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Title:
The citric acid cycle of _thiomicrospira crunogena_ : an oddity amongst the proteobacteria
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English
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Quasem, Ishtiaque
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Thiomicrospira crunogena
Central carbon metabolism
Citric acid cycle
Proteobacteria
Chemolithoautotroph
Dissertations, Academic -- Biology -- Masters -- USF   ( lcsh )
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Abstract:
ABSTRACT: Thiomicrospira crunogena, a deep-sea hydrothermal vent chemolithoautotroph, uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately autotrophic Gammaproteobacterium would use a 'wishbone' version of the citric acid cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the fully oxidative version to minimize carbon loss as carbon dioxide. However, upon examination of its complete genome sequence, it became apparent that this organism did not fulfill this expectation. Instead of a wishbone pathway, T. crunogena appears to run a fully oxidative CAC. The cycle is 'locked' in the oxidative direction by replacement of the reversible enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both oxidoreductases was confirmed via assays on T. crunogena cell extracts. To determine whether this peculiar CAC was novel, complete genome sequences of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated Microbial Genome database. Genes catalyzing steps in the CAC were collected from each organism and vetted for paralogs that had adopted an alternative, 'non-CAC' function through genome context and cluster analysis. Alignments were made with the remaining sequences and were verified by comparing them to curated alignments at Pfam database and examination of active site residues. Phylogenetic trees were constructed from these alignments, and instances of horizontal gene transfer were determined by comparison to a 16S tree. These analyses verified that the CAC in T. crunogena is indeed unique, as it does not resemble any of the canonical cycles of the six classes of proteobacteria. Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes encoded by genes that are likely to have been acquired via horizontal gene transfer. The gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with those present in the Cyanobacteria, while those encoding oxoglutarate: acceptor oxidoreductase cluster among the Firmicutes, and malate: quinone oxidoreductase clusters with the Epsilonproteobacteria.
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Thesis (M.S.)--University of South Florida, 2009.
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by Ishtiaque Quasem.
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The Citric Acid Cycle of Thiomicrospira crunogena : An Oddity Amongst the Proteobacteria by Ishtiaque Quasem A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Integrative Biology College of Arts and Sciences University of South Florida Major Professor: Kathleen M. Scott, Ph.D. Jim Garey, Ph.D. Degeng Wang, Ph.D. Date of Approval: November 2, 2009 Keywords: Thiomicrospira crunogena, central carbon metabolism, citric acid cycle, Proteobacteria, chemolithoautotroph Copyright 2009, Ishtiaque Quasem

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Acknowledgments I would like to begin by acknowledging the USDA-CSREES program and NSFMCB, which have provided the funds for my research assistantship. Their support was valuable in helping me progress as a researcher and independent thinker. I would also like to thank the University of South Florida Biology Department and the College of Arts and Science for a travel stipend to attend and present my research at the ASM meeting. Most importantly, I would like to thank my committee for their advice on my project and help through the years. Dr. Jim Garey has taught me the fine art of molecular phylogenetics. Through thick and thin, his expertise was invaluable in helping me understand the phylogeny related to my bacterial genes and the many mechanations of how to properly interpret such data. Dr. Degeng Wang was also invaluable in the techniques he had taught me in bioinformatics. Thanks to his thorough demonstrations of the many different ways to acquire and compare amino acid sequences, it allowed my data collection to be sensible and complete. My years as a graduate student could not have been more productive nor as pleasant, had it not been for my persistent and wise major professor, Dr. Kathleen Scott. I would like to thank her for not only convincing me to take on a project entailing the genomic screening of the entire Proteobacterial phylum, but also directing and supporting me towards completing such a task. In addition, her experience and advice led me to not only complete this thesis, but also proposals, grants, and conference posters related to this project. It was through her compassion, understanding, and encouragements that helped me get through even the darkest of graduate school days. I am humbled and grateful for all the help and moral support she has given me during the time I have worked with her.

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i Table of Contents List of Tables ................................................................................................................ ..... iii List of Figures ............................................................................................................... ..... iv Abstract ...................................................................................................................... ..........v Chapter 1 – Introduction ...................................................................................................... 1 1.1 Hydrothermal Vent Habitat ................................................................................1 1.2 Physiology..........................................................................................................2 1.3 Phylogeny ..........................................................................................................3 1.4 Citric Acid Cycle Function ................................................................................4 1.5 Steps of the Citric Acid Cycle ...........................................................................8 1.5.1 Pyruvate to Acetyl-coA .......................................................................8 1.5.2 Acetyl-coA and Oxaloacetate to Citrate ...........................................10 1.5.3 Citrate to Isocitrate ............................................................................11 1.5.4 Isocitrate to 2-oxoglutarate ...............................................................11 1.5.5 2-oxoglutarate to Succinyl-coA ........................................................12 1.5.6 Succinyl-coA to Succinate ................................................................12 1.5.7 Succinate to Fumarate .......................................................................13 1.5.8 Fumarate to Malate ...........................................................................14 1.5.9 Malate to Oxaloacetate .....................................................................14 1.6 The Citric Acid Cycle in T. crunogena ............................................................15 1.7 Purpose .............................................................................................................16 Chapter 2 – Methods ..........................................................................................................1 7 2.1 Finding Citric Acid Cycle Genes in Proteobacteria .........................................17 2.2 Removing Genes with Apparent Alternative Function ...................................18 2.3 Multiple Sequence Alignment and Phylogenetic Analysis ..............................18 2.4 Enzyme Assays ................................................................................................20 Chapter 3 – Results and Discussion ...................................................................................24 3.1 Steps 1 – 4: Pyruvate to Oxoglutarate .............................................................25 3.2 Steps 5 – 9: Oxoglutarate to Oxaloacetate ......................................................34 3.3 Conclusion ......................................................................................................42 References .................................................................................................................... ......43

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ii Appendices .................................................................................................................... .....50 Appendix 1: Biochemically Characterized Genes of the Citric Acid Cycle Used as Query Sequences for BLAST Searches of IMG .........51 Appendix 2: Paralogous Genes with an Alternative Function ...............................57 Appendix 3: Active Site Residues for Alignment Verification .............................60 Appendix 4: Pfams Whose HMM Logos Were Used to Verify Alignments ........63 Appendix 5: The Citric Acid Cycle of T. crunogena .............................................66 Appendix 6: The Canonical Citric Acid cycle of Gammaproteobacteria .............67 Appendix 7: The Canonical Citric Acid cycle of Betaproteobacteria ..................68 Appendix 8: The Canonical Citric Acid cycle of Alphaproteobacteria ................69 Appendix 9: The Canonical Citric Acid cycle of Epsilonproteobacteria ..............70 Appendix 10: The Canonical Citric Acid cycle of Delta and Acido-Proteobacteria .............................................................................................71 Appendix 11: Citric Acid Cycle Genes (Pyruvate to 2-Oxoglutarate) Present for Each Species in the Gammaproteobacteria .....................................................72 Appendix 12: Citric Acid Cycle Genes (Pyruvate to 2-Oxoglutarate) Present for Each Species in the Betaproteobacteria ...........................................................75 Appendix 13: Citric Acid Cycle Genes (Pyruvate to 2-Oxoglutarate) Present for Each Species in the Alphaproteobacteria ........................................................77 Appendix 14: Citric Acid Cycle Genes (Pyruvate to 2-Oxoglutarate) Present for Each Species in the Epsilon-, Delta-, and Acido-Proteobacteria ...................79 Appendix 15: Citric Acid Cycle Genes (2-Oxoglutarate to Oxaloacetate) Present for Each Species in the Gammaproteobacteria ........................................80 Appendix 16: Citric Acid Cycle Genes (2-Oxoglutarate to Oxaloacetate) Present for Each Species in the Betaproteobacteria ..............................................83 Appendix 17: Citric Acid Cycle Genes (2-Oxoglutarate to Oxaloacetate) Present for Each Species in the Alphaproteobacteria ............................................85 Appendix 18: Citric Acid Cycle Genes (2-Oxoglutarate to Oxaloacetate) Present for Each Species in the Epsilon-, Delta-, and Acido-Proteobacteria .......87 Appendix 19: Phylogeny of Homodimeric Pyruvate Dehydrogenase Sequences .....................................................................................88 Appendix 20: Phylogeny of Isocitrate Dehydrogenase Sequences ........................92 Appendix 21: Phylogeny of Succinyl-coA Synthetase Sequences ........................96 Appendix 22: Phylogeny of Succinate Dehydrogenase/ Fumarate Reductase sequences ............................................................................103 Appendix 23: Phylogeny of Class II Fumarase Sequences .................................109

