA01_00216), and the UbiD- like/UbiX-like decarboxylases (PA01_00217 and PA01_00218), respectively. The CoA- transferases are suggested to be involved in o-phthalate activation to o-phthalyl-CoA, and UbiD-like/UbiX-like decarboxylases in decarboxylating o-phthalyl-CoA to benzoyl-CoA [Junghare et al., 2016]. Our results are consistent with the phthalate-induced genes 21725 (UbiD-like) and 21730 (UbiX-like) that were also shown to be involved in the o-phthalyl- CoA decarboxylation in the phthalate-degrading ‘T. chlorobenzoica’ [Ebenau-Jehle et al., 2016]. In addition to this, terephthalate-fermenting Syntrophoharbdus aromaticivorans strain UI contains two genes SynarDRAFT_0373 and SynarDRAFT_0374 that were suggested to be involved in terephthalyl-CoA decarboxylation to benzoyl-CoA [Nobu et al., 2015]. Interestingly, in all cases these genes involved in decarboxylation were originally annotated as proteins homologous to UbiD-like/UbiX-like decarboxylases (except gene 0374 from S. aromaticivorans strain UI that was annotated as hypothetical protein).

UbiD-like and UbiX-like proteins are widely distributed among prokaryotes, e.g. Escherichia coli with the carboxy-lyase activity converting 4-hydroxy-3-octaprenyl benzoic acid to 2- octaprenylphenol, an early step in ubiquinone biosynthesis [Meganathan, 2001; Liu and Liu,

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase

2006; Gulmezian et al., 2007; Zhang and Javor, 2000]. Even though 4-hydroxy-3-octaprenyl benzoic acid and o-phthalyl-CoA are structurally quite different from each other, the genes PA01_00217 and PA01_00218 that are homologous to UbiD-like/UbiX-like proteins are suspected to be involved in the anaerobic decarboxylation of phthalate by Azoarcus sp. strain PA01 [Junghare et al., 2016]. However, their specific roles still remained unclear, especially the question how these two proteins function together to catalyse the decarboxylation of o- phthalyl-CoA to benzoyl-CoA. The aim of the present study was to clone, express and characterize both o-phthalate-induced proteins PhtDa (60 kDa) and PhtDb (23 kDa) that are involved in the decarboxylation of o-phthalyl-CoA to benzoyl-CoA during the anaerobic degradation of o-phthalate by Azoarcus sp. strain PA01.

Materials and methods

Materials and reagents All chemicals used in this study were of analytical grade. Coenzyme A (CoA) trilithium salt, ampicillin, flavin mononucleotide (FMN), flavin dinucleotide (FAD), γ,γ-dimethylallyl monophosphate (DMAP), phthalate isomers and phthalic anhydride were from Sigma- Aldrich (Germany). o-Phthalyl-CoA’s used in this study were produced as described previously [Junghare et al., 2016]. Materials and equipment for protein purification were obtained from GE Healthcare (Munich, Germany).

Strains and cultivation conditions Azoarcus sp. strain PA01 was cultivated anoxically in non-reduced, freshwater mineral medium containing o-phthalate (2 mM) or benzoate (2 mM) as the growth substrate supplemented with 10 - 12 mM nitrate as electron acceptor as described previously [Junghare et al., 2015b; Junghare et al., 2016]. Cells were harvested by centrifugation (10,000 x g for

10 min at 10ºC) at an optical density (OD600nm) 0.2 - 0.3 and stored at -20 °C until further use. E. coli OneShot TOP10, E. coli BL21 star (DE3) and E. coli Rosetta 2 (DE3)pLysS (Invitrogen) cells were grown either in Luria Bertani (LB) [Sambrook et al., 1989] or in terrific broth (TB) [Tartoff et al., 1987] media (unless otherwise mentioned) supplemented with 100 µg/ml of ampicillin.

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase

Gene amplifications, plasmid construction and sequencing The genes encoding o-phthalyl-CoA decarboxylase, i.e., PA01_00217 (1,584 bp) and PA01_00218 (600 bp) sequence can be found at the Integrated Microbial Genomes (IMG) under gene ID 2597201524 and 2597201525, (https://img.jgi.doe.gov/cgi-bin/m/main.cgi) respectively, in the draft genome sequence of Azoarcus sp. strain PA01 [Junghare et al., 2015b]. For PCR amplification of the respective protein (PhtDa and PhtDb) coding genes, primers were synthesized (Microsynth, Switzerland). To facilitate directional cloning of the genes into champion pET100/D-TOPO expression vector which introduces a C-terminal 6x- His-tag (3 kDa), overhang of four nucleotides (CACC as underlined) was added to 5’ end of each forward primer of the respective genes. The gene PA01_00217 coding for PhtDa (60 kDa) was amplified using the primers: 217f, 5’-CACCATGAACGATCTGGCAACGA-3’ and 217r, 5’-CTTTGTGGTGGCAGCAGC-3’, and PA01_00217 coding for PhtDb (22 kDa) was amplified using primers: 218f, 5’-CACCATGAAAAGACTGATAGTGGGG-3’ and 218r, 5’-TGCGATTACTTCTGCGGC-3’, respectively. Gene amplification was performed using proof reading Phusion High Fidelity (HF) DNA polymerase (Finnzymes) in the PCR reaction (50 µl) containing: 2 µl of gDNA template (approx. 50 - 100 ng); 2 µl of each primer

(100 µM); 1 µl of 50 mM MgCl2 (unless otherwise mentioned); 10 µl of 5x Phusion HF buffer; 20 µl of dNTPs (500 µM); 0.5 µl of Phusion HF DNA polymerase (0.25 Units) and

12.5 µl of molecular grade water (for amplification of gene PA01_00218, MgCl2 was not added). A personal DNA thermal cycler (Eppendorf) was used for PCR reactions: initial denaturation at 98 °C for 5 min followed by 35 cycles of 98 °C for 30 s, 65 °C (70 °C for PA01_00217) for 40 s, 72 °C for 90 s and final extension at 72 °C for 10 min. PCR reactions were analysed by agarose (1 % w/v) gel electrophoresis and stained with ethidium bromide. Analysed PCR products were directly purified using DNA Clean and Concentrator kit (Zymo Research). DNA concentrations were determined using a nano UV spectrophotometer (Thermo Scientific).

The purified PCR products were cloned by mixing with the champion pET100/D-TOPO expression vector and ligated according to the manufacturer’s protocol (Invitrogen). The ligated vector containing the gene of interest (recombinant plasmid) was transformed into chemically competent E. coli OneShot TOP10 cells and grown on LB agar plates containing ampicillin (100 µg/ml). Positive clones containing the gene of interest were screened using the T7 primer pair as: T7, 5´-TAATACGACTCACTATAGGG-3´ and T7 reverse, 5´-

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase

TAGTTATTGCTCAGCGGTGG-3´, provided in the champion directional TOPO expression kit. Positive clones of E. coli TOP10 cells were propagated in LB medium (ampicillin, 100 µg/ml) and used for recombinant plasmid DNA isolation (Zypy Plasmid Miniprep, Zymo Research). Prior to plasmid transformation to expression host E. coli cells, the correct integration and sequence identity of the inserted gene was individually verified by sequencing the recombinant plasmids across the cloning site using the T7 primers (as mentioned above). DNA sequencing was performed at GATC-Biotech (Constance, Germany) and sequences were compared with the original gene sequences from the genome of Azoarcus sp. strain PA01 [Junghare et al., 2015b] for any mutation or base change using the BLAST (http://www.ncbi.nlm.nih.gov).

Optimization of protein expression and purifications Chemically competent OneShot E. coli BL21 Star (DE3) and E. coli Rosetta 2 (DE3)pLysS cells were transformed with the purified recombinant vector. Transformant’s were grown either in 10 ml of LB liquid or onto the LB solid agar medium (for maintenance) containing ampicillin (100 µg/ml). Over-expression of recombinant proteins PhtDa and PhtDb were performed in LB medium or in TB medium containing 100 µg/ml of ampicillin supplemented with 1 % glucose and 3 % ethanol (unless otherwise mentioned). Pre-cultures were grown initially at 37 °C under shaking conditions (200 rpm). Cells were induced for protein expression at an optical density (OD600nm) between 0.5 to 0.6 by adding 0.3 - 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) and grown at different temperatures (at 37 °C for about 4 h; 25 °C for about 7 h and at 15 °C for about 17 h). Cells were harvested by centrifugation (10,000 x g for 10 min at 8 °C) and stored frozen at -20 °C until further use or analysed for soluble protein expression. Protein expression optimization experiments were performed in small scale cultures using 10 to 100 ml of growth media.

For large scale protein expression, transformed cultures were grown in 500 or 1000 ml media. About 2 to 3 g wet cell material was suspended in 5 ml lysis buffer containing 50 mM Tris- HCl (pH 7.6), 200 mM NaCl, 100 mM KCl, 60 mM imidazole, 10 % glycerol and 5 mM dithiotritol designated as buffer A. Prior to cell lysis, the cell suspension was supplemented with 1 mg/ml lysozyme (Sigma), 0.01 mg/ml DNase I (Sigma), 50 mM L-glutamate, 50 mM L-arginine-HCl, 1 mg of protease inhibitor cocktail (Roche) plus dimethyl allyl monophosphate (DMAP) together with 1 mM flavin mononucleotide (FMN) or 1 mM flavin

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase dinucleotide (FAD). The cell suspension was incubated on ice for about 30 min for enzymatic (lysozyme) cell lysis. Finally, cells were passed through a French pressure cell operated at 139 kPa at least for three times. Cell lysate was centrifuged at 27,000 x g for 30 min at 4 °C to remove the cell debris. The supernatant (cell-free extract) was either used directly for enzyme activity measurements or subjected to further enzyme purification.

His-tagged recombinant enzyme purification was performed using the Ni2+ ion affinity chromatography. E. coli supernatant was directly applied onto a 1-ml Ni2+ ion chelating poly- histidine affinity column (HisTrap HP, GE Healthcare) equilibrated with buffer A (50 mM Tris-HCl, pH 7.6; 200 mM NaCl; 100 mM KCl; 60 mM imidazole; 10 % glycerol and 5 mM dithiotritol). After washing the column with 10 ml of buffer A, the His-tagged proteins were finally eluted with 2 - 3 ml of elution buffer B (buffer A supplemented with 0.5 M imidazole final concentration). Imidazole was removed by passing the eluted protein fraction over a desalting NAPTM-25 column (GE Healthcare) equilibrated with buffer A and eluted with 2.5 ml of the same buffer. Fifty percent glycerol was added to the purified protein to a final concentration of 25 % (v/v) and stored at -20 °C. Protein contents were determined using the Bradford assay (using BAS as the standard).

Molecular weight determination by SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the purity and molecular weight of the over-expressed proteins. SDS-PAGE was also used to determine the expression level of soluble protein in E. coli cell-free extracts or cell pellet, according to Laemeli method using the established protocol [Junghare et al., 2016]. Protein samples were mixed with sample loading buffer (0.05 % bromophenol blue, 5 % β-mercaptoethanol, 10 % glycerol, 1 % SDS in 0.25M Tris-HCl buffer pH 6.8) and denatured by heating at 99 ºC for 10 min. Samples were loaded onto the one-dimensional SDS gel (12 % resolving and 4 % stacking gel) and electrophoresis was performed using a BioRad Mini-Protean cell (8 cm x 6 cm x 1.0 mm) at constant voltage of 120 V for about 2 h. For molecular weight (MW) estimation of protein bands, 10 µl of the prestained protein ladder of MW range 10 - 180 kDa was used (Page Ruler, Thermo Fisher). Protein bands were visualised with 0.25 % Coomassie Brilliant Blue R-250 dye dissolved in 10 % acetic acid and 50 % methanol, followed by de-staining in 10 % acetic acid and 50 % methanol in distilled water. The detected protein band size was compared with standard protein MW markers for

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase size estimation (Pre-stained protein molecular size marker 15 – 100 kDa, Themo Scientific). Expected size protein bands were excised from the SDS gel and given for peptide identification by electrospray mass spectrometry (MS) analysis at the Proteomics Facility, University of Konstanz. The MS spectra of peptides were analysed using the Mascot search engine (version 2.2.03, Matrix Science) to confirm the identity of the over-expressed proteins by comparison with the deduced amino acid sequence of the respective genes from the genome of the Azoarcus sp. strain PA01 [Junghare et al., 2015b].