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iii List of Tables Table 1 Canonical Citric Acid Cycle Enzymes of Proteobacterial Classes ............24 Table 2 Activities of Citric Acid Cycle Enzymes in T. crunogena ........................26

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iv List of Figures Figure 1 Proteobacteria Phylogeny ............................................................................3 Figure 2 16S RNA Tree of Gammaproteobacteria ....................................................3 Figure 3 Oxidative Citric Acid Cycle.........................................................................5 Figure 4 Reverse Citric Acid Cycle ...........................................................................6 Figure 5 Reductive Citric Acid Pathway ....................................................................7 Figure 6 Phylogeny of siCitrate Synthase Sequences ..............................................27 Figure 7 Effect of NADH on the Activities of Citrate Synthase in T. crunogena and E. coli .......................................................................29 Figure 8 Phylogeny of Aconitase Sequences ...........................................................31 Figure 9 Phylogeny of 2-Oxoglutarate: Acceptor Oxidoreductase Sequences ........35 Figure 10 Phylogeny of Malate: Quinone Oxidoreductase Sequences ......................39

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v The Citric Acid Cycle of Thiomicrospira crunogena : An Oddity Amongst the Proteobacteria Ishtiaque Quasem ABSTRACT Thiomicrospira crunogena, a deep-sea hydrothermal vent chemolithoautotroph, uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately autotrophic Gammaproteobacterium would use a ‘wishbone’ version of the citric acid cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the fully oxidative version to minimize carbon loss as carbon dioxide. However, upon examination of its complete genome sequence, it became apparent that this organism did not fulfill this expectation. Instead of a wishbone pathway, T. crunogena appears to run a fully oxidative CAC. The cycle is ‘locked’ in the oxidative direction by replacement of the reversible enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both oxidoreductases was confirmed via assays on T. crunogena cell extracts. To determine whether this peculiar CAC was novel, complete genome sequences of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated

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vi Microbial Genome database. Genes catalyzing steps in the CAC were collected from each organism and vetted for paralogs that had adopted an alternative, ‘non-CAC’ function through genome context and cluster analysis. Alignments were made with the remaining sequences and were verified by comparing them to curated alignments at Pfam database and examination of active site re sidues. Phylogenetic trees were constructed from these alignments, and instances of horizontal gene transfer were determined by comparison to a 16S tree. These analyses verified that the CAC in T. crunogena is indeed unique, as it does not resemble any of the canonical cycles of the six classes of proteobacteria. Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes encoded by genes that are likely to have been acquired via horizontal gene transfer. The gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with those present in the Cyanobacteria while those encoding oxoglutarate: acceptor oxidoreductase cluster among the Firmicutes and malate: quinone oxidoreductase clusters with the Epsilonproteobacteria

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1 Chapter 1 Introduction Thiomicrospira crunogena was among the first obligate chemolithoautotrophic bacteria to have its genome sequenced [1]. One of the peculiarities identified during genome annotation was its bizarre citric acid cycle (CAC), which, unlike the wishboneshaped citric acid pathway anticipated based on its autotrophic lifestyle, was locked in an oxidative direction with noncanonical enzymes [1]. The work conducted here explores the CAC of this organism in greater depth using genome comparisons, phylogenetic analyses, and enzyme assays. 1.1 Hydrothermal Vent Habitat Thiomicrospira crunogena is a deep sea vent chemolithoautotrophic gammaproteobacterium [1], originally isolated from the East Pacific Rise [2] and detected through molecular methods in deep sea vents in both the Pacific and Atlantic oceans [3]. At these vents, warm hydrothermal vent fluid rich in redox substrates and dissolved inorganic carbon (DIC) is emitted [1]. The temperature difference of this warm vent fluid with the surrounding seawater (~2C) creates turbulent eddies as they mix; therefore the vent habitat fluctuates between dominance of the cold oxic bottom water and the warm anoxic vent fluid in short periods of time [1, 4]. Nonetheless, the deep sea hydrothermal vent is an ecosystem teaming with life, primarily due to chemolithoautotrophic microorganisms, like T. crunogena producing

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2 the bulk of the organic material. These primary producers are able to harness the energy released from the oxidation of reduced inorganic compounds (e.g., H2S, CH4, H2, Fe+2) emitted from the vent and use it for carbon fixation [1, 5-8]. 1.2 Physiology T. crunogena is one of the most rapidly growing obligate autotrophs known, with a doubling time of approximately 1 hr under mesophilic conditions (20-30C) [2] This organism does not demonstrate an ability to use extracellular organic carbon compounds as electron donors or carbon sources [2]; growth on, and assimilation of, acetate, succinate, or glucose are not detectable (K. Scott, unpubl. data). Instead, it is capable of using reduced sulfur compounds (H2S, S2O4 -2, S) as electron donors, likely via a membrane-associated Sox system. Oxygen is the only electron acceptor supporting growth by T. crunogena [1]; growth is most rapid under low oxygen conditions, and its genome encodes a cbb3-type cytochrome c oxidase, which has a high affinity for oxygen, facilitating this microaerophilic lifestyle [1, 9]. This organism uses the energy from sulfur oxidation to fuel carbon fixation via the Calvin-Benson-Bassham cycle [1]. Despite the periodically high concentrations of dissolved inorganic carbon (DIC) present at hydrothermal vents, T. crunogena can grow rapidly even when the DIC concentration is as low as 20 M, apparently due to this organism’s ability to accumulate intracellular DIC concentrations that are more than 100X higher than extracellular [10].

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3 Figure 1: A generalized phylogenetic tree of the Proteobacterial classes, adapted from [11, 12]. 1.3 Phylogeny T. crunogena is a member of the Gammaproteobacteria one of the six classes into which the Proteobacteria are divided ( Alpha, Beta, Gamma, Delta, Epsilon, and Acidobacteria ; Fig. 1 [11, 12]). Based on 16S sequences, T. crunogena does not fall within any of the other clusters of Gammaproteobacteria whose genomes have been sequenced at this point (Fig. 2). Figure 2: 16S RNA tree of Gammaproteobacteria whose genomes have been sequenced The inferred evolutionary relatedness is based on 16S rRNA gene sequences. Complete 16S sequences were aligned via RNACAD, implemented via RDP 2, version 10, using a model for secondary structure based on bacterial 16S [13]. Neighbor-joining trees were constructed with MEGA 4.0, with bootstrap values based on 1000 replicate samplings of the alignments, pairwise deletions for gaps and missing data, a maximum composite likelihood model, and the assumption of uniform rates among sites [14]. Only those bootstrap values larger than 50 are incuded on this tree. To simplify the figure, the Enterobacteria and some monogeneric clades have been collapsed and are listed in the tree as a genus name. Enterobacteria Haemophilus Vibrio Shewanella Idiomarina loihiensis L2TR Pseudoalteromonas haloplanktis Psychromonas ingrahamii 37 Pseudoalteromonas atlantica T6c Colwellia psychrerythraea 34H Pseudomonas Saccharophagus degradans 240 Hahella chejuensis KCTC 2396 Chromohalobacter salexigens DSM 3043 Alcanivorax borkumensis SK2 Marinobacter aquaeolei VT8 Marinomonas sp. MWYL1 Psychrobacter Acinetobacter Xanthomonas Xylella Dichelobacter nodosus VCS1703A Legionella Methylococcus capsulatus str. Bath Coxiella Nitrosococcus oceani ATCC 19707 Halorhodospira halophila SL1 Alkalilimnicola ehrlichei MLHE1 Thiomicrospira crunogena XCL2 Candidatus Vesicomyosocius okutanii HA Candidatus Ruthia magnifica str. Cm Francisella Candidatus Carsonella ruddii PV Sulfurovum sp. NBC371 Campylobacter jejuni subsp. jejuni Wolinella succinogenes DSM 1740 Sulfurimonas denitrificans ATCC 33889 Epsilon Outgroup 100 100 100 100 100 100 100 100 100 100 100 100 100 53 99 58 80 74 64 56 77 50 85 52 94 68 83 69 95

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4 1.4 Citric Acid Cycle Function Organisms with different physiologies have adopted different modes of the CAC to meet their metabolic needs. In the oxi dative CAC (Fig. 3), oxaloacetate and acetylcoA are condensed to synthesize citrate, which is oxidized and decarboxylated twice to regenerate oxaloacetate, and produce NADH, which can feed electrons into the electron transport chain (ETC) [15]. In the process of running this cycle, metabolic intermediates necessary for biosynthesis are generated [16, 17]. Oxoglutarate is formed, which is necessary for glutamate synthesis [18]. Succinyl-coA is also produced, which in many organisms is necessary for porphyrin synthesis [19]. Oxaloacetate is also synthesized, which is necessary for aspartate and nucleobase synthesis [17]. In some autotrophic bacteria and archaea, the CAC can also operate in a reverse, reductive, CO2-fixing direction (Fig. 4) as a primary carbon fixation pathway [20, 21]. Many cyanobacteria and other autotrophs using the Calvin-Benson-Bassham cycle for carbon fixation have a wishbone shaped version of the citric acid cycle [22], which minimizes decarboxylations. In this wishbone-shaped reductive citric acid pathway (Fig. 5), the metabolic requirements for oxoglutarate are met by decarboxylation of citrate, as in the oxidative CAC, while succinyl-coA and oxaloacetate are synthesized from pyruvate by carboxylation and reduction [1, 15, 22]; by circumventing the decarboxylation of oxoglutarate, these organisms conserve the energy they have expended in forming this carbon-carbon bond.

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5 Figure 3: The oxidative citric acid cycle. The numbers correspond to the following enzymes: 1. Pyruvate dehydrogenase 2. Citrate synthase 3. Aconitase 4. Isocitrate dehydrogenase 5. 2-oxoglutarate dehydrogenase 6. Succinyl-coA synthetase 7. Succinate dehydrogenase 8. Fumarase 9. Malate dehydrogenase NAD(P)+ NAD(P)H, CO2

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6 Figure 4: The reverse citric acid cycle, adapted from [23], which is utilized, instead of the Calvin-BensonBassham cycle, by some autotrophs to fix carbon. The numbers correspond to the following enzymes: 1. ATPdependent citrate lyase; 2. Pyruvate: acceptor oxidoreductase, 3. Phosphoenolpyruvate synthase, 4. Phosphoenolpyruvate carboxykinase, 5. Malate dehydrogenase, 6. Fumarase, 7. Fumarate reductase, 8. Succinyl-coA ligase, 9. Oxoglutarate: acceptor oxidoreductase, 10. Isocitrate dehydrogenase, 11. Aconitase. 1 2 3 4 5 6 7 8 9 10 11 ATP ADP Fdre d Fdox CO2 Fdox Fdre d

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7 Figure 5: The wishbone shaped reductive citric acid pathway, adapted from [24]. This pathway provides no net reduction or oxidation, and allows lithoautotrophic organisms which do not use organic carbon as their major electron donor to still produce intermediates necessary for biosynthesis. The numbers represent enzymes catalyzing each reaction, and are as listed in Fig. 3. In this pathway, the interconversion of succinyl-coA and 2oxoglutarate does not occur. CO2 CO2

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8 1.5 Steps of the citric acid cycle 1.5.1 Pyruvate to Acetyl-coA. Three complexes are capable of interconverting pyruvate and acetyl-coA (pyruvate dehydrogenase, pyruvate: acceptor oxidoreductase, and pyruvate: formate lyase). The pyruvate dehydrogenase complex (PDH) catalyzes the irreversible decarboxylation of pyruvate to form acetyl-coA. This complex exists in homodimeric and heterodimeric forms. The homodimeric PDH consists of the enzymes pyruvate dehydrogenase/decarboxylase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) and is octahedral in symmetry [25-27]. Heterodimeric PDH does not have an E1 subunit homologous to the one present in homodimeric PDH. Instead, the evolutionarily distinct heterodimeric PDH E1 consists of an and subunit, and holoenzyme is a heterotetramer (E1 and E1 E2, E3) with icosahedral symmetry [26]. The subunits of the PDH complex act in concert to produce acetyl-coA. E1 or E1 decarboxylates pyruvate and links the remaining two carbon unit to thiamine pyrophosphate. The E2 subunits transfer the two carbon unit from thiamine pyrophosphate to coA via lipoic acid prosthetic groups. The E3 subunit reoxidizes the E2-bound lipoic acid [27]. The E2 subunit forms the structural core of the complex [2729], while the E1 and E3 subunits form a shell around it [29]. The genetic organization of this complex varies across species, but in general the E1/E1 and E2 subunits are present in an operon [25]. E3 can be shared by other complexes and thus the gene is sometimes present on the genome far from the PDH operon [25]. PDH is particularly useful for aerobic organisms, since unlike the other enzymes capable of converting pyruvate to acetyl-coA, it is not sensitive to oxygen and can contribute electrons to the

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9 ETC [30]. Pyruvate: Acceptor Oxidoreductase (PAOR) catalyzes the reversible conversion of pyruvate to acetyl-coA. Thus far, four forms of this enzyme have been described: single subunit, two-subunit, four subunit, and five subunit [31, 32]. The four types are phylogenetically homologous; the ancestral form is believed to have had four subunits, and the other forms may have arisen from them via gene fusion [31-34]. The fifth subunit of the five-subunit form appears to be unique to that form [32]. The PAOR complex contains 1–3 iron-sulfur clusters and thiamine pyrophosphate [35], which aid in the oxidative decarboxylation of pyruvate. In general, PAOR is oxygen sensitive, although the homodimeric PAOR from Desulfovibrio africanus is not [36]. The PAOR reaction is reversible because the cellular electron acceptors this complex uses have extremely low midpoint potentials (e.g., ferredoxin and flavodoxin; [32, 37, 38]. The reversibility of this enzyme lends it a degree of versatility [37]. For instance, in autotrophs that utilize the reductive citric acid cycle as their primary carbon fixation pathway, PAOR functions to carboxylate acetyl-coA [35]. In many anaerobes, the PAOR complex catalyzes the decarboxylation of pyruvate as the first step of the phosphoroclastic system, in which acetyl-coA that PAOR produces is converted to acetyl-phosphate via a phosphotransacetylase, which in turn can be used to phosphorylate ADP via substrate-level phosphorylation [32, 37]. Furthermore, sulfate reducers, nitrogen fixers and acetogenic bacteria can use the extremely low-potential electrons produced by the PAOR complex for a variety of biosynthetic processes [37]. The third complex, pyruvate formate lyase (PFL), is found in many organisms

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10 growing anaerobically and aids in fermentative glucose metabolism [39]. It is a homodimer and, like PAOR, catalyzes a reversible decarboxylation of pyruvate. Unique to PFL, a glycyl radical intermediate is used, and the one-carbon unit removed from pyruvate is released as formate instead of carbon dioxide. Therefore, unlike both PAOR and PDH, no reduced cellular electron carrier (e.g., NADH or ferredoxin) is produced [40, 41]. This oxygen-sensitive enzyme is particularly useful for organisms growing without an ETC, as it does not produce reduced electron carriers. 1.5.2 Acetyl-coA and Oxaloacetate to Citrate. The condensation of acetyl-coA and oxaloacetate to form citrate is unique as it is the only reaction in the CAC that results in the formation of a carbon-carbon bond [42]. Two physiologically dissimilar enzymes catalyze this irreversible reaction: the ubiquitous si-citrate synthase (siCS) and the less common re-citrate synthase (reCS) [43]. The prefixes refer to the stereospecificity of the carboxymethyl group of oxaloacetate to which the acetyl-coA is incorporated [43]. Two forms of siCS exist: Type 1 and Type 2. In general, Type 1 is dimeric, regulated by ATP, and prevalent among eukaryotes, archaea, and gram-positive bacteria, whereas Type 2 is hexameric and common among the gram-negative bacteria [44-46]. Most Type 2 siCS enzymes are allosterically inhibited by NADH [44]. However, cyanobacterial siCS enzymes are insensitive to NADH [47-49], as is that of Acetobacter aceti [50]. This irreversible enzyme differs structurally across domains of life, but studies suggest that all forms (Types 1 & 2) have similar active sites [44, 50]. Due to its oxygen insensitivity, it could be useful for aerobic organisms that have an ETC for ATP generation. Facultative anaerobes allosterically inhibit the activity of this enzyme when