Decarboxylase activity assays and cofactor requirement o-Phthalyl-CoA decarboxylase activity of PhtDa and PhtDb enzymes overexpressed in E. coli cells was assayed according method described before [Junghare et al., 2016]. The decarboxylase activity was assayed directly with the E. coli cell lysate containing overexpressed protein PhtDa and PhtDb for conversion of o-phthalyl-CoA to benzoyl-CoA. Standard anoxic enzyme assays (150 µl) contained: 100 µl tri-ethanolamine buffer (0.1M, pH 7.6), 10 - 20 µl cell lysates (PhtDa and PhtDb) and 1 mM dithionite. Enzyme reaction was started by the addition of 10 µl o-phthalyl-CoA and incubated at 30 °C for about 30 min. Samples (30 µl) were removed at different time intervals and proteins were precipitated with methanol (60 µl). Samples were centrifuged and the supernatants were analysed for benzoyl- CoA formation by liquid chromatography coupled with electrospray ionization-MS (LC-ESI- MS) as described previously [Junghare et al., 2016]. The substrate specificity of the decarboxylase activity was assayed for decarboxylation of isophthalyl-CoA, terephthalyl- CoA or fluoro-o-phthalyl-CoA to benzoyl-CoA (fluoro-benzoyl-CoA in case fluoro-o- phthalyl-CoA). Effect of oxygen exposure on the stability and activity of PhtDa and PhtDb was assayed by performing enzyme assay under oxic condition.

The flavin dependent activity of recombinant PhtDb (PA01_00218) was determined using reconstitution experiments. Heterologously expressed PhtDb in E. coli cells were suspended in cell lysis buffer A (as mentioned above) and supplemented with 1 mM flavin dinucleotide (FAD) or flavin mononucleotide (FMN) plus about 10 µl of di-methyl allyl mono phosphate (DMAP). Cell were incubated on ice for about 30 min (enzymatic cell lysis) and finally opened by French Press treatment as described above. Cell lysate was either used directly for enzyme activity determinations or processed further by centrifugation (27,000 x g for 30 min at 4 ºC) for His-tag purification of recombinant protein. UV-visible spectrum (300nm to

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500nm wavelength) of the purified PhtDb enzyme was recorded in the UV-vis spectrophotometer (Jasco V-630, spectrophotometer) for determination of FMN content. Spectrum of 1 µM FMN in elution buffer was used as reference.

Native protein PAGE analysis Estimation of protein complex size and protein-protein interaction, native protein polyacrylamide gel electrophoresis (PAGE) was performed with the cell-free extract of Azoarcus sp. strain PA01 cells grown with o-phthalate and benzoate as the control. Additionally, native protein gel was also performed with the partially His-tag purified recombinant PhtDa (60 kDa) and PhtDb (22 kDa) proteins. Approximately, each of 30 µg of purified PhtDa and PhtDb proteins or soluble proteins from the cell-free extract of Azoarcus sp. strain PA01 cells were mixed with the native protein sample loading buffer (60 mM Tris- HCl, pH 6.8; 30 % glycerol; 0.01 % (w/v) bromophenol blue). PhtDa and PhtDb protein- protein interaction were investigated by incubating these two proteins together plus FMN and DMAP addition. Electrophoresis was performed using BioRad Protean Mini cell and the PreCast gel slab (BioRad) at 130 V for about 3 h. Protein bands were visualised by straining the gels with Coomassie Brilliant Blue R-250 as described above. Protein-protein interaction and protein complex sizes were estimated by comparing the stained protein bands with the native protein molecular weight markers range between 60 to 690 kDa (Amersham, GE Healthcare Life Sciences). Protein bands excised from the stained native protein gels were identified by MS analysis.

Sequence alignment and structure homology modelling Protein similarity searches were performed using the BLASTp search tool available at the NCBI web page (http://www.ncbi.nlm.nih.gov/BLAST/). A phylogenetic tree was constructed to determine sequence divergence and the evolutionary positions using the MEGA 7 software package [Kumar et al., 2016]. Amino acid sequences of the PhtDb and PhtDa of Azoarcus sp. strain PA01 and the characterized the UbiX-like and UbiD-like enzymes from bacterial and fungal species were considered for tree construction. Phylogenetic tree was calculated using neighbor-joining method and the bootstrap (1000 replicates) method was followed for corroborating the consistency of each node in the tree. For determination of conserved amino acid regions, the T-Coffee Expresso multiple sequence alignment package with the CLUSTAL W (1.83) alignment tool (http://tcoffee.crg.cat/apps/tcoffee/do:expresso) was used

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[Notredame et al., 2000]. Aligned sequences were edited in the ESPript 3.0 program [(http://espript.ibcp.fr/ESPript/ESPript/index.php); Robert and Gouet, 2014]. Homology-based 3-dimensional (3-D) models were constructed for PhtDa and PhtDb proteins to understand structural aspects and functional homology to the related UbiD-like and UbiX-like enzymes. The predicted structural models for PhtDa and PhtDb were generated through the SWISS- MODEL workspace in an automated mode [Arnold et al., 2006; Biasini et al., 2014]. PhtDa structure was predicted using the template putative aromatic acid decarboxylase (Pad1) crystal structure (PDB accession code 4iws.1.A) from Pseudomonas aeruginosa [Jacewicz et al., 2013], whereas PhtDb structure was predicted using the FMN-dependent Ubix-like decarboxylase (PDB code 1sbz.1.A) from Escherichia coli O157:H7 [Rangarajan et al., 2004]. Predicted structural model visualization and superimpositions analyses with the respective template structures were performed with the UCSF Chimera package [Pettersen et al., 2004].

Results

Gene amplifications, cloning of PhtDa and PhtDb Genomic DNA used for amplification of full length o-phthalyl-CoA decarboxylase genes resulted in a 1.58 kb amplicon for PA01_00217 and 0.6 kb amplicon for PA01_00218 (Figure 1A). PCR products were cloned into the ampicillin resistance pET100/D-TOPO expression vector which adds 6His-tag (3kDa), Xpress Epitope to the N-terminal end of the overexpressed protein. Recombinant plasmid was used to transform E. coli TOP10 cells and grown on LB media containing ampicillin (100 µg/ml). Several positive clones were found with a gene of interest inserted into the pET100/D-TOPO expression vector. Recombinant clones were picked and screened based on the PCR product size of amplified PCR products using T7 primers (as discussed in method section) and confirmed by comparing the size of the respective genes plus additional 276 bp from cloning site of pET100/D-TOPO vector (Figure 1AB). The gene coding for PhtDa is 1,584 bp long plus 276 and PhtDb is 600 bp plus 276 and expected size PCR products (using T7 primers) from positive clones are shown in Figure 1B.

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Selected positive clones of E. coli TOP10 cells containing recombinant plasmids were propagated in LB media (ampicillin, 100 μg/ml) for plasmid DNA isolation. Purified recombinant plasmid DNAs were sequenced across the cloning site using T7 primers and inserted gene sequences were determined (data not shown). The sequence of recombinant genes displayed correct insertion of genes into the vector and showed 100 % sequence identity with the original gene sequences available from the published draft genome sequence of Azoarcus sp. strain PA01 [Junghare et al., 2015b].

Figure 1. Electrophoretic analysis of PCR-amplified genes in 1.0 % agarose gel stained with ethidium bromide: A) PCR amplification of genes PA01_00217, 1.58 kb and PA01_00218, 0.6 kb from gDNA of Azoarcus sp. strain PA01; B) screening of the positive clones for insertion of the respective genes into the pET100/D-TOPO expression vector PCR amplified using T7 primers (expected size of the PCR amplicon is calculated from original gene length plus 276 bp from cloning site of vector); and C) recombinant plasmid DNA isolation. 1 kb gene ruler (Thermo Fischer) was used to determine the molecular size of the PCR products on the gel.

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Heterologous expression, purification and protein identification The genes PA01_00217 and PA01_00218 (Figure 1A), putatively coding for the enzymes PhtDa and PhtDb responsible decarboxylation of o-phthalyl-CoA to benzoyl-CoA, i.e., o- phthalyl-CoA decarboxylase, were individually expressed in E. coli BL21 star (DE3) and E. coli Rosetta 2 (DE3)pLysS host cells with a N-terminal His-tag introduced from pET expression vector. The purified recombinant pET100/D-TOPO plasmids (Figure 1C) containing genes coding for PhtDa (60 kDa) and PhtDb (60 kDa) were used to transform the chemically competent E. coli BL21 star (DE3) and E. coli Rosetta 2 (DE3)pLysS as the expression host cells and grown on LB media with ampicillin selection (100 µg/ml). Complete open reading frames of the cloned genes PA01_00217 and PA01_00218 are expected to encode the proteins of 527 (PhtDa) and 199 (PhtDb) amino acids in length with the computed molecular weight of about 59.54 kDa (̴60 kDa) and 21.99 kDa (̴22 kDa), respectively, plus 3 kDa 6His-tag.

In order to find the optimal expression conditions for obtaining soluble proteins, both the transformed E. coli expression host strains were grown under different combinations of growth conditions, including temperatures (37, 25 and 15 ºC), culture media (LB and TB medium) varying IPTG concentrations (0.1 to 1 mM) and with or without glucose (1 %) plus ethanol (3 %), respectively, (data not shown). Protein expression optimization experiments revealed, E. coli Rosetta 2 (DE3)pLysS host cells were found to be suitable for optimal expression of soluble protein after induction with 0.5 mM IPTG (at OD between 0.5 - 0.6) and grown at 15 ºC for about 18 - 20 h in the TB medium supplemented with 3 % ethanol and 1 % glucose (Figure 2A and 2B). Although, under these growth conditions protein over- expression was also observed at 37 ºC and 25 ºC, but expressed proteins formed inclusion bodies (insoluble protein). Finally, E. coli Rosetta 2 (DE3)pLysS cells cultured in TB media was selected for the large scale protein expression and purifications. Protein expression was up-scaled to 500 - 1000 ml culture volume.

His-tag on the recombinant proteins (PhtDa and PhtDb) allowed quick and easy purification from the supernatant. About 6 - 8 ml of supernatant prepared from 3 - 4 g of wet cell material lysate was used to load the Ni2+ ion chelated column (40 mg His-tagged protein binding capacity). By washing the Ni2+ ion chelated column with about 10 ml buffer A (as discussed above), his-tagged proteins were eluted with buffer B containing higher concentrations of

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase imidazole (0.5 M). Eluted fractions were subjected to SDS-PAGE analysis and simultaneously, purified protein contents were determined using the Bradford assay. SDS- PAGE analysis of Ni2+ ion affinity chromatography purified proteins resulted the dense single protein bands at the expected size of about 62 kDa for PhtDa and 23 kDa for PhtDb that include plus 3 kDa 6His-tag (Figure 2C). Protein bands excised from the SDS gel and identified peptides by MS analysis, showed the expected molecular mass and high identity with the deduced amino acid sequence of genes PA01_00217 and PA01_00218 from the draft genome of the Azoarcus sp. strain PA01 (data not shown).

Figure 2. Sodium dodecyl sulfate-polyacrylamide (12 % resolving) gel electrophoretic analysis of proteins expressed in E. coli Rosetta 2 (DE3)pLysS cells grown in different culture media (3 % ethanol and 1 % glucose) and induction with 0.5 mM of IPTG at varying temperatures: A) expression level of PhtDa protein (̴60 kDa); and B) expression level of PhtDb protein (̴ 23 kDa): lane 1 - 2, cell-extract and pellet of cells grown at 37 ºC for 4 h; lane 3 - 4, cell-extract and pellet of cells grown at 25 ºC for 7.5 h; lane 5 - 6, cell-extract and pellet of cells grown at 15 ºC for 20 h; and C) partial purification of PhtDb (̴ 60 kDa ± 3) and PhtDb (̴ 22 kDa ± 3) proteins using the Ni2+ ion affinity column. The pre-stained protein molecular size marker (range 15 – 100 kDa) was used to estimate the size of the protein bands.