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11 growing anaerobically to prevent excess NADH formation [50]. Although the subunit composition and molecular mass have not yet been reported in reCS, the unusual stereospecificity of its reaction, need for Mn+2 or Co+2, and sensitivity to oxygen make it quite distinct from siCS [43]. These characteristics restrict this enzyme to anaerobes such as Clostridia and Desulfovibrio [43]. 1.5.3 Citrate to Isocitrate. The interconversion of citrate to isocitrate proceeds via an intermediate dehydration product (cis-aconi tate) [51]. This reversible isomerization reaction is catalyzed by aconitase [52], of which three forms exist. Aconitase A (AcnA) and aconitase B (AcnB) are distantly related iron-sulfur proteins [53]. AcnA is relatively oxygen-stable, while AcnB is oxygen-sensitive [53, 54]. Some organisms have genes encoding both enzymes, and express them under conditions reflecting their oxygen sensitivity [53, 54]. A third form, aconitase X (AcnX), was predicted via comparative genomics [55]. In some species of Archaea, genes encoding aconitase A and B are absent, but some form of aconitase must be active for the cells to be viable. A bioinformatics-informed examination of their genomes uncovered a gene predicted to bind citrate that was distantly related to AcnA and AcnB [55]. Ca talytic activity by aconitase X has yet to be measured. 1.5.4 Isocitrate to 2-oxoglutarate. The interconversion of isocitrate to 2oxoglutarate is catalyzed by isocitrate dehydrogenase (IDH), a reversible enzyme. Thus far, only NADP-dependent IDH’s have been det ected in bacteria [56], and of these there are two forms: monomeric and multimeric. The monomeric IDH is approximately 80-

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12 100kDa, while the multimeric form (usually found as a dimer) is approximately 4075kDa [56, 57]. Analyses of the sequences of these two types reveal conserved primary structures [57]. 1.5.5 2-oxoglutarate to Succinyl-coA. The interconversion of 2-oxoglutarate to succinyl-coA can be catalyzed by two enzyme complexes. One enzyme that can accomplish this feat is the irreversible 2-oxoglutarate dehydrogenase (2-OGDH) enzyme. It belongs to a family of multienzyme complexes that includes PDH [58]; accordingly, they possess an E1, E2, and E3 subunit with the structural and catalytic core in E2 [5860]. These subunits can be found in a gene cluster, although the E3 subunit can be found in other operons. Gene sequences encoding the subunits of 2-OGDH do not appear to diverge from eachother as substantially as those encoding PDH [59]. The other complex capable of converting oxoglutarate to succinyl-coA is the 2oxoglutarate: acceptor oxidoreducatase (OAOR). Unlike 2-OGDH this enzyme is reversible [61]. This complex is homologous to PAOR, and is similarly sensitive to oxygen [62] but occurs in only three forms: two subunit, four subunit, and five subunit [63, 64]. 1.5.6 Succinyl-CoA to Succinate. The interconversion of succinyl-coA to succinate is a reversible reaction catalyzed by the succinyl-coA synthetase enzyme, which couples the removal of coA to the phosphorylation of ADP [65]. Due to the reversible nature of this enzyme it can also function in the other direction for anabolic purposes [65]. This enzyme exists as a heterotetramer of two different subunits, and [66], which are usually found in an operon. The -subunit binds ATP and catalyzes

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13 phosphoryl group transfer, while the -subunit contains at least part of the coA and succinate binding sites [67]. 1.5.7 Succinate to Fumarate. The interconversion of succinate to fumarate is catalyzed by enzymes generally referred to as the succinate dehydrogenases/fumarate reductases (Sdh-Frd). These enzymes either oxidize succinate to fumarate, and transfer the electrons to quinone [68], or do the reverse reduction/oxidation. They consist of two hydrophilic subunits (A and B) as well as membrane-spanning hydrophobic subunits (C and D). Subunits A and B contain the catalytic core of the enzyme; subunit A carries a covalently linked FAD, and subunit B has three Fe-S clusters [69, 70]. Subunits C and D transfer electrons to, or accept electrons from, the membrane quinone/quinol pool [69, 70]. There are five types of this enzyme complex (A-E) which can be distinguished by their C and D subunits, which vary in their prosthetic groups and copy number per complex [70]. Types A, B, and C contain 2, 2, and 1 haem groups respectively. Types A, C, D, and E have two anchor subunits, while B has only one. The anchor subunits from type E complexes are particularly unusual, as they resemble heterodisulphide reductase from methanogenic archaea [68]. A phylogenetic analysis of the A and B subunits is consistent with clustering these complexes based on their C and D subunit structure [70]. The five types of Sdh-Frd complex are also distinguished by their catalytic directionality. Types A and E act as irreversible succinate dehydrogenases, while Type D is only capable of catalyzing fumarate reduction. Types B and C can operate bidirectionally [68]. Organisms with different physiologies tend to have (Sdh-Frd) complexes that are

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14 consistent with their lifestyles. For example, E. coli which is a facultative anaerobe, expresses two different forms of Sdh/Frd; one during aerobic growth for succinate oxidation (Type C), and the other during anaerobic growth for fumarate reduction (Type D) [71]. 1.5.8 Fumarate to Malate. Fumarase catalyzes the reversible interconversion of fumarate and malate. Two classes of fumarase enzymes have been described so far (I and II) [72]. Dimeric class I fumarases are oxygen sensitive, heat inactivated, and irondependent, whereas tetrameric class II fumarases are iron-independent and thermo-stable [72]. Accordingly, oxygen sensitivity restricts class I to anaerobes, while aerobes possess class II fumarases [73-77]. Facultative anaerobes Bacillus stearothermophilus, E. coli and Bradyrhizobium japonicum all possess genes encoding both forms of this enzyme [78]. In E. coli, class II fumarase is expressed as part of the soxR regulatory system, which operates during oxidative stress to maintain cellular redox balance [78]. 1.5.9 Malate to Oxaloacetate. The interconversion of malate and oxaloacetate acts to replenish the oxaloacetate necessary to complete the citric acid cycle. Oxaloacetate formation from malate can be carried out in bacteria by two enzymes: malate dehydrogenase (MDH) and malate: quinone oxidoreductase (MQO). MDH is a cyctoplasmic enzyme while MQO is membrane associated [79]. MDH is a multimeric enzyme that can be present as dimers or tetr amers of identical subunits [80]. This enzyme couples malate oxidation to NAD reduction, and is reversible [81], enabling it to play a role in the wishbone-shaped citric acid pathway [82]. MQO, which also has a single subunit, uses quinones instead of NAD as the

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15 electron acceptor [79, 81]. These quinones are subsequently oxidized in the ETC [81]. Unlike MDH, malate oxidation catalyzed by MQO is irreversible, as the relatively positive midpoint potential of the quinols produced preclude the reverse reaction under physiological conditions [83]. 1.6 The Citric Acid Cycle in T. crunogena Based on the initial genome annotation, the T. crunogena genome encodes enzymes that could catalyze a complete citric acid cycle [1]. Particularly surprising was the presence of 2-oxoglutarate oxidoreductase genes as well as those encoding malate: quinone oxidoreductase. A citric acid cycle constructed from the enzymes encoded by these genes would be ‘locked’ in the oxidative direction by malate: quinone oxidoreductase, as the transfer of electrons from malate to quinones (ubiquinone for T. crunogena) is irreversible under physiological conditions due to the relatively positive midpoint potential of quinones [83]. T. crunogena also lacks pyruvate carboxylase and phosphoenolpyruvate carboxylase genes, leaving malate oxidation as the only route available in this organism for oxaloacetate biosynthesis [1]. Consequently, the biosynthesis of this key metabolic intermediate requires three oxidative decarboxylations (via pyruvate dehydrogenase, isocitrate dehydrogenase, and oxoglutarate dehydrogenase), which seems rather inefficient for an obligate autotroph, which expends considerable cellular energy fixing and reducing carbon via the Calvin-Benson-Bassham cycle.

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16 1.7 Purpose The purpose of this study was to verify and expand upon the results from the initial annotation of the T. crunogena genome. The genome was exhaustively searched for enzymes encoding each step of the citric acid cycle. Genes predicted to encode CAC enzymes were scrutinized via alignments, cluster analysis, and genome context to strengthen function predictions (and verified, when particularly surprising, via enzyme assays: citrate synthase allosteric regulation; presence of OAOR and MQO; absence of MDH). To examine the possible acquisiti on of genes via horizontal gene transfer, placement of T. crunogena CAC genes within phylogenetic trees was compared to T. crunogena’s 16S-based phylogeny. Lastly, the citric acid cycle assembled from these genes was compared to the citric acid cycles encoded by the genomes of 340 proteobacteria to determine whether any other organism has a CAC similar to the peculiar CAC present in T. crunogena.

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17 Chapter 2 Methods 2.1 Finding Citric Acid Cycle Genes in Proteobacteria In order to strengthen predictions of CAC gene function in T. crunogena, as well as to elucidate patterns of CAC gene presence and absence among a phylogenetically and physiologically diverse group of bacteria, complete genomes of 340 Proteobacteria present in the IMG database (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi), were searched for genes encoding enzymes catalyzing steps of the citric acid cycle. Several tools present at the IMG website were used to query each genome. BLAST searches were undertaken using the amino acid sequences of biochemically characterized enzymes as query sequences (Appendix 1). The list of sequence matches (‘target genes’) resulting from these BLAST searches were then supplemented with sequences retrieved via searches of the target genomes for members of the appropriate COG (Clusters of Orthologous Groups of Proteins, Appendix 1) and KEGG pathways (Kyoto Encyclopedia of Genes and Genomes). Genomes were also queried using E.C. numbers and keyword searches. For multisubunit enzymes, the genes encoding the largest, most informative subunits were used for BLAST and COG-based queries of IMG. For those Thiomicrospira crunogena genes that did not fall clearly within the Gammaproteobacteria a BLAST-based query of all genomes (draft and complete; bacterial, archaeal, and eukaryotic) was undertaken to find genes most closely related to those from T. crunogena

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18 2.2 Removing Genes with Apparent Alternative Function Paralogous genes with apparent alternative function were removed from the list of target genes based on genome context, phylogenetic cluster analysis, and protein size. Gene context was visualized using the “View Neighborhood” function in IMG, and surrounding genes in an apparent operon were examined to determine whether the target gene might play a role outside of the citric acid cycle (Appendix 2). Genes were also vetted via phylogenetic cluster analysis. Amino acid sequences predicted from paralogous genes with alternative function were aligned with those predicted from target genes and biochemically characterized genes using the Clustal W program implemented in MEGA 4.0 (Molecular Evolutionary Genetics Analysis software, [14]). A neighbor-joining tree [14] was then constructed by MEGA 4.0 using default parameters, and target genes were cl assified as likely to have an alternative function if they clustered with the paralogous genes of alternative function. Predicted protein size was also used to identify pseudogenes. Genes predicted to encode “oversized” proteins were scrutinized to determine whether they might be fusion proteins in multisubunit enzymes or bifunctional enzymes. 2.3 Multiple Sequence Alignment and Phylogenetic Analysis Amino acid sequences predicted from the vetted genes were re-aligned via Clustal W implemented in MEGA 4.0 (Blosum62 matrix, default parameters). Alignments were verified by checking them for aligned active site residues (Appendix 3). To further verify the alignments, alignment logos were generated from them using Weblogo

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19 (http://weblogo.berkeley.edu/, [84]), and were compared with curated PFAM HMM logos (http://pfam.sanger.ac.uk/, [85]) to ensure that other conserved regions besides active site residues were appropriately aligned (Appendix 4). For multisubunit enzymes, alignments for each subunit were generated, and then concatenated. The neighbor-joining method was then used to construct phylogenetic trees in MEGA 4.0 from each alignment. Bootstrap values for each clade resulted from resampling the alignments 1000 times [14]. To maximize the number of sites available for analysis and account for gaps and missing data, pairwise deletion was used [14]. When possible, paralogous genes with apparent alternative function were used as outgroups. If this was not possible, orthologous genes from distant relatives were used instead. Apparent horizontal gene transfer events were identified as incongruences between the trees generated from enzyme-e ncoded genes and those generated from 16S sequences. To highlight patterns of citric acid cycle enzyme presence/absence among Proteobacteria, as well as to identify possi ble instances of horizontal gene transfer, phylogenetic trees of 16S genes were constructed. 16S rRNA sequences were collected from IMG and aligned via RNACAD, implemented via RDP 2 (http://rdp.cme.msu.edu/), version 10, using a model for secondary structure based on bacterial 16S [86]. Neighborjoining trees were constructed with MEGA 4.0, with bootstrap values based on 1000 replicate samplings of the alignments, pairwise deletions for gaps and missing data, a maximum composite likelihood model, and the assumption of uniform rates among sites.

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20 2.4 Enzyme Assays Enzyme activities were measured in T. crunogena, as well as Escherichia coli when warranted as a control. T. crunogena was grown in thiosulfate-supplemented artificial seawater medium in 5L chemostats under ammonia limitation [2, 10]. E. coli cells were cultivated in Luria broth supplemented with 50 mg/L kanamycin. Cells from both species were harvested via centrifugation (5000 g, 4C, 10 min), and stored at -80C until use. Citrate synthase activity and allosteric inhibition were measured in cell extracts prepared from T. crunogena and E. coli. Wild-type E. coli have two enzymes capable of condensing oxaloacetate and acetyl-coenzyme A: citrate synthase, as well as methylcitrate synthase [87]. In order to prevent any methylcitrate synthase, which can also catalyze the citrate synthase reaction, from interfering with the assay, the E. coli strain used, JW0324-1 ([88]; obtained from the E. coli Genetic Stock Center at Yale University), has its methylcitrate synthase gene interrupted with a kanamycin resistance cartridge. To prepare cell extracts, frozen cell pellets were thawed and suspended in 5ml PBS (0.5 M NaCl, 0.01 M Na2HPO4, pH 7.5) supplemented with lysozyme (1 mg/ml). B-PER II detergent (5 ml; Thermo Scientific) was mixed in to the suspended cells, and the lysate was sonicated with glass beads on ice with 15 sec pulses until viscousity was reduced. Cell lysate was then centrifuged (10,000 g, 4C, 15 min) to remove cell debris. To remove cofactors, supernatant (=cell extract) was desalted using PD-10 columns (GE Healthcare) equilibrated with PBS. Citrate synthase activity was measured spectrophotometrically at 412 nm via 5-thio-2-nitrobenzoic acid (DTNB) reduction, using a

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21 citrate synthase assay kit (Sigma-Aldrich, Inc.) as per the manufacturer’s recommendations, with measurements every 60 sec over a 5 min timecourse. Potential allosteric affectors were added to the assay as necessary (ATP: 1 mM, 10 mM; NADH: 2.5 mM; 5 mM; 10 mM; oxoglutarate: 1 mM; 10 mM). Substrate-free control reactions (lacking either oxaloacetate or acetyl-coenzyme A) were also run to uncover any background DTNB reduction present in the cell extracts. To prepare cell extracts for oxoglutarate: acceptor oxidoreductase (OAOR) activity measurements, frozen T. crunogena and E. coli cell pellets were resuspended in 5 ml assay buffer (100 mM Tris/HCl, 4 mM dithioerthritol, 5 mM MgCl2, pH 7.8; Hugler et al., 2003) supplemented with 1 mg/ml lysozyme. Suspended cells were refrozen for 20 min at -80C, thawed under a stream of N2 gas, and sonicated and centrifuged as described above for citrate synthase assays. Assay conditions were similar to [89], but OAOR activity was measured in the reducing, rather than the oxidizing, direction via 14CO2 incorporation. Assay buffer was supplemented with methylviolagen (4 mM) to act as an electron donor, and 0.9 ml portions were injected into 2 ml glass autosampler vials sealed with gastight silicon septa. Cell-free extract (0.1 ml) and succinyl-coenzyme A (to test for OAOR activity) or acetyl-coenzyme A (to test for pyruvate: acceptor oxidoreductase activity) were added to a concentration of 1 mM. Vial headspace was sparged three times for 20 sec with N2 gas, and dithionite was injected to a final concentration of 0.5 mM to reduce the methylviolagen and remove dissolved O2. Reactions were begun by injecting 0.01 ml H14CO3 (55mCi/mmol). Samples of 0.2 ml were removed at 1 min intervals over a 4 min timecourse and injected into scintillation