Enzyme activity, substrate specificity and cofactor requirement Decarboxylase activity for conversion of o-phthalyl-CoA to benzoyl-CoA was determined with recombinantly expressed PhtDa and PhtDb in E. coli. Enzyme assay were performed directly with the E. coli transformant’s cell lysate or cell-free extracts containing respective expressed proteins PhtDA and PhtDb. Decarboxylation of o-phthalyl-CoA was demonstrated

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase with E. coli cell-free extracts expressing PhtDa and PthDb proteins and LC-MS analysis of samples collected at the different time intervals revealed an increase of benzoyl-CoA (Figure 3). Decarboxylase activity was unaffected by oxygen exposure and was independent of oxygen. Neither one of these proteins could decarboxylate o-phthalyl-CoA to benzoyl-CoA alone. As expected, no decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA [Junghare et al., 2016]. Furthermore, decarboxylase activity was observed only when, PhtDb was previously incubated with flavin mononucleotide (FMN) and dimethylallyl monophosphate (DMAP). No activity was observed when FMN was replaced with the flavin dinucleotide (FAD). This result strongly indicates that PhtDb is a FMN- binding/dependent protein that is essential for the decarboxylation of o-phthalyl-CoA to benzoyl-CoA. In addition to this, UV-visible scanned spectrum of FMN-DMAP incubated and His-tag purified PhtDb revealed the possible binding and modification of the FMN molecule to a PhtDb protein (Figure 4). UV-visible scanning of PhtDa and PhtDb showed the same absorption maxima at the wavelength of about 340 nm but at different intensity.

Figure 3. LC-MS/MS ion traces following benzoyl-CoA formation from the o-phthalyl-CoA decarboxylation in an enzyme assay performed with cell-free extracts of recombinantly expressed PhtDa and PhtDb in E. coli. A) Time course of formation of benzoyl-CoA from o- phthalyl-CoA monitoring the specific ion traces m/z 365 of the MS/MS fragmentation of benzoyl-CoA (quasimolecular ion m/z 872); and B) ESI-MS/MS of the quasimolecular ion m/z 872 of benzoyl-CoA.

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Figure 4. UV-visible scanned spectrum of: a) flavin mono nucleotide (FMN) in elution buffer; b) FMN-DMAP incubated and His-tag purified PhtDb showing absorption peak maxima at a wavelength of about 340 nm, 380 nm and 458 nm possible indicating binding of FMN molecules (preliminary results). PhtDa without FMN was scanned as the control.

Phylogenetic position of PhtDa and PhtDb A phylogenetic tree was constructed using MEGA 7.0 software [Kumar et al., 2016] based on the neighbor-joining method to determine the evolutionary relationship of PhtDa and PhtDb of Azoarcus sp. strain PA01 with the other known bacterial and fungal species UbiX- like/UbiD-like decarboxylases whose crystal structure are available. Amino acid sequence of total 13 different proteins were aligned with clustal W method and a single phylogenetic tree was generated for the evolutionary analysis (Figure 5). Among all the UbiD-like and UbiX- like enzymes from the bacterial and fungal species, the PhtDa enzyme (PA01_00217) with expected decarboxylase activity formed a discrete group with UbiD-like and a putatively flavin (FMN)-binding PhtDb (PA01_00218) grouped with UbiD-like enzymes, respectively (Figure 5). The proteins PhtDa and PhtDb were clearly separated from each other in the distinct clade and showed only about 30 % sequence identity to one another. Among the UbiD-like enzymes clade in the phylogenetic tree, PhtDa of Azoarcus sp. strain PA01 was found to be more closely related with the putative aromatic acid decarboxylase (Pad1, 4IP2_C) of P. aeruginosa and the ferulic acid decarboxylase (Fdc1, AHY75481) of S. cerevisiae showing about 23 % of sequence similarity. Whereas, the PhtDb FMN-binding protein of Azoarcus sp. strain PA01 shared highest 64 % of amino acid sequence similarity with UbiX-like enzyme (P69772) of E. coli strain O157:H7 (Figure 5). The other closely

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase related UbiX-like enzymes includes the phenolic acid decarboxylase (β-subunit, P94404) of B. subtilis, fungal phenylacrylic acid decarboxylases (Pad1/PadA1) of S. cerevisiae (P33751) and A. niger (A3F715) were positioned relatively distant with around 50 % amino acid sequence similarity, but all grouped together UbiX-like enzymes (Figure 5). However, the UbiX (Q9HX08) flavin prenyltransferase enzyme of P. aeruginosa strain PAO1 showed only about 44 % sequence identity with PhtDb and was distantly placed compared other UbiX (Figure 5).

Figure 5. Phylogenetic tree showing the relationship of PhtDa and PhtDb proteins of Azoarcus sp. strain PA01 that are involved in the decarboxylation of o-phthalyl-CoA to benzoyl-CoA and the other known UbiX-like and UbiD-like enzyme from bacterial and fungal species. Bar indicates 2 % sequence divergence.

Multiple sequence alignment and homology-based structure modelling A BLAST search for proteins PhtDa and PhtDb found several sequences sharing significant similarity (data not shown). However, the BLAST results contain the enzymes with known functions that shared less similarity with PhtDa and PhtDb. For example, the phylogenetic analysis of PhtDa suggested, it shared only about 23 % sequence identity with the UbiD-like decarboxylase (Pad1) of P. aerugonisa (PDB code 4IP2_C, UniProt Q9I6N5) whose crystal structure is currently known [Jacewicz et al., 2013]. Therefore, due to very less sequence similarity (23 %) of PhtDa to enzyme with known function, multiple sequence alignment and

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase secondary structure prediction for PhtDa was not performed. On the other hand, BLAST search and phylogenetic analysis of PhtDb revealed about 64 % sequence identity with FMN- dependent UbiX-like decarboxylase of E. coli O157:H7 (UniProt P69772) whose crystal structure (PDB code 1sbz) is currently resolved [Rangarajan et al., 2004]. Other known UbiX-like enzymes of bacterial and fungal species also shared significant identity with PhtDb (Figure 5). Therefore, multiple amino acid sequence alignment and secondary structure predictions (using PDB code 1sbz as template) of PhtDb revealed highly conserved regions not only with the FMN-dependent UbiX-like decarboxylase of E. coli (P69772), but also with other UbiX-like enzymes from bacterial and fungal species (Figure 5). We have analyzed more closely the top 5 sequences, which all had lengths of ∼200 residues and had over 50 % sequence identity to PhtDb of Azoarcus sp. strain PA01. Among the ∼200 amino acid residues in the sequences, there are ∼50 residues conserved in all sequences, as highlighted (red) as shown in Figure 6. The majority of these enzymes sequences are the known FMN- binding prenyltransferase belonging to the superfamily of UbiX-like enzymes.

The three-dimensional (3-D) structures of many UbiD-like and UbiX-like enzymes are presently known. The 3-D structure prediction and modelling of PthDa and PhtDb were computed in SWISSMODEL workspace (https://swissmodel.expasy.org/interactive) using the deduced amino acid sequences of the PhtDa (PA01_00217) and PhtDb (PA01_00218) to find the best templates. Although, amino acid sequence of ferulic acid decarboxylase (Fdc1) from S. cerevisiae and putative aromatic acid decarboxylase (Pad1) of P. aeruginosa shared ∼23% amino acid sequence similarity with PhtDa (Figure 5). The homodimer crystal structure of Pad1 (PDB code 4iws) of P. aeruginosa [Jacewicz et al., 2013] was the best template and chosen for homology or template based structural modelling of PhtDa (Figure 7A). Whereas, the homo 12-mer (dodecamer) crystal structure (PDB code 1sbz) of FMN-binding UbiX flavoprotein (Pad1) in E. coli O157: H7 [Rangarajan et al., 2004] was the best template for PhtDb and chosen for predicting the 3-D structure of PhtDb (Figure 7B). The predicted 3-D structures of PhtDa (homodimer) and PhtDb (homo 12-mer) in Azoarcus sp. strain PA01 were visualised (Figure 7AB), and compared and superimposed with the respective templates (PDB code 4zac and 1sbz) using the UCSF-Chimera package (Figure 7CD). PhtDb showed a high degree of secondary structure and motif conservation with the template (Figure 7D), compared to overlapping of PhtDa with its template (Figure 7C).

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Figure 6. Multiple sequence alignments of PhtDb (PA01_00218) of Azoarcus sp. strain PA01 with other UbiX-like enzymes from selected bacterial or fungal species: E. coli O157:H7 (P69772), B. subtilis bsdB (P94404), S. cerevisiae Pad1 (P33751), A. niger PadA1 (A3F715) and P. aeruginosa UbiX (Q9HX08). Conserved amino acid residues that putatively involved in flavin mononucleotide nucleotide (FMN), phosphate, and nucleotide binding are indicated by labelled arrows. The predicted secondary structure elements of PhtDb are shown based on the ESPrip 3.0 online tool. Alpha (α) helices and 310-helices (denoted as ղ) are shown as squiggles, β-strands as arrows and β-turns as TT. Conserved amino acid sequences of PhtDb are presented as highlighted regions and red denotes highly conserved amino acids regions.

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Figure 7. Homology-based molecular modelling of PhtDa (PA01_00217) and PhtDb (PA01_00218) proteins with the decarboxylase activity from Azoarcus sp. strain PA01 were constructed using SWISSMODEL workspace: A) a 3-D model (homodimer) structure of PhtDa based on the homodimer crystal structure (PDB code 4iws) of Pad1 in P. aeruginosa [Jacewicz et al., 2013]; B) a 3-D model (homo 12-mer) of PhtDb based on the crystal structure (PDB code 1sbz) of UbiX flavoprotein (Pad1, homo 12-mer) in E. coli O157:H7 as a template [Rangarajan et al., 2004]. Superimposition of predicted 3-D model structure of: C) PhtDa, and D) PhtDb with their respective templates (4iws and 1sbz) are shown in faint blue color performed using UCSF-Chimera package (https://www.cgl.ucsf.edu/chimera/).

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Native PAGE and protein identification by MS analysis In order to investigate the protein complexes in phthalate-induced proteins by Azoarcus sp. strain PA01, especially to determine the PhtDa and PthDb protein-protein interactions and oligomer state. Native-protein gel electrophoresis was performed with cell-free extracts of o- phthalate versus benzoate-grown cells. Interestingly, only few native protein bands were visible on o-phthalate-grown cells compared to benzoate-grown cells (Figure 8A). Specifically phthalate-induced protein bands were estimated between 400 to 140 kDa that include protein bands labeled as 2, 3, 4 and 5 (Figure 8A). These spots were excised from the native gel and identified by MS. Protein identification of protein bands 2 and 3 (approximate estimated molecular size ranging between 390 to 350 kDa) resulted the identification majorly two proteins, namely PhtDa (60 kDa) and PhthDb (22 kDa) as shown in Table 1. Among the protein identification of the lower molecular weight protein bands (4, 5, 6 and 7) PhtDa was also identified, including the CoA-transferases (PhtSa and PhtSb protein) that are involved in the activation of o-phthalate to o-phthalyl-CoA reported in our recent findings [Junghare et al., 2016].