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22 vials primed with 0.2 ml glacial acetic acid to halt the reaction and remove H14CO3. After sparging each sample overnight with air, 3 ml scintillation cocktail was added and oxoglutarate was quantified via scintillation counting. To measure malate: quinone oxidoreductase (MQO) activity, T. crunogena cells were fractionated to generate a cytoplasmi c and membrane fraction, as MQO, while not an integral membrane protein, is membrane-associated (e.g., [90-92]). Frozen T. crunogena cells were thawed in 10 ml MQO sonication buffer (50 mM HEPES, 10 mM potassium acetate, 10 mM CaCl2, 5 mM MgCl2, pH 7.5; [91]) supplemented with 1 mg/ml lysozyme. Cells were frozen, thawed, and sonicated as described above for the OAOR assay. Lysate was gently centrifuged to remove cell debris (6000 g, 4C, 30 min). Supernatant was then centrifuged to pellet the membranes (75,000 g, 4C, 1 hr). The high-speed supernatant was removed, and the pellet (=membrane fraction) was resuspended in 10 ml MQO sonication buffer, washed twice, and resuspended in 0.5 ml 0.05 mM HEPES, 10 mM CaCl2, 5 mM MgCl2, pH 7.5. To prepare to assay MQO activity spectrophotometrically via dichlorophenolindophenol (DCPIP) reduction, 0.95 ml assay buffer (100 mM K2HPO4, 10 mM KCN, 1 mM phenazine methosulfate,0.1 mM 0.1 mM DCPIP, 10 M FAD, 50 M ubiquinone) was added to a cuvette, followed by 0.05 ml membrane fraction. Malate was added to a final concentration of 10 mM to commence the reaction, and the A600 was monitored over 3 min timecourse [90, 91]. Reactions were also conducted in the absence of malate, to measure any background DCPIP reduction that might be present in the membrane fraction. Malate dehydrogenase activity was assayed on T. crunogena and E. coli cell

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23 extracts prepared as for OAOR activity. Enzyme activity was assayed spectrophotometrically by measuring malate-stimulated NADH oxidation at 340 nm [92]. All enzyme activities were normalized to the protein concentration present in the assay. Protein concentrations were quantified via the RC DC Protein Assay (Biorad, Hercules, CA).

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24 Chapter 3 Results and Discussion T. crunogena has a unique CAC (Table 1; Appendix 5), unlike the “canonical” cycles of the six classes of Proteobacteria (Table 1; Appendix 6-10), which were constructed based on the most common enzyme(s) catalyzing each step in each class. Furthermore, no individual bacterial species sampled here had a CAC similar to that present in T. crunogena. Genes found in each individual genome are presented in Appendices 11 – 18. Table 1: Canonical Citric Acid Cycle Enzymes of Proteobacterial Classes Steps of the CAC T.crunogena GammaProteobacteria BetaProteobacteria AlphaProteobacteria EpsilonProteobacteria Delta-, AcidoProteobacteria Pyruvate to Acetyl-coA Homodimeric PDH Homodimeric PDH Homodimeric PDH Heterodimeric PDH 4-subunit & Single subunit PAOR Heterodimeric PDH & Single subunit PAOR Acetyl-coA, Oxaloacetate to Citrate siCitrate Synthase Type 2 siCitrate Synthase Type 2 siCitrate Synthase Type 2 siCitrate Synthase Type 2 siCitrate Synthase Type 2 siCitrate Synthase Type 2 Citrate to Isocitrate Aconitase B Aconitase A & B Aconitase A & B Aconitase A Aconitase B Aconitase A & B Isocitrate to Oxoglutarate Monomeric IDH Monomeric & Multimeric IDH Monomeric & Multimeric IDH Multimeric IDH Monomeric IDH Multimeric IDH Oxoglutarate to Succinyl-coA 2-subunit OAOR 2-OGDH 2-OGDH 2-OGDH 4-subunit OAOR 2-OGDH & 4-subunit OAOR Succinyl-coA to Succinate Succinyl-coA Synthetase Succinyl-coA Synthetase Succinyl-coA Synthetase Succinyl-coA Synthetase Succinyl-coA Synthetase Succinyl-coA Synthetase Succinate to Fumarate SDH-FRD Type C SDH-FRD Type C & D SDH-FRD Type C SDH-FRD Type C SDH-FRD Type B & E SDH-FRD Type B Fumarate to Malate Fumarase Class II Fumarase Class I & II Fumarase Class I & II Fumarase Class II Fumarase Class II Fumarase Class I & II Malate to Oxaloacetate MQO MDH & MQO MDH MDH MDH & MQO MDH To simplify, the steps of the CAC in T. crunogena will be discussed in two parts; steps 1 – 4 (pyruvate to oxoglutarate) will be discussed first, followed by steps 5 – 9 (oxoglutarate to oxaloacetate).

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25 3.1 Steps 1 – 4: Pyruvate to Oxoglutarate In T. crunogena the conversion of pyruvate to acetyl-coA is likely to be catalyzed by homodimeric PDH, as genes encoding this enzyme were present in the genome, but none of the others were (e.g., heterodimeric PDH, PFL, PAOR). Genes encoding homodimeric PDH are present in many other Gammaproteobacteria (Appendix 11). The top hits resulting from a BLAST-based query of IMG using the T. crunogena PDH E1 subunit gene were all gammaproteobacterial sequences. In addition, phylogenetic analysis places the T. crunogena gene within a clade with other members of Gammaproteobacteria (Appendix 19), and the overall topology of the tree is similar to the 16S tree, suggesting that the genes encoding this enzyme have primarily been vertically transmitted within the Proteobacteria. Interestingly, homodimeric PDH is also widely distributed among the Betaproteobacteria but only a few representatives of this enzyme were found amongst members of the Alpha-, Delta-, or Epsilonproteobacteria, or Acidobacteria (Appendix 12, 13, 14), which suggests that homodimeric PDH may have been acquired by the shared ancestor of the Beta and Gammaproteobacteria T. crunogena has a gene encoding a Type II siCS to catalyze the condensation of acetyl-coA and oxaloacetate to form citrate, and has moderate activities of citrate synthase in cell extracts (Table 2). However, it does not appear that the Type II gene present in the T. crunogena genome shares a long history within the Gammaproteobacteria A BLAST-based query of IMG using the amino acid sequence predicted from the T. crunogena Type II siCS gene had top hits dominated by members of the phyla Cyanobacteria and Aquificales Consistent with the distribution of these top hits,

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26 phylogenetic analysis placed the T. crunogena gene, as well as those from several other chemolithoautotrophic and methylotrophic bacteria, within a clade dominated by cyanobacteria (Fig. 6). Furthermore, this clade was outside of the clade of proteobacterial Type II CSs, which is consistent with acquisition of this gene within the lineage leading to T. crunogena via horizontal transfer, perhaps from a member of the Cyanobacteria A possible selective advantage resulting from the acquisition of a cyanobacterial-like siCS gene is suggested from patterns of allosteric regulation of cyanobacterial citrate synthases. In these organisms, type II siCS enzymes are insensitive to NADH [48, 49]. This insensitivity is a boon to organisms that use the citric acid cycle primarily for biosynthetic purposes, as inhibition by excess cellular reductant is counterproductive under these circumstances. Indeed, T. crunogena citrate synthase was found to be insensitive to NADH (Fig. 7), as well as ATP and oxoglutarate (data not shown). Table 2: Activities of Citric Acid Cycle Enzymes Measured in T. crunogena Activities (mol/min mg protein) Enzyme Fraction + substrate substrate* Citrate synthase Cell-free extract 0.41 0.01 Oxoglutarate: acceptor oxidoreductase Cell-free extract 2.3 E-05 0.5 E-05 Malate: quinone oxidoreductase Membrane 0.19 0.01 Malate dehydrogenase Cell-free extract ND** N/A*** *Negative controls in which substrate was omitted were included in each assay. For citrate synthase assay, the omitted substrate was oxaloacetate. Similar results were measured in the absence of acetyl-coA. For OAOR, the omitted substrate was succinyl-coA. For MQO and MDH, the omitted substrates were malate and oxaloacetate, respectively. **ND=none detected. Assays of E. coli cell-free extract had activities of 8 mol/min mg protein. *** N/A = not applicable.