Furthermore, native-protein gel analysis of heterologously expressed and partially purified PhtDa and PhtDb, were performed individually and incubating together for about 1 h at room temperature to determine the protein-protein interactions. Surprisingly, when PhtDa (60 kDa) and PhtDb (22 kDa) were incubated together with FMN and DMAP, only a single band at ca. 400 kDa was visible on the native protein gel (Figure 8B). Furthermore, individual native protein gel analysis of purified PhtDa and PhtDb revealed the formation of two distinct bands visible at the estimated molecular size between ca. 280 to 420 kDa. Nevertheless, SDS analysis of purified PhtDa (60 kDa) and PhtDb (22 kDa) showed only a single bands at the expected size for the respective proteins (Figure 2C). These results strongly indicate that these high molecular size protein bands in native gel are most likely the multimeric complexes of PhtDa and PhtDb. However, at the moment it is still unclear whether the single band observed in the lane of the PhtDab incubated mixture (with FMN plus DMAP) is due to the PhtDa and PhtDb multimeric complex, because MS confirmation of the proteins is elusive.

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Figure 8. Native-PAGE analysis of: A) the soluble protein fraction from cell-free extracts of Azoarcus sp. strain PA01 cells grown with a) o-phthalate and b) benzoate; and B) partially purified PhtDa, PhtDb and PhtDab expressed in E. coli. Protein separation was performed using the Mini-PROTEAN TGX native gel slab (BioRad) and bands were visualized by staining with Coomassie Brilliant Blue G250. Lane Mr: native high molecular weight protein marker (Amersham, GE Healthcare life sciences). The arrow in figure B highlights the probable protein complex of PhtDa and PhtDb of estimated size of about 390 kDa (PhtDa 60 kDa x dimer = 120 kDa plus PhtDb 22 kDa x 12 mer = 264 kDa).

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase

Table 1 Protein identification by mass spectrometry of the spots excised from native PAGE from the o-phthalate-grown cells compared to benzoate grown cells of Azoarcus sp. strain PA01.

Band MW on gel Locus tag Major proteins identified Calculated Coverage % Score No. (kDa) by MS analysis MW (kDa)

2 390 00217 PhtDa, UbiD-like 58.8 74.57 15680 00218 PhtDb, UbiX-like 22.0 53.27 616 3 350 00217 PhtDa, UbiD-like 58.8 70.02 8169 00218 PhtDb, UbiX-like 22.0 56.28 418

4 210 00217 PhtDa, UbiD-like 58.8 59.96 5812 5 140 00217 PhtDa, UbiD-like 58.8 61.29 10500 00216 PhtSb, CoA-transferase 43.6 14.74 194 00215 PhtSa, CoA-transferase 43.8 14.04 114 6 100 00217 PhtDa, UbiD-like 58.8 49.72 2101 00215 PhtSa, CoA-transferase 43.8 20.20 261 00216 PhtSb CoA-transferase 41.3 13.65 88.85 7 80 00215 PhtSa, CoA-transferase 43.8 53.69 2019 00216 PhtSb CoA-transferase 41.3 47.77 1096 00217 PhtDa, UbiD-like 58.8 33.97 661

Note- From the each spot different proteins were identified. However, in this table the results of four proteins (four subunits) that are involved in the initial steps of o-phthalate degradation were shown. *SPA stands for spots from o-phthalate-grown gel, SBA stands for spots from benzoate-grown gel and MMPA are spots from membrane proteins of o-phthalate-grown Azoarcus sp. PA01

Discussion Present work demonstrates that PhtDa is the enzyme responsible for catalysing decarboxylation of o-phthalyl-CoA to benzoyl-CoA, and the decarboxylase activity of PhtDa depends on the FMN-dependent PhtDb protein constituting a two-enzyme component system. PhtDa (60 kDa) and PhtDb (22 kDa) were cloned into pET100/D-TOPO and overexpressed as N-terminal His-tagged proteins in E. coli Rosetta (DE3)pLysS host. Both proteins were purified to homogeneity by utilizing Ni2+ ion affinity chromatography (Figure 2C). The molecular weight of the recombinant proteins was determined by SDS PAGE analysis. Alternatively, proteins were identified by MS analysis and revealved that both protein bands showed expected molecular masses and high identity with the deduced amino acid sequenes of both gene from the genome sequence of Azoarcus sp. strain PA01 [Junghare et al., 2015b].

Enzyme assays with cell lysate or cell-free extracts of E. coli strains expressing PhtDa and PhtDb could readily convert o-phthalyl-CoA to benzoyl-CoA (Figure 3). Interestingly, none of these proteins possess decarboxylase activity alone. Although, PhtDa and PhtDb do not share sequence similarity with each other, but they belong to a new class of enzyme family

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase and are homologous to UbiD-like and UbiX-like decarboxylases found in a wide range of bacterial and fungal species (Figure 5, 6 and 7). UbiD and UbiX are known to act in the decarboxylation of aromatic substrates [Zhan and Javor, 2003] but individually UbiX or UbiD were unable to perform decarboxylation [Jacewicz et al., 2013; Rangarajan et al., 2004; Kopec et al., 2011]. Our results showing the involvement of PhtDa and PhtDb for decarboxylation reaction are in agreement with the previous studies. Because decarboxylation of phenylacrylic acid in S. cerevisae required Fdc1 (UbiD-like) and Pad1 (FMN dependent UbiX-like). Similarly, in E. coli O157:H7 or P. aeruginosa decarboxylation of 3-octaprenyl- 4-hydroxybenzoate to 2-octaprenylphenol is also required two enzymes as UbiX-like (FMN- dependent) and an UbiD-like aromatic acid decarboxylase [Rangarajan et al., 2004; Jacewicz et al., 2013].

Furthermore, heterologously overexpressed PhtDa and PhtDb in E. coli only led to benzoyl- CoA formation, when PhtDb was pre-incubated with FMN and dimethylallylphosphate (DMAP). No decarboxylase activity was observed when FMN was replaced against flavin dinucleotide (FAD). These results strongly suggest that decarboxylase activity of PhtDa, is dependent on FMN-dependent PhtDb. Phylogenetically, PhtDb is closely related to the FMN- dependent UbiX-like enzyme which covalently modifies FMN molecule that is needed for the decarboxylase activity by UbiD-like decarboxylase in E. coli [White et al., 2015; Lin et al., 2015; Rangrajan et al., 2015; Payne et al., 2015]. Multiple sequence alignment of PhtDb with the known UbiX flavin-binding proteins from bacterial and fungal species (Figure 5 and 6) revealed highly conserved amino acid residues. PhtDb showed highest sequence similarity (64 %) with the FMN-dependent UbiX-like enzyme of E. coli O157:H7 for which the FMN- binding site at the amino acid positions Ser36 and Arg122, and a nucleotide binding site at Thr9, Gly10, Ala11, Ser87 and Met88, respectively, has been established recently [White et al., 2015]. Interestingly, PhtDb also possesses the conserved amino acid residues at these positions, suggesting a probable binding site for FMN and DMAP along with the similar FMN dependent enzymes (Figure 6). The addition of DMAP and FMN to PhtDb caused distinct changes in the FMN absorbance profile (Figure 4), which indicated possible binding and modification of FMN due to the formation of PhtDa:FMN:DMAP complex. UV-VIS measurement of purified PhtDb after incubation with FMN and DMAP showed absorption peak maxima at 384 nm and 458 nm (Figure 4B), which are the characteristic peaks for the FMN molecule [Lin et al., 2015]. However, slight changes were observed in PhtD:FMN

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase spectrum that consisted of a decrease in the absorbance intensity at 380 nm and ̴ 450 nm and the developed flat shoulders at 380 and 450 nm (Figure 4B). This strongly indicates an interaction of FMN with the PhtDb. In addition to this, structural modelling of both PhtDa and PhtDb suggested that only PhtDb possesses the FMN-binding domain and forms a dodecameric complex (264 kDa; 12 FMN per 12 PhtDb), whereas PhtDa is a homodimer based on the known template structure [Rangarajan et al., 2004; (Figure 7B and 5C)]. Superimposition of the model structure of both PhtDa and PhtDb with the respective templates of known crystal structures such as homodimer Pad1 in P. aeruginosa [(PDB code 4iws); Jacewicz et al., 2013] showed high overlapping and similarity in the overall fold of the secondary structure elements with the predicted model of PhtDa (Figure 7C). Whereas superimposition of the homo 12-mer crystal structure of FMN-dependent UbiX-like enzyme (PDB code 1sbz) in E. coli O157:H7 [Rangarajan et al., 2004] exhibited very high overlapping of predicted homo-12mer 3-D model of PhtDb (Figure 7D). Therefore, this accurately reflects that subunits in both PhtDa and PhtDb are arranged in a similar pattern with the oligomeric structures of their respective templates and thus it is most likely to have functional homology (Figure 7). Native protein analysis of cell-free extract of Azoarcus sp strain PA01 as well as purified PhtDa and PhtDb revealed the formation of a protein complex of the expected molecular weight in analogy to the oligomeric structure of the related proteins as described in Figure 7. Based on the 3-D structure modelling, PhtDa (60 kDa) forms a 120 kDa homodimer while the PhtDb (22 kDa) is homo dodecamer forming a 264 kDa oligomer complex. Thus, both together (PhtDa and PhtDb) are expected to form a 384 kDa (approx. 390 kDa) protein complex. Native protein analysis of cell-free extract of Azoarcus sp. strain PA01 cells grown in o-phthalate showed the protein bands, particularly at approximate 380 and 350 kDa size (Figure 8A). MS analysis results (Table 1) of these two bands (2 and 3) surprisingly showed the identification of mainly PhtDa and PhtDb, with significant sequence coverage and score (Table 1) among the other proteins detected (data not shown). However, MS identification of the following lower molecular weight size protein bands (between 140 to 80 kDa), resulted in the identification of mainly PhtSa (43 kDa) and PhtSb (41 kDa), the expected subunits of the succinyl-CoA dependent CoA-transferase that is involved in the activation of o-phthalate to o-phthalyl-CoA [Junghare et al., 2016]. The CoA-transferase are expected to form a tetramer (approximately 170 kDa) homologous to succinyl-CoA:(R)-benzylsuccinate CoA-transferase

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Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase involved in toluene activation by T. aromatica [Leutwein and Heider, 2001]. In addition to this, native protein analysis of partially purified PhDb incubated with FMN plus DMAP showed a single protein band at 390 kDa size, although the native protein analysis of PhtDa and PhtDb individually formed two distinct protein bands of 400 to 350 kDa (Figure 8B). Yet, the SDS-PAGE analysis of the purified PhtDa and PhtDb exhibited single bands at the expected sizes of 60 and 22 kDa (Figure 2C). In conclusion, our experiments demonstrate that PhtDa and PhtDb constitute a two- component decarboxylase system for o-phthalyl-CoA decarboxylation similar to the UbiX and UbiD two-component decarboxylase system in which UbiX with its prenylated FMN cofactor is essential for the decarboxylase UbiD [Lin et al., 2015; White et al., 2015]. Therefore, we strongly suggest that the prenylated FMN consequently plays a crucial role in the decarboxylation of o-phthalyl-CoA to benzoyl-CoA. However, how exactly prenylated FMN facilitates the decarboxylation of o-phthalyl-CoA to benzoyl-CoA is yet to be established.

Acknowledgments MJ acknowledges the University of Konstanz for financial support. MJ greatly acknowledges Jasmin Frey for helpful suggestions and discussions during the cloning and heterologous protein expression experiments.

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CHAPTER 5

Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate- reducing bacterium isolated from a wastewater treatment plant

Madan Junghare and Bernhard Schink

Published in International Journal of Systematic and Evolutionary Microbiology 2015; 65:

77-84.

Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer

Abstract A strictly anaerobic, mesophilic, sulfate-reducing bacterium, strain KoBa311T, isolated from the wastewater treatment plant at Konstanz, Germany, was characterized phenotypically and phylogenetically. Cells were Gram-stain-negative, non-motile, oval to short rods, 3 - 5 mm long and 0.8 - 1.0 mm wide with rounded ends, dividing by binary fission and occurring singly or in pairs. The strain grew optimally in freshwater medium and the optimum temperature was 30 °C. Strain KoBa311T showed optimum growth at pH 7.3 - 7.6. Organic electron donors were oxidized completely to carbon dioxide concomitant with sulfate reduction to sulfide. At excess substrate supply, substrates were oxidized incompletely and acetate (mainly) and/or propionate accumulated. The strain utilized short-chain fatty acids, alcohols (except methanol) and benzoate. Sulfate and DMSO were used as terminal electron acceptors for growth. The genomic DNA G+C content was 52.3 mol % and the respiratory quinone was menaquinone MK-5 (V-H2). The major fatty acids were C16:0, C16:1ω7c/ω6c and T C18:1ω7c. Phylogenetic analysis based on 16S rRNA gene sequences placed strain KoBa311 within the family Desulfobulbaceae in the class Deltaproteobacteria. Its closest related bacterial species on the basis of the distance matrix were Desulfobacterium catecholicum DSM 3882T (93.0 % similarity), Desulfocapsa thiozymogenes (93.1 %), Desulforhopalus singaporensis (92.9 %), Desulfopila aestuarii (92.4 %), Desulfopila inferna JS_SRB250 LacT (92.3 %) and Desulfofustis glycolicus (92.3 %). On the basis of phylogenetic, physiological and chemotaxonomic characteristics, strain KoBa311T was distinct from any related type species. Therefore, strain KoBa311T is considered to represent a novel species of a new genus, for which the name Desulfoprunum benzoelyticum gen. nov., sp. nov. is proposed. The type strain of Desulfoprunum benzoelyticum is KoBa311T (= DSM 28570T = KCTC 15441T).

The 16S rRNA gene nucleotide sequence of Desulfoprunum benzoelyticum KoBa311T reported in this study is submitted at NCBI under accession number KJ766003.

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer

Introduction Anaerobic processes find wide applications in the anaerobic treatment of domestic and industrial wastewaters. A major development has been to apply anaerobic processes for the treatment of aromatic compounds in industrial wastewaters [Li et al., 1995]. In the past decade, studies have demonstrated that simple aromatic compounds such as phenol and benzoate can be effectively degraded by anaerobic processes [Li et al., 1995; Fang et al., 1996]. Benzoate is one of the central intermediates in the degradation of many naturally or chemically synthesized aromatic compounds. In addition, several syntrophically fermenting bacteria degrade benzoate, such as Syntrophus buswellii [Mountfort et al., 1984] and Syntrophus gentianae HQGÖ1T [Szewzyk and Schink, 1989]. However, most wastewaters contain aromatic compounds along with large amounts of sulfate. Therefore, sulfate reduction becomes a crucial process under anoxic conditions and contributes to biogeochemical sulfur cycling in different environments. Sulfate-reducing bacteria (SRB) comprise a diverse group of species [Castro et al., 2000; Kuever et al., 2006]. More than 10 sulfate-reducing cultures that can grow with various aromatic substrates have been reported since 1980 [Beller et al., 1996], and these exhibit a broad metabolic diversity [see detailed review by Plugge et al., 2011]. Some species of SRB oxidize benzoate with sulfate as electron acceptor [Widdel, 1987]. Also, a thermophilic, benzoate-degrading, sulfate-reducing bacterium, Desulfotomaculum thermobenzoicum, has been reported [Tasaki et al., 1991]. In the present study, we report the isolation of strain KoBa311T, a benzoate-degrading, sulfate-reducing bacterium from a wastewater treatment plant.

Material and methods

Enrichment, isolation and cultivations Enrichment cultures with o-phthalate as substrate were inoculated with activated sewage sludge from the municipal wastewater treatment plant at Konstanz, Germany. Although strain KoBa311T was isolated from an enrichment with o-phthalate, it did not grow with this substrate: perhaps benzoate is a side product of phthalate degradation in the enrichment culture and supports growth of strain KoBa311T by cross feeding. Therefore, the strain described in this study was grown with benzoate as sole carbon and energy source. Isolation, cultivation and growth experiments were performed in anoxic, bicarbonate-buffered, sulfide-

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer reduced freshwater mineral medium containing (g/l, except where indicated): NaCl, 1;

MgCl2. 6H2O, 0.4; KH2PO4, 0.2; NH4Cl, 0.25; KCl, 0.5; CaCl2. 2H2O, 0.15; NaHCO3, 2.5;

Na2S. 9H2O, 1 mM; Na2SO4, 20 mM [Widdel and Bak, 1992]. The medium (excluding Na2S.

9H2O and NaHCO3) was autoclaved at 121 °C for 25 min and cooled under an oxygen-free mixture of N2/CO2 (80 : 20) gas phase. Sterile stocks of 1 ml trace element solution SL-10 [Widdel et al., 1983], 1 ml selenate tungstate [Tschech and Pfennig, 1984] and 1 ml seven- vitamin solution [Pfennig, 1978] were added from concentrated stocks. The initial pH of the medium was adjusted to 7.3±0.1 with sterile 1 M NaOH or 1 M HCl. Cultivation and transfer of the strain were performed under N2/CO2 (80 : 20) atmosphere. The strain was cultivated in the dark at 30 °C. Pure cultures were obtained by repeated application of deep-agar (1 %) shake dilutions as described by Widdel and Bak (1992). The medium was supplemented with 1 mM sodium benzoate plus sulfate (20 mM). The agar shake tubes were incubated in inverted positions for 2 - 3 weeks until visible colonies appeared. The strain was routinely checked for purity under the light microscope (Zeiss west Germany). Stock cultures were transferred every 4 - 5 weeks and stored in liquid medium at 4 °C. Each electron donor was added at a final concentration of 5 mM unless indicated otherwise. The isolate was examined by phase-contrast microscopy (Axiophot Zeiss) and photographs were taken using the agar slide technique [Pfennig and Wagener, 1986]. Gram staining was determined using a staining kit (Difco Laboratories) according to the manufacturer’s instructions, and by the KOH test [Gregersen, 1978].

Growth optimization and substrate utilizations The effects of temperature, pH and salinity were studied to optimize the growth conditions. The temperature range for growth was performed by growing the strain at 10, 15, 20, 25, 30, 37, 40, 45 and 50 °C. The initial pH range for growth was determined over the pH range 4 - 9 at intervals of 0.5 pH units. The salt (NaCl) optimization of strain was determined in the absence of NaCl and with 0.01, 0.02, 0.05, 0.10, 0.5, 1.0, 1.5 and 2 % (w/v) NaCl. The ability to utilize various electron acceptors, namely elemental sulfur, ferric hydroxide (10 mM), thiosulfate (10 mM), sulphite (10 mM), sulfate (20 mM), DMSO (10 mM) and nitrate (5 mM), was studied with succinate (8 mM) as electron donor. The medium with sulfate served as a positive control and medium without sulfate as a negative control. The following substrates were tested for utilization, based on the list of compounds metabolized by the

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer different genera of SRB [Widdel and Bak, 1992] with sulfate as electron acceptor: acetate, propionate, butyrate, valerate, pyruvate, lactate, succinate, malate, fumarate, glucose, benzoate, nicotinate, betaine, choline chloride, formate, methanol, ethanol, propanol, butanol, alanine, glutamate, 2-oxoglutarate, oxaloacetate, citrate (all 10 mM), o-phthalate (1 mM), iso- phthalate (1 mM), tere-phthalate (1 mM), 3-hydroxybenzoate (1 mM), 4-hydroxybenzoate (1 mM) and 3,4-dihydroxybenzoate (1 mM). Utilization of each electron acceptor or donor was analysed via turbidity and sulphide production. Organic acids were quantified with an HPLC system (LC-prominence; Shimadzu) equipped with an Aminex HPX-87H ion-exclusion column (Bio-Rad) and analysed at 60 °C, with 10 mM H3PO4 as the mobile phase and UV detection at 200 nm. The mobile phase was used at a flow rate of 1 ml per min. Growth experiments were carried out in duplicate and terminated after 3 weeks of incubation.

Chemotaxonomy and phylogenetic analysis Genomic DNA G+C content analysis [Cashion et al., 1977] was performed by HPLC at the Identification Service of the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany). Fatty acid methyl esters of cellular fatty acids (CFAs) [Kämpfer and Kroppenstedt, 1996] and respiratory quinones [Tindall, 1990] were also identified by DSMZ.

The genomic DNA of the isolate was extracted with a genomic DNA extraction kit (Cat. No. 19060; Qiagen) according to the manufacturer’s instructions, and amplification of the 16S rRNA gene was performed by PCR as described by Junghare et al. (2012) with bacterial universal primers 27F and 1492R [Lane, 1991]. Micro-organisms and environmental sequences closely related to strain KoBa311T were determined by BLAST search against the nonredundant GenBank database [Altschul et al., 1990] and the EzTaxon-e tool [Kim et al., 2012]. The 16S rRNA gene sequences of strain KoBa311T (1358 bp) and closely related taxa obtained from the GenBank database were aligned using the SINA sequence alignment program [(http://www.arb-silva.de/aligner/); Pruesse et al., 2012; Quast et al., 2013]. The ARB program package was used for phylogenetic tree reconstruction. Phylogenetic trees were reconstructed based on 1116 nt unambiguously aligned sequence positions and a 50 % conservation filter using the RAxML method [Stamatakis et al., 2008]. The results achieved

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer using the maximum-parsimony [Fitch, 1971], neighbour joining [Saitou and Nei, 1987] and maximum-likelihood [Felsenstein, 1981] methods also supported the same branching pattern and phylogenetic placement of strain KoBa311T (data not shown). Confidence in the resultant tree topology was evaluated by resampling 100 bootstrap trees [Tamura et al., 2011] using the RAxML algorithm [Stamatakis et al., 2008].

Results and discussion

Cells of strain KoBa311T were oval to short rods, 3 - 5 µm long and 0.8 - 1.0 µm wide, with rounded ends, occurring singly or in pairs (Figure 1). The cells divided by binary fission and were non-motile. Cells were Gram-stain-negative as determined both by Gram staining and by the KOH test. No spore formation was observed.

Figure 1. Phase-contrast micrograph of cells of strain KoBa311T grown with benzoate plus sulfate. Bar, 10 µm.

Strain KoBa311T reduced sulfate to sulfide, with benzoate and succinate as electron donors, and produced sulphide close to the theoretical molar ratio of 1:3.75 (benzoate/sulfide) and 1:1.75 (succinate/sulfide), as shown in Table 1. Thus, strain KoBa311T showed complete oxidation of electron donors. However, succinate at excess concentration led to accumulation of acetate as an intermediate product in the medium. This could be due to inhibition of acetate utilization by accumulating sulfide.

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer

Table 1. Stoichiometry of benzoate and succinate degradation by strain KoBa311T with sulfate as electron acceptor.

Substrate Substrate utilized Sulfide produced (mM) VFAs produced (mM) (mM) Acetate Propionate Benzoate 0.98 3.1 0.03 0

Succinate 3.7 4.9 0.02 0.01 3.8 (5.8)* 5.7 4.08 0.01 *excess substrate concentration.

The strain grew even in the absence of added NaCl. Optimum growth was at 0.01 - 0.10 % (w/v) NaCl in the medium. Growth was inhibited at higher NaCl concentrations. Optimum temperature for growth was 30 °C; little growth was observed at 20, 25 and 37 °C. The optimum initial pH was 7.3 - 7.6.

Strain KoBa311T used sulfate and DMSO as electron acceptors. Slow growth was observed with thiosulfate. However, the strain could not grow with elemental sulfur, Fe(OH)3 or sulfite. Nitrate was not reduced to nitrite. Electron donors utilized by the strain included pyruvate, butanol, H2, ethanol, benzoate, propionate, propanol, fumarate, malate, succinate, acetate (slow growth), formate, lactate, butyrate and oxaloacetate. It could not grow with 2- oxoglutarate, citrate, valerate, nicotinate, choline chloride, 3,4-dihydroxybenzoate, methanol, 3-hydroxybenzoate, glucose, glutamate, alanine or betaine. The strain could not ferment substrates in the absence of an electron acceptor.