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27 Figure 6: Phylogeny of si-citrate synthase sequences with 2-methylcitrate synthase as the outgroup. Branches in blue indicate chemolithoautotrophs, while green branches indicate cyanobacteria. The red dot indicates T. crunogena while the branches in green indicate cyanobacteria. Type 1 Citrate Synthase Type 2 Citrate Synthase 637464909 Photorhabdus luminescens su... 56 22 43 54 98 Alpha-, Beta-, and Deltaproteobacteria. Alpha-, Beta-, Gammaand Epsilonproteobacteria.

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28 Figure 6 (continued): 643741788 Sulfurihydrogenibium azoren... 643744047 Persephonella marina EX-H1 642657486 Sulfurihydrogenibium sp. YO... 642222754 Gemmata obscuriglobus UQM 2... 639758686 Candidatus Ruthia magnifica... 640538426 Candidatus Vesicomyosocius ... 637785059 Thiomicrospira crunogena XCL-2 637739109 Nitrosococcus oceani ATCC 1... 643531416 Acidithiobac illus ferrooxid... 644408215 Halothiobac illus neapolitan... 637460416 Gloeobacter violaceus PCC 7421 637876306 Synechococcus sp. JA-2-3Ba(... 637440583 Prochlorococcus marinus sub... 637448737 Prochlorococcus marinus str... 642882935 Cyanothece sp. PCC 7822 unf... 643482314 Cyanothece sp. PCC 7424 642897453 Cyanothece sp. PCC 8802 unf... 637011760 Synecho cystis sp. PCC 6803 641612633 Synechococcus sp. PCC 7002 641251255 Acaryochloris marina MBIC11017 641253084 Acaryochloris marina MBIC11017 637315038 Thermosynechococcus elongat... 643584664 Cyanothece sp. PCC 7425 638106723 Trichodesmium erythraeum IM... 640015187 Lyngbya sp. PCC 8106 unfini... 643167709 Arthrospira maxima CS-328 u... 637799023 Synechococcus elongatus PCC... 644370569 Nostoc azollae 0708 unfinis... 637230580 Nostoc sp. PCC 7120 637718058 Anabaena variab ilis ATCC 2 9413 640028055 Nodularia spumigena CCY9414... 642604547 Nostoc punctiforme PCC 73102 Q8ZWP2 Pyrobaculum aerophilum* Q9WYC6 Thermotoga maritima* 637984292 Acidobacteria bacterium Ell... 639684321 Solibacter usitatus E llin6076 637481228 Bdellovibrio bacteriovorus ... 641263660 Candidatus Desulfococcus ol... 639702872 Syntrophobacter fumaroxidan... 639702299 Syntrophobacter fumaroxidan... 637483028 Bdellovibrio bacteriovorus ... 2-methylcitrate synthase 100 100 100 99 41 82 100 100 100 42 48 33 72 44 33 82 56 15 17 100 16 96 100 96 60 99 100 34 74 83 68 59 60 53 72 88 94 51 93 34 0.2

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29 Figure 7: Effect of NADH on the activity of citrate synthase from T. crunogena and E. coli 0 0.5 1 1.5 2 0246810Citrate synthase activity ( mol/mg protein min)NADH (mM) T. crunogena E. coli

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30 A gene encoding the more oxygen-sensitive version of aconitase, AcnB, is present in the T. crunogena genome, suggesting that this enzyme is used to catalyze the conversion of citrate to isocitrate, as is the case for many Gamma-, Betaand Epsilonproteobacteria (Appendix 11, 12, 14). T. crunogena lives in a microaerophilic environment, and is likely to be subject to lower levels of oxidative stress. AcnA is more resistant to oxidative damage; under the low-oxygen conditions under which T. crunogena grows, the higher catabolic efficiency of AcnB may provide more of a selective advantage [53]. Similarly to siCS, a BLAST-based query of IMG using the amino acid sequence predicted from the T. crunogena AcnB gene had top hits primarily from the cyanobacteria and falls among sequences of other chemolithoautotrophs and methylotrophs. A phylogenetic analysis clustered the T. crunogena gene separate from a clade dominated by cyanobacteria (Fig. 8); both clades however were nested deep within the clade of proteobacterial AcnB. Thus, a di rect link of descent cannot be established between the T. crunogena gene and the cyanobacterial genes due to low statistical significance (poor bootstrap values) and because the clade was not separate from the clade of proteobacterial AcnB.

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31 Acn A Acn A 100 57 53Figure 8: Phylogeny of aconitase sequences, with isopropylmalate synthase as the outgroup. Branches in blue indicate chemolithoautotrophs, while branches in orange indicate methylotrophs, and green branches indicate cyanobacteria. The red dot indicates T. crunogena Gamma-, beta-, and deltaproteobacteria. Gammaproteobacteria.

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32 641348188 Sorangium cellulosum So ce 56 Alphaproteobacteria 639852103 B Verminephrobacter eisenia... 641263600 B Candidatus Desulfococcus ... Deltaproteobacteria 640026969 Nodularia spumigena CCY9414... 642601132 Nostoc punctiforme PCC 73102 644374051+644374052 Nostoc azollae 0708 637231633 Nostoc sp. PCC 7120 637715897 Anabaena variabilis ATCC 29413 638105963 Trichodesmium erythraeum IM... 641250733 Acaryochloris marina MBIC11017 643586182 Cyanothece sp. PCC 7425 637875987 Synechococcus sp. JA-2-3Ba(... 637313308 Thermosynechococcus elongat... 637799320 Synechococcus elongatus PCC... 637012460 Synechocystis sp. PCC 6803 641611693 Synechococcus sp. PCC 7002 642884700 Cyanothece sp. PCC 7822 unf... 643171773 Arthrospira maxima CS-328 u... 640020093 Lyngbya sp. PCC 8106 unfini... 642897877+642897878 Cyanothece sp. PC... 637442303 Prochlorococcus marinus sub... 637448966 Prochlorococcus marinus str... 637938733 B Methylobac illus flagellat... 641096998 Beggiatoa sp. PS unfinished... 644407455 Halothiobac illus n eapolitan... 637738578 B Nitrosococcus oceani ATCC... 637785863 B Thiomicrospira crunogena ... 639722308 B Magnetococcus sp. MC-1 637171626 B Methylococcus capsulatus ... Gamma-, Betaproteobacteria Gammaproteobacteria 100 99 26 41 94 20 23 42 79 40 35 48 23 76 23 63 88 12 70 13 13 36 48 60 46 94 99 5 21 6 4 9 5 21 100 100Figure 8 (continued): Aconitase B clade

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33 639758611 B Candidatus Ruthia magnifi... 640538351 B Candidatus Vesicomyosociu... Epsilonproteobacteria 3-isopropylmalate dehydrogenase 100 84 6 100 0.2Figure 8 (continued):

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34 A gene encoding monomeric IDH is present in the T. crunogena genome, whose product could catalyze the interconversion of isocitrate to 2-oxoglutarate. An abundance of gammaproteobacterial top hits from a BLAST search of IMG, as well as phylogenetic analysis (Appendix 20), are consistent with vertical transmission within the Gammaproteobacteria 3.2 Steps 5 – 9: Oxoglutarate to oxaloacetate Unlike most Gammaproteobacteria in which OGDH catalyzes the conversion of 2-oxoglutarate to succinyl-coA, in T. crunogena, genes encoding a 2-subunit OAOR are present (Appendix 15). Indeed, a BLAST query of IMG using the T. crunogena OAOR alpha and beta subunit amino acid sequence produced top hits dominated by members of the phylum Firmicutes and phylogenetic analysis of the concatenated alpha-beta subunit sequence placed the T. crunogena gene apart from the clade containing most of the proteobacterial sequences. Instead, it falls within a small clade of proteobacterial facultative and obligate autotrophs ( Rhodospirillum rubrum, Alkali ehrlichei, Rhodopseudomonas palustris, Magnetospirillum magneticum ), with some support (60% bootstrap value) for inclusion in a clade with Firmicutes (Fig. 9). Given the rarity of OAOR genes among other proteobacteria (Appendices 16-18; Fig. 9) and the clustering of this gene near a clade of firmicutes, it is possible that the OAOR genes in T. crunogena are a recent acquisition from another lineage. It is interesting that T. crunogena an obligate autotroph, has an enzyme that catalyzes this step (Table 2) because many obligate autotrophs, such as cyanobacteria, use the wishbone pathway,