The predominant cellular fatty acids (CFAs) were C16:0 (20.6 %), C16:1 ω7c/ω6c (35.0 %) and

C18:1 ω7c (23.5 %). Branched-chain fatty acids contributed about 72.6 % of total fatty acids. However, only 24.1 % of the CFAs were even-numbered straight-chain fatty acids. No cyclo- fatty acids were found. The detailed fatty acids pattern of strain KoBa311T and two other members of the family Desulfobulbaceae, Desulfopila aestuarii [Suzuki et al., 2007] and Desulfopila inferna [Gittel et al., 2010] are shown in Table 2. The total contribution of about

20.6 % of C16:0 fatty acid was equal among the members of the family Desulfobulbaceae. T Strain KoBa311 had about 23.5 % of C18:1 ω7c and this component is not observed in the above two type strains of the family Desulfobulbaceae (Table 2). The major respiratory quinone was menaquinone MK-5 (V-H2). The G+C content of the genomic DNA of strain KoBa311T was 52.3 mol %.

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer

Table 2. Cellular fatty acids (CFA) composition of strain KoBa311T and other related strains. Strains: 1, strain KoBa311T; 2, Desulfopila aestuarii [Suzuki et al., 2007]; 3, Desulfopila inferna [Gittel et al., 2010]; Values are percentages by weight of total fatty acids.

Fatty acid composition (%) KoBa311T D. aestuarii D. inferna

C14:0 0.87 1.4 1.1 C14:0 3OH - 1.8 - C15:0 0.87 - 1.6 C15:1 w6c 0.29 1.1 - C15:1 w9c - 11.7 - C16:0 20.57 33.6 23.3 C16:0 3OH 0.36 1.6 - C16:1 w5c 5.03 17.1 11.4 C16:1 w7c - - 18.3 C16:1 w9c - - 0.7 C16:1 w7c/16:1 w6c 34.97 6 - C17:0 1.51 3.4 8.3 C17 cyclo - - 15.3 C17:1 w6c 3.9 13.7 - C17:1 w8c 0.55 - - C18:0 2.63 2.5 11.5 C18:1 w5c 0.78 2.7 - C18:1 w7c 23.45 1.7 8.6 C18:1 w9c - 1.3 - C18:1 w7c 11 methyl 0.72 - - C19:0 10 methyl 0.53 - - C20:1 w7c 1.41 - - Note: −, Not present.

The almost-complete 16S rRNA gene sequence of strain KoBa311T (1458 bp) was obtained. 16S rRNA gene sequence analysis revealed that strain KoBa311T is a member of the family Desulfobulbaceae [Kuever et al., 2005]. However, it showed only 93.0, 93.1, 92.9, 92.3, 92.4 and 92.3 % similarity to the most closely related type species, Desulfobacterium catecholicum DSM 3882T [Szewzyk and Pfennig 1987], Desulfocapsa thiozymogenes Bra2(T) [Janssen et al., 1996], Desulforhopalus singaporensis T1(T) [Lie et al., 1999], Desulfofustis glycolicus PerGlyS(T) [Friedrich et al., 1996], Desulfopila aestuarii MSL86(T) [Suzuki et al., 2007] and Desulfopila inferna JS_SRB250LacT [Gittel et al., 2010], respectively. Phylogenetic analysis using the maximumparsimony algorithm revealed that strain KoBa311T clustered with uncultured bacterial clone sequences obtained from polyaromatic hydrocarbon-contaminated soil, which represent a separate lineage (Figure 2) and also form a clade separate from the existing type species in the family Desulfobulbaceae.

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer

The differential morphological and biochemical characteristics of strain KoBa311T and other type species are listed and compared in Table 3. The physiological characteristics of strain KoBa311T are quite distinct from these type species. Therefore, strain KoBa311T needs to be accommodated in a new genus in the family Desulfobulbaceae. Based on these data, we suggest that strain KoBa311T represents a novel species of a new genus, for which the name Desulfoprunum benzolyeticum gen. nov., sp. nov. is proposed.

Figure 2. Maximum-likelihood tree showing the phylogenetic placement of the 16S rRNA gene sequences of strain KoBa311T and related taxa as generated using the RAxML algorithm [Stamatakis et al., 2008]. Members of Syntrophobacteraceae were used as anoutgroup. Numbers at nodes represent bootstrap percentages. Bar, 10 % estimated sequence divergence.

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Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-negative benzoate-degrading sulfate-reducer

Description of Desulfoprunum gen. nov.

(De.sul.fo.pru’num. L. pref. de- off, from; L. n. sulfur sulfur, L. neut. n. prunum plum. N. L. neut. n. Desulfoprunum a plum-shaped sulfate-reducing bacterium).

Cells are Gram-stain-negative, strictly anaerobic, non-motile, oval to short rods. The predominant respiratory quinone is menaquinone MK-5 (V-H2). Major fatty acids are C16:0,

C16:1ω7c/v6c and C18:1ω7c. Mostly utilizes short-chain fatty acids, alcohols and benzoate as carbon and energy source. Growth is mesophilic; grows optimally in freshwater medium. A member of the family Desulfobulbaceae of the class Deltaproteobacteria; the type species is Desulfoprunum benzoelyticum sp. nov.

Description of Desulfoprunum benzoelyticum gen. nov., sp. nov.

Desulfoprunum benzoelyticum sp., nov. [ben.zo.e.ly’ti.cum. N.L. n. benzoe (from arab. luban dschawi), benzoic resin, N.L. adj. lyticum (from Gr. adj. lytikos) dissolving; N.L. neut. adj. benzoelyticum, degrading benzoate].

Has the following characteristics in addition to those given for the genus. Cells are non- sporulating and 3 - 5 mm long and 0.8 - 1.0 mm wide when grown with benzoate or succinate. Growth occurs at 20 - 30 °C, with an optimum at 30 °C. Grows at pH 7 - 8 (optimum growth at pH 7.3±0.2). Optimal growth occurs with 0.01 - 0.1 % (w/v) NaCl; higher concentrations inhibit growth. Sulfate is reduced to sulfide with complete oxidation of the electron donors. Besides sulfate, grows with DMSO as terminal electron acceptor and thiosulfate (slow growth). Elemental sulfur, ferric hydroxide and sulfite are not utilized as electron acceptor, and nitrate is not reduced to nitrite. The following compounds are utilized as electron donors: H2, pyruvate, butanol, propanol, ethanol, benzoate, propionate, formate, fumarate, malate, succinate, acetate, lactate, butyrate and oxaloacetate. The following compounds are not utilized: 2-oxoglutarate, citrate, valerate, nicotinate, choline chloride, 3,4- dihydroxybenzoate, methanol, 3-hydroxybenzoate, glucose, glutamate, alanine and betaine. Does not ferment any substrate in the absence of electron acceptors. The type strain is KoBa311T (= DSM 28570T = KCTC 15441T), which was isolated from the wastewater treatment plant at Konstanz, Germany. The DNA G+C content of the type strain is 52.3 mol %.

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Table 3. Physiological and chemotaxonomic characteristics for differentiation of strain KoBa311T from related species within the family Desulfobulbaceae. Strains: 1, KoBa311T (this study); 2, Desulfocapsa thiozymogenes [Janssen et al., 1996]; 3, Desulfobacterium catecholicum DSM 3882T [Szewzyk and Pfennig 1987]; 4, Desulfopila aestuarii [Suzuki et al., 2007]; 5, Desulforhopalus singaporensis [Lie et al., 1999]; 6, Desulfofustis glycolicus [Friedrich et al., 1996] and 7, Desulfopila inferna [Gittel et al., 2010].

Characteristics KoBa311T D. D. D. aestuarii D. singaporensis D. glycolicus D. inferna thiozymogenes catecholicum Isolation source Wastewater Fresh water lake Anoxic mud Estuarine Sulfide rich black Marine Tidal sediment treatment plant sediment from a bay sediment marine mud sediment Cell shape Rod Rod Oval to lemon Rod Rod Rod Rod Motility Non-motile Motile Motile Non-motile Non-motile Motile Non-motile

Optimum growth NaCl (% w/v) 0.01-0.1 1.5 0.1* 1 ND 2 2-3 Temperature (0C) 30 20-30 28 35 31 28 28 pH 7.3-7.5 7.3-7.5 6.9-7.1 7.5-7.6 7.4 7.3 ND Metabolism Complete Incomplete Complete Incomplete Complete Incomplete Complete oxidation oxidation oxidation oxidation oxidation oxidation oxidation Electron donors Acetate + - + + - - - Propionate + - + - ND - - Butyrate + - + - + - + Fumarate + - + + + + + Malate + - + - ND + - Succinate + - + ? + + + Lactate + - ND ND + + + Oxaloacetate + - ND ND ND ND ND Pyruvate + - ND ND + ND + Methanol - ND + - ND - ND Ethanol + + ND ND + - + Propanol + + + + ND - + Butanol + + + + + - + Glycerol ND ND ND + ND ND + Glycine ND - ND - ND - - Alanine ND - ND - ND ND - Glutamate - - + - ND ND ND H2 + ND + ND - + + Formate (acetate) + ND ND ND - - ND α-keto glutarate - ND ND ND ND ND ND Citrate - ND ND ND ND ND ND Valarate - ND ND ND ND - ND Glucose - - ND ND ND - ND Benzoate + ND ND ND ND - - Nicotinate ND ND ND ND ND ND ND Choline-Cl - ND ND ND ND - - 3,4 diOH benzoate - ND ND ND ND ND ND 3 OH benzoate - ND ND ND ND ND ND Betaine - ND ND ND ND - - Electron acceptors Sulfate + ND ND ND + + + Sulfite - + + + + + + Thiosulfate - + + + + + + S0 - ? ? ? ? ? ? DMSO + ND ND ND ND ND ND Nitrate - ND ND ND + - -

DNA G+C content 52.3 50.7 52.4 54.4 50.6 56.6 50.3 (%mol) Respiratory quinone MK-5 (V-H2) ND ND MK-8 (H4) ND MK-5 (H2) ND

16S rRNA gene NA 93.1 93 92.4 92.9 92.3 92.3 similarity (%) *Strain NZva20T grows best in freshwater medium containing 0.1% (w/v) NaCl and does not grow in medium with NaCl concentration exceeding 0.5 % (w/v) NaCl. ND=no data available. NA=not applicable.

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Acknowledgments

We are very grateful to Dr. Michael Pester (University of Konstanz, Germany) for helping to construct the phylogenetic tree. We also acknowledge Antje Wiese for preparing media. Authors greatly thank the German Academic Exchange Service (DAAD), Germany, for providing a fellowship to M. J.

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CHAPTER 6

Degradation of phthalate isomers by enrichment cultures under nitrate-reducing, sulfate-reducing and fermenting conditions.

Degradation of phthalate isomers by enrichment cultures under nitrate-/sulfate-/fermenting conditions

1. Introduction Anaerobic degradation of phthalate and phthalate esters was reported for numerous pure and mixed cultures under nitrate- or sulfate-reducing or methanogenic conditions [Liang et al., 2008; Qiu et al., 2004’ Cheung et al., 2007; Qiu et al., 2006; Kleerebezem et al., 1999a; Ejlertsson and Svensson, 1996; Lykidis et al., 2011]. Recently, we have identified the genes, enzymes and intermediates involved in the anaerobic decarboxylation of o-phthalate to benzoate/benzoyl-CoA by the newly isolated o-phthalate degrading, nitrate-reducing bacterium, Azoarcus sp. strain PA01 [Junghare et al., 2015b; Junghare et al., 2016; discussed in chapter 3 and 4]. However, the biochemistry of anaerobic phthalate isomer degradation, especially their decarboxylation to benzoate/benzoyl-CoA in sulfate-reducing or fermenting bacteria is still unknown. In this chapter, I describe different enrichment cultures that degrade phthalate isomers under nitrate-reducing, sulfate-reducing or fermenting conditions. Enrichments were mainly started from sludge samples collected from wastewater treatment plants.

1.1 Phthalate isomer degradation under denitrifying conditions Enrichment cultures degrading phthalate isomers under nitrate-reducing conditions were performed in non-reduced, freshwater mineral water medium (as described in chapter 2 and 3) containing 2 mM isophthalate. Cultures were incubated over 3 - 4 months at 30 ºC and sub-cultured after every 4 - 5 months. An enrichment culture designated as ‘E1iso’ was used for the isolation and purification of a defined culture. After, several attempts, 3 pure strains were identified based on the 16S rRNA gene sequencing (Table 1). Morphologically the cells were short rods (Figure 1) and motile. Strains E1iso4 and E1iso3 exhibited very poor and slow growth (flocculation) with 2 mM isophthalate under denitrifying conditions. Therefore, these cultures were not further investigated. But in general, isophthalate and terephthalate (data not shown) degradation was observed to be slower compared to phthalate degradation. The mixed culture ‘E1iso’ degraded 2 mM isophthalate completely under nitrate-reducing conditions. Interestingly, culture E1iso readily adapted for benzoate utilization. Preliminary enzyme assays performed with the cell-free extract prepared from E1iso culture grown with 2 mM isophthalate showed no conversion of isophthalyl-CoA to benzoyl-CoA by LC-MS analysis (data not shown). However, it is still doubtful whether isophthalate degradation

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Degradation of phthalate isomers by enrichment cultures under nitrate-/sulfate-/fermenting conditions occurs analogous to phthalate degradation by strain PA01 (chapter 3 and 4) and further experiments are needed.

Table 1. Defined pure cultures identified from the phthalate-isomer degrading enrichment culture.

Culture 16s rRNA similarity Growth Morphological features Designation % substrate

E1f pht2 Diaphorobacter Phthalate Cells of strain E1f phth2 were rods, 0.5- 0.8 µm nitroreducens (99.85 in diameter, 2.8 -3.1 µm in length and motile and %) divide by binary fission (Figure A).

E1 iso4 Diaphorobacter Isophthalate Cells of strain E1iso4 were rods, 0.4- 0.8 µm in nitroreducens (99.64%) diameter, 2.4 -2.9 µm in length, motile and divide by binary fission (Figure B).

E1 iso3 Pseudomonas Isophthalate Cells of strain E1f iso3 were rods, 0.3- 0.6 µm in xanthomarina (98.33 diameter, 2.8 -3.1 µm in length and motile %) (Figure C).

Strains isolated from the enrichment culture, but not investigated further due to poor growth.

Figure 1. Phase-contrast microscopic photographs: A) D. nitroreducens strain E1f pht2; B) D. nitroreducens E1 iso4; and P. xanthomarina E1 iso4 isolates purified from phthalate- degrading enrichment culture. Bar length: 10 µm.

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Degradation of phthalate isomers by enrichment cultures under nitrate-/sulfate-/fermenting conditions

1.2 Phthalate degradation under sulfate-reducing/fermenting conditions An enrichment culture degrading o-phthalate designated as ‘KOPA’ was originally enriched from a sample collected from a wastewater treatment plant (Konstanz, Germany) in freshwater, sulfide-reduced mineral medium supplemented with 10 mM of sulfate and 2 mM of o-phthalate as sole carbon source as described before [Junghare and Schink, 2015; chapter 5]. The enrichment culture was incubated at 30 ºC and sub-cultured for several passages. Several attempts were made to obtain defined pure cultures of sulfate-reducing/fermenting bacteria using anoxic agar shake techniques. But all the attempts were unsuccessful to obtain a phthalate-degrading pure culture. Therefore, the enrichment culture KOPA was further partially investigated. Growth of the enrichment culture was monitored for o-phthalate utilization and product formation using HPLC analysis. Hydrogen or methane production was analyzed by GC analysis.

1.3 Partial characterization of enrichment culture KOPA Microbial community analysis of the mixed culture KOPA degrading o-phthalate under sulfate-reducing conditions was performed using molecular techniques. Community DNA was prepared from about 0.1 - 0.3 g (wet cell mass) using the modified CTAB method as described [Junghare et al., 2015b]. Total bacterial community 16S rRNA genes were PCR- amplified using a set of the universal primer 8F (5′-AGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACCTTGTTACGACTT-3′), and the archaea-specific primers for the small subunit unit (SSU) rRNA genes were amplified using the Ar109F (5’-ACKGCTCAGTAACACGT- 3‘) and Ar912rt (5’-GTGCTCCCCCGCCAATTCCTTTA-3’), respectively. PCR products were cloned into pCR™4-TOPO®TA vector and transformed into competent E. coli cells as per the manufacturer’s instructions (Invitrogen, Germany). Cloned insert DNAs (16S rDNA/SSU) were amplified using the vector specific M13 primers (Invitrogen) and sequenced (GATC Biotech AG, Konstanz, Germany).

Sequencing of the cloned 16S rRNA genes resulted in the identification of the majority of the sequences that were similar (98 - 99 %) to the recently isolated benzoate-degrading sulfate- reducing bacterium D. benzoelyticum from the same enrichment [Junghare et al., 2015a; chapter 5]. Therefore, it is the pre-dominant bacterium in the enrichment culture. Other cloned bacterial sequences include the known aromatic compound degrading, sulfate-

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Degradation of phthalate isomers by enrichment cultures under nitrate-/sulfate-/fermenting conditions reducing bacteria Desulfatiglans catecholica, Desulfatiglans anilini, and Desulfatiglans parachlorophenolica (90 to 95 % similarity). Additionally, a sequence showing similarity to the known phthalate-fermenting bacterium Syntrophorhabdus aromativorans was identified. On the other hand, the archaeal community analysis identified mainly sequences belonging to the genus Methanosaeta sp., i.e., acetoclastic methanogens which convert acetate to methane. Growth experiments with the ‘KOPA’ culture revealed that phthalate was utilized from about 2 mM to 0.6 mM within the first 15 days of incubation. Further incubation resulted in no or very little utilization of phthalate (Figure 2). This strongly suggests that sulfate-reducers were inhibited due to accumulation of toxic hydrogen sulfide produced in the medium by sulfate reduction during phthalate degradation.

Figure 2. Utilization of phthalate at different time points by KOPA culture under sulfate- reducing conditions.

HPLC analysis of the supernatants of the KOPA culture identified mainly acetate as intermediate. Also very small amounts of propionate, succinate and formate were detected (data not shown). Head space analysis of the enrichment culture revealed the formation of hydrogen and methane. Furthermore, addition of 5 mM molybdate to the enrichment culture completely inhibited phthalate utilization, and no growth or other metabolites were detected. The KOPA enrichment culture degraded o-phthalate degradation without inhibition (Figure

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Degradation of phthalate isomers by enrichment cultures under nitrate-/sulfate-/fermenting conditions

2). Thus, these results indicate that sulfate reducing bacteria are key players in the o-phthalate degrading enrichment culture. Apart from these, several other enrichment cultures were established that could degrade either one of the phthalate isomers. The list of enrichment cultures degrading phthalate isomers under different conditions is shown in table 2. Table 2. List of enrichment cultures degrading phthalate isomers under different conditions.

Culture Growth conditions Growth substrate Designation E1iso Nitrate-reducing Isophthalate/benzoate

KOPA Sulfate-reducing Phthalate

Wo-PA Fermenting condition/methanogenic conditions Phthalate/isophthalate/terephthalate KN-Ter Mo-Iso Mo-PA Mo-Ter

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CHAPTER 7

General discussion and concluding remarks

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General discussion

General discussion

The aim of this dissertation was to reveal the biochemistry of anaerobic degradation of phthalate, especially it illustrates enzymes and intermediates involved in the difficult step of anaerobic phthalate decarboxylation by the newly isolated nitrate-reducing bacterium Azoarcus sp. strain PA01. It is well established that in aerobic phthalate degradation, decarboxylation of phthalate is facilitated by introducing molecular oxygen to the phthalate ring by oxygenases followed by decarboxylation to protocatechuate [discussed in chapter one, section 4.3; figure 5]. In aerobic catabolism of aromatic compounds, oxygen is used to de•aromatize and cleave the ring. However, due to the absence of molecular oxygen, anaerobic bacteria cannot use the oxygenases and have to use oxygen-independent strategy for accomplishing decarboxylation of phthalate to benzoate during the anaerobic degradation of phthalates.

Activation of o-phthalate by Azoarcus sp. strain PA01

The use of CoA esters in an aerobic aromatic degradation pathway are important not only for retaining CoA•bound intermediates (membrane impermeable) in the cell, but CoA-thioesters also mechanistically facilitate various reactions [Teufel et al., 2010; Teufel et al., 2011], such as the electron•withdrawing character of CoA thioesters facilitates the reduction of the aromatic ring. Moreover, CoA•bound intermediates can also be rapidly recognized and bound through CoA•binding motifs of the enzymes [Rather et al., 2011]. For investigation of the reaction mechanism that is involved in the initial activation of phthalate by nitrate-reducing bacteria, Azoarcus sp. strain PA01 has been used as a model microorganism. Previously it was assumed that phthalate decarboxylation occurs through the initial formation of phthalyl- CoA by an ATP dependent acetyl-CoA synthatase in denitrifying Pseudomonas sp. strain P136 which is then converted to benzoyl-CoA by specific decarboxylase [Nozawa and Maruyama, 1988ab; Niazi et al., 2001]. But, the authors were unable to demonstrate the formation of phthalyl-CoA and this hypothesis could not be proven.

In general anaerobic bacteria uses different peripheral pathways for the activation of a wide variety of aromatic compounds (ATP-dependent or ATP-independent) lead to a few central aromatic intermediates, such as benzoyl-CoA most common intermediate (discussed in chapter 1). For instance, the ATP-mediated activation of benzoate to benzoyl-CoA is a

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General discussion general feature in the most anaerobic bacteria [Fuchs, 2008]. However, enzyme assays performed with cell-free extract of Azoarcus sp. strain PA01 grown with o-phthalate did not result in the formation of o-phthalyl-CoA with o-phthalate plus ATP [Junghare et al., 2016; discussed in chapter 3]. Therefore, hypothetical ATP-dependent activation of o-phthalate to o-phthalyl-CoA was ruled out. However, the current view was still that phthalate degradation occurs through the benzoate/benzoyl-CoA degradation pathway, i.e., phthalate decarboxylation is the essential step. Because, the phthalate degrading anaerobic cultures reported so far, including Azoarcus sp. strain PA01 could readily adapt for benzoate utilization and thus phthalate decarboxylation to benzoate was considered to be a key step in anaerobic phthalate degradation.

In the past, studies with aerobic aromatic compound degradation, the use of a CoA transferase rather than an ATP-dependent CoA ligase for substrate activation has already been reported, e.g., in the 3-hydroxybenzoate degradation by the fermenting bacterium S. hydroxybenzoicum [Müller and Schink, 2000] or in the peripheral toluene degradation by denitrifying bacteria [Leutwein and Heider, 2001]. Moreover, the use of a CoA transferase rather than a CoA ligase (ATP-consuming) for substrate activation has important energetic implications at least for fermenting bacteria with poor energy budget [Müller and Schink, 2000]. Although, denitrifying bacteria are energy rich, we still hypothesized an ATP- independent activation of o-phthalate to o-phthalyl-CoA by a CoA-transferase enzyme that could use succinyl-CoA, acetyl-CoA or free CoA as the possible CoA donor. The genome of Azoarcus sp. strain PA01 was sequenced and annotated during the course of this study [Chapter 2]. Indeed, our hypothesis was supported by the fact that differential two- dimensional protein profiling of o-phthalate versus benzoate grown cells of Azoarcus sp. strain PA01 identified several proteins that were induced only under o-phthalate grown conditions [Chapter 3, Figure 1, 2]. These proteins were identified using the annotated genome of Azoarcus sp. strain PA01 and were homologous to a solute transporter (PA01_00214), the CoA-transferases (PA01_00215 and PA01_00216), and the UbiD- like/UbiX-like (PA01_00217 and PA01_00218) decarboxylase [Chapter 3; Table 1). Interestingly, the proteins which are up-regulated during the metabolism of o-phthalate are located in a single gene cluster that are also found in the genomes of other aromatic compounds degrading, nitrate-reducers, e.g., T. chlorobenzoica [Chapter 3; Figure S4].

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General discussion

From the proteomic and genomic results, we suggested that two CoA-transferase genes PA01_00215 which code for PhtSa (43.8 kDa), whereas PA01_00216 code for PhtSb (41.3 kDa) are involved in the o-phthalate activation to o-phthalyl-CoA [Junghare et al., 2016]. Interestingly, both PhtSa and PhtSb are homologous (30 %) to BbsE and BbsF the two subunits of a succinyl-CoA:(R)-benzylsuccinate CoA-transferase that activates benzylsuccinate to its CoA thioester in anaerobic toluene degradation by T. aromatica [Leutwein and Heider 2001]. Furthermore, enzyme assays with cell-free extract of Azoarcus sp. strain PA01 showed formation of o-phthalyl-CoA, specifically with o-phthalate and succinyl-CoA as the CoA donor as revealed by LC-MS [Chapter 3; Figure 5]. The enzyme activity was found to be substrate specific and no isophthalate or terephthalate could be converted to corresponding phthalyl-CoA-derivatives. Acetyl-CoA and free-CoA could not serve as the CoA donor for phthalate activation [Junghare et al., 2016].

Decarboxylation of activated o-phthalate: a two component enzyme system

Among the phthalate induced proteins, there are two genes coding for proteins which are homologous to UbiD-like (PA01_00217) and UbiX-like (PA01_00218) decarboxylases that were identified in the 2-D differential proteomics [Chapter 3; Figure 3 and table 1]. UbiD/UbiX-like decarboxylases that are found in a wide range of bacteria and catalyse the decarboxylation of 4-hydroxy-3-oactprenyl benzoaic acid to 2-octaprenyl phenol an intermediate step in ubiquinone biosynthesis [Meganathan, 2001; Zhang and Javor, 2000; Gulmezein et al., 2007]. Enzyme assays with cell-free extract showed a high conversion of o- phthalyl-CoA to benzoyl-CoA, specifically in phthalate-grown cells of Azoarcus sp. strain PA01 [Chapter 3]. Further, no decarboxylase activity was detected with isophthalyl-CoA or terephthalyl-CoA. Therefore, these two genes PA01_00217 which code for PhtDa (60 kDa) and PA01_00218 code for PhtDb (22 kDa) were considered to be involved in decarboxylation of o-phthalyl-CoA to benzoyl-CoA by Azoarcus sp. strain PA01 [Junghare et al., 2016].

Cloning and heterologous expression of PhtDa (60 kDa) and PhtDb (22 kDa) in E. coli confirmed that these two proteins are certainly responsible for the decarboxylation of o- phthalyl-CoA to benzoyl-CoA during anaerobic degradation of o-phthalate by Azoarcus sp. strain PA01. Alone, PhtDa and PhtDb did not exhibit the decarboxylation and thus, each protein has a different catalytic function [discussed in chapter 4]. Our results revealed the

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General discussion high similarity with the decarboxylase reaction catalysed by the UbiD-like and UbiX-like decarboxylases together, in which UbiX is a FMN-dependent protein which does not possess decarboxylase activity. But rather it generates the prenyl-FMN containing cofactor that is required by UbiD-like aromatic acid decarboxylase [White et al., 2015; Lin et al., 2015; Payne et al., 2015]. Interestingly, heterologously expressed PhtDa (UbiD-like) and PhtDb (UbiX-like) together converted o-phthalyl-CoA to benzoyl-CoA, only if PhtDb was prior incubated with FMN and dimethylallyl phosphate (DMAP). Replacement of FMN with FAD led to no conversion. In addition to this, amino acid sequence alignments and homology based 3-D structure modelling revealed that, only PhtDb possesses the binding site for FMN and DMAP analogous to the FMN-dependent UbiX-like enzyme in E. coli O157:H7 [discussed in chapter 4]. At the moment, the mechanistic details of FMN in the enzymatic decarboxylation of o-phthalyl-CoA to benzoyl-CoA is not fully understood. However, it is known from PhtDa and PhtDb homologous enzymes that the FMN-containing cofactor of the UbiX-like enzyme in E. coli assists in the decarboxylation of 4-hydroxy-3-octaprenyl benzoic acid to 2-octaprenyl phenol catalysed by UbiD-like aromatic acid decarboxylase constituting a two component enzyme system.

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Concluding remarks and general outlook

Concluding remarks and general outlook

The difficult step of anaerobic phthalate decarboxylation to benzoate has been understood in greater detail by using denitrifying bacterium Azoarcus sp. strain PA01. Our results are consistent with the recent study reported on the identification genes and enzymes involved in phthalate degradation by denitrifying anaerobic bacteria [Ebenau-Jehle et al., 2016]. However, there are still several open questions that need to be answered. For instance, it is still unclear how the other isomers of the phthalate are decarboxylated. It can be assumed that isophthalate or terephthalate are also likely to be activated analogous to phthalate, to the corresponding phthalyl-CoAs by specific CoA-transferase and which can be then subsequently decarboxylated to benzoyl-CoA. Because, the phthalate isomers degrading strain could utilize benzoate readily. However, this need to be confirmed by experiments. We have already identified the two o-phthalate induced genes CoA-transferases (PA01_00215 and PA01_00216) that are involved in the phthalate activation to o-phthalyl-CoA in Azoarcus sp. strain PA01. Even though I have already cloned both CoA-transferases genes, but no conclusive results were generated at the time of writing this thesis. Therefore, the role of each enzyme in the activation reaction needs to be confirmed. For the phthalyl-CoA decarboxylase enzyme, there are already several indications and lines of evidences for the involvement of a FMN-dependent enzyme in the decarboxylation activation, nevertheless its exact function in decarboxylation reaction need to be confirmed. Further, it would be also interesting to know whether the strictly anaerobic bacteria such as sulfate-reducing bacteria or fermenting bacteria strategy for phthalate activation analogous to nitrate reducers, i.e., Azoarcus sp. strain PA01.

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Personal record of achievements

Unless stated otherwise, all the wet-lab experiments were developed, planned and executed by myself under the guidance of Prof. Dr. Bernhard Schink and Prof. Dr. Dieter Spiteller. Occasionally, cultivation media were prepared by Antje Wiese. Few enrichments used in this research study were already initiated before the beginning of my doctoral studies. Isolation, purification, identification and characterization of bacterial strains reported in this work was performed myself. Dr. Michael Pester explained and assisted for calculating phylogenetic tree used in the chapter five. Cloning, expression and protein purification were performed and optimized by me. Genome sequencing and assembly was performed at the GATC Biotech Konstanz, and annotation was performed by the Integrated Microbial Genome, JGI state of the art. Gene annotation used in this study were manually checked by me. Enzyme assays analysis performed by me and phthalyl-CoA were synthesized by Prof. Dr. Dieter Spiteller. All the protein identifications were performed by Mr. Marquat at the Proteomics Facility, University of Konstanz. All the manuscripts were solely written by me, and corrected by Prof. B. Schink, Prof. D. Spiteller and the co-authors of the respective publications if any. Unless stated otherwise, all the results presented in my PhD dissertation analyzed by myself. The manuscript mentioned in chapter 4, has to be improved significantly with some additional experiments and will be completed soon.

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List of publications

Published

1. Patil, Y., Müller N., Schink, B. et al., and Junghare, M. (2017). High-quality-draft genome sequence of the fermenting bacterium Anaerobium acetethylicum type strain GluBS11T (DSM 29698). Standards in Genomic Sciences (Accepted).

2. Patil, Y., Junghare, M. and Müller, N. (2016). Fermentation of glycerol by Anaerobium acetethylicum and its potential use in biofuel production. Microbial Biotechnology, doi: 10.1111/1751-7915.12484

3. Junghare M, D Spiteller, and B Schink (2016). Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01. Environmental Microbiology, 18(9); 3175-3188. doi:10.1111/1462-2920.13447

4. Junghare, M., Patil, Y., and Schink, B. (2015). Draft genome sequence of a nitrate- reducing, o-phthalate degrading bacterium Azoarcus sp. PA01T. Standards in Genomic Sciences, 10; 90. http://doi.org/10.1186/s40793-015-0079-9

5. Patil, Y., Junghare, M., Pester, M., Müller, N., and Schink, B. (2015). Anaerobium acetethylicum gen. nov., sp. a strictly anaerobic, gluconate-fermenting bacterium isolated from a methanogenic bioreactor. International Journal of Systematics and Evolutionary Microbiology, 65(10); 3289-3296. http://www.ncbi.nlm.nih.gov/pubmed/26297346

6. Junghare, M., and Schink, B. (2015). Desulfoprunum benzoelyticum gen. nov., sp. nov., a Gram-stain-negative, benzoate-degrading, sulfate-reducing bacterium isolated from a wastewater treatment plant. International Journal of Systematics and Evolutionary Microbiology, 65(1); 77-84. http://www.ncbi.nlm.nih.gov/pubmed/25278560

Un-published

1. Junghare, M., Spiteller, D. and Schink, B. (2017). Cloning, heterologous expression and characterization of a novel o-phthalyl-CoA decarboxylase from Azoarcus sp. strain PA01. (Manuscript to be submitted).

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Workshops and training courses attended

1. Practical workshop on “Proteomics” held on 28-30 Sep, 2015 at the Proteomics Facility

Centre, University of Konstanz, Germany.

2. Theory course on "Gene Expression and Protein Purification Strategies" held on 9 Feb,

2015 at the Proteomics Facility Centre, University of Konstanz, Germany.

3. Practical workshop on “Microbial Genomics & Metagenomics” held on 21-25 July, 2014 at the University of Valencia, Spain (organised by Joint Genome Institute, USA).

4. Theory course on “Good Scientific Practice” 26-27 Jun, 2014 (organised by academic staff development) at the University of Konstanz, Germany.

5. Theory course on the “English skills in academia-Dissolving fear of teaching in English and inspiring the English atmosphere in the classroom” 25 Jun, 2014 (organised by academic staff development) at the University of Konstanz, Germany.

6. Theory course on’ “Conference presentation-How to engage the listener in your talk”

21-22 Nov, 2013 (organised by academic staff development) at the University of Konstanz,

Germany.

7. Workshop on the “Aquatic Microbial and Molecular Ecology” 05-23 Aug, 2013 at the

Institute of Biology, Southern University of Denmark, Denmark.

8. Theory course on “Writing successful grant proposals” 4-5 Jul, 2013 (organised by academic staff development) at the University of Konstanz, Konstanz.

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References

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Abbreviations

µl Micro liter µM Micro molar 3D Three dimensional bp Base pair CDSs Coding DNA sequences CFA Cellular fatty acid CoA Coenzyme A COG Clusters of Orthologous Groups CTAB Cetyl Trimethyl Ammonium Bromide Da Dalton DMAP Dimethylallyl monophosphate DNA Deoxyribonucleic acid DTT Dithiothreitol EDTA Ethylene diamine tetraacetic acid FAD Flavin adenine dinucleotide Fdc1 Ferulic acid decarboxylase FMN Flavin mononucleotide g Gram GC Gas chromatography GOLD Genomes Online Database HGAP3 Hierarchical Genome Assembly Process HPLC High performance liquid chromatography IA Isophthalic acid (isophthalate) IFE Iso electric focusing IMG Integrated Microbial Genomes IMG-ER Integrated Microbial Genomes-Expert Review IPTG Isopropyl βHDH1Hthiogalactopyranoside kb Kilo base L Litre LB Luria bertani medium M Molar min Minute ml mili liter mM Milli molar MS Mass spectrometry N Normality NCBI National Center for Biotechnology Information OD600 Optical density at 600nm

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ORF Open reading frame PA Phthalic acid (phthalate) Pad1 Phenylacrylic acid decarboxylase PAEs Phthalic acid esters PCR Polymerase chain reaction PDB Protein data bank pKa Acid dissociation constant rpm Revolutions per minute s/sec Seconds SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SRB Sulfate-reducing bacteria TA Terephthalic acid (terephthalate) TB Terrific broth medium UbiD 3-prenyl-4-hydroxybenzoate decarboxylase UbiX 3-prenyl-4-hydroxybenzoate decarboxylase UV Ultraviolet w/v Weight per volume

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