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35 Bacillus thuringiensis serovar tochig... Bacillus anthracis str. A 1055 A1055 Bacillus cereus MM3 MM3 Bacillus wei henstephanensis KBAB4 KBAB4 Bacillus mycoides DSM 2048 DSM 2048 Bacillus cereus Rock3-44 Rock3-44 Bacillus ps eudomycoides DSM 12442 DSM... Bacillus mycoides Rock3-17 Rock3-17 Bacillus cytotoxicus NVH 391-98 NVH 3... Anoxybacillus flavithermus WK1 WK1 Geobac illus sp. WCH70 WCH70 Geobac illus therm odenitrificans NG80-... Geobac illus sp. G11MC16 G11MC16 Geobac illus kaust ophilus HTA426 HTA426 Geobac illus sp. Y 412MC61 Y412MC61 Geobac illus sp. Y 412MC52 Y412MC52 Bacillus sp. NRRL B14911 NRRL B-14911 Bacillus sp. B 14905 B14905 Bacillus hal odurans C-125 C-125 Exiguobacterium sibiricum 255-15 255-15 Paenibac illus sp. JDR-2 JDR-2 Macrococcus caseolyticus JCSC5402 JCS... Staphylococcus carnosus subsp. carnos... Staphylococcus aureus subsp. aureus C... Staphylococcus saprophyticus subsp. A... Staphylococcus haemolyticus JCSC1435 ... Staphylococcus warneri L37603 L37603 Staphylococcus epidermidis ATCC 12228... Veillonella parvula DSM 2008 DSM 2008 Alkaliphilus oremlandii OhILAs OhILAs Natrialba magadii ATCC 43099 ATCC 43099 Halorhabdus utahensis DSM 12940 DSM 1... Haloarcula marismortui ATCC 43049 ATC... Halomicrobium mukohataei DSM 12286 DS... Halorubrum lacusprofundi ATCC 49239 A... Halogeometricum borinquense DSM 11551... Haloquadratum walsbyi DSM 16790 strai... Picrophilus torridus DSM 9790 DSM 9790 Thermoplasma acidophilum DSM 1728 DSM... Thermoplasma volcanium GSS1 GSS1 BAB21494.1 2-subunit Hydrogenobacter ... Sulfurihydrogenibium sp. YO3AOP1 YO3AOP1 639722491 Magnetococcus sp. MC-1: NC ... Chloroflexus aggregans DSM 9485 DSM 9485 Chloroflexus aurantiacus J-10-fl J-10-fl Chloroflexus sp. Y-400-fl Y-400-fl Opitutus terrae PB90-1 PB90-1 637786446 Thiomicrospira crunogena XC... 637826831 Rhodospir illum rubrum ATCC ... 638125446 Alka lilimnicola ehrlichei M... 637961727 Rhodopseudomonas palustris ... 639318228 Rhodopseudomonas palustris ... 637820757 Magnetospir illum m agneticum... Thauera sp. MZ1T MZ1T Thauera aromatica 637864563 Anaeromyxobacter dehalogena... 640844519 Anaeromyxobacter sp. Fw109-... 637982603 Acidobacteria bacterium Ell... 639687177 Solibacter usitatus E llin60... 100 80 100 100 100 100 100 100 100 100 96 94 93 51 100 77 59 68 100 100 100 100 91 57 92 60 32 61 66 56 92 68 91 100 100 97 100 100 91 100 100 100 100 99 64 86 97 41 57 95 100 100 99 100 86 99 100 80which lacks this step [22]. Figure 9: Phylogeny of 2-oxoglutarate: oxidoreductase sequences, with pyruvate: acceptor oxidoreductase as the outgroup. Firmicutes A rchaea P roteobacteria

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36 638139814 Maricaulis maris MCS10: NC ... 637528164 Desulfotalea p sychr ophila L... 640931916 Shewanella sediminis HAW-EB... 641632620 Shewanella woodyi ATCC 5190... 637609855 Azoarcus sp. EbN1: NC 006513 637627466 Azoarcus sp. EbN1 plasmid 2... 637917617 Rhodoferax ferrireducens T1... 640582781 Sphingomonas wittichii RW1:... 637374332 Bradyrhizobium japonicum US... 640523317 Bradyrhizobium sp. ORS278: ... 640561015 Bradyrhizobium sp. BTAi1: N... 637130888 Legionella pneumophila subs... 637579704 Legionella pneumophila str .... 637582754 Legionella pneumophila str .... 640570634 Legionella pneumophila str .... 637863500 Anaeromyxobacter dehalogena... 640843400 Anaeromyxobacter sp. Fw109-... 637609007 Azoarcus sp. EbN1: NC 006513 638125538 Alka lilimnicola ehrlichei M... 638009916 Sphingopyxis alaskensis RB2... 637856683 Erythrobacter litoralis HTC... 640444498 Novosphingobium aromaticivo... Natronomonas pharaonis DSM 2160 PAOR outgroup 100 44 73 100 100 100 85 100 100 100 100 69 82 100 80 100 100 100 91 98 91 100 100 64 0.2Figure 14 (continued): P roteobacteria

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37 Genes encoding succinyl-coA synthetase are present in the T. crunogena genome, suggesting that this enzyme catalyzes the interconversion of succinyl-coA to succinate in this organism. Genes encoding this enzyme are ubiquitous among proteobacteria (Appendices 15-18). Based on a BLAST search of IMG and phylogenetic analysis, both the alpha and beta subunit genes for this enzyme from T. crunogena cluster among succinyl-coA synthetase genes from other Gammaproteobacteria (Appendix 21), indicating that these genes were primarily vertically transmitted within the Gammaproteobacteria Like many proteobacteria (Appendices 15-18), the T. crunogena genome encodes the four subunits of a type C SDH-FRD, which could catalyze the interconversion of succinate to fumarate, as well as a class two fumarase, which is likely to convert fumarate to malate. SDH-FRD Type C, unlike SDH type A, D, or E, is a reversible enzyme [70] found in many Gammaproteobacterial facultative anaerobes (Appendix 15). SDH-FRD Type B, the other reversible enzyme catalyzing this step [70], is predominant among the microaerophiles in our sample. It is interesting that T. crunogena, which is an obligate microaerophile, does not follow this trend. A BLAST search of IMG using the sequences of alpha and beta subunits of SDHFRD Type C and fumarase, as well as phylogenetic analysis of the genes encoding both enzymes (using concatenated alpha and beta subunits for the SDH-FRD Type C) indicates that these sequences are most closely affiliated with those from other Gammaproteobacteria (Appendices 22-23), consistent with vertical transmission within the Gammaproteobacteria

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38 Based on gene presence and supported by enzyme assays, T. crunogena uses MQO, and not malate dehydrogenase, to convert malate to oxaloacetate (Table 2). Although this enzyme, which catalyzes the irreversible oxidation of malate to oxaloacetate via reduction of quinone [83], is common among the Gammaproteobacteria (Appendix 9), BLAST searches of IMG as well as phylogenetic analysis suggests relatively recent acquisition by the lineage leading to T. crunogena from the Epsilonproteobacteria where it is also widely distributed (Fig. 15; Appendix 12). Furthermore, this enzyme is not widely distributed among methylotrophic and autotrophic proteobacteria; the few that have it ( Methylobacterium extorquens, Methylobacterium radiotolerens, Methylobacillus flagellates, Rhodobacter palustris, Sulfurimonas denitrificans ) also have genes encoding malate dehydrogenase. Given the irreversibility of MQO, as well as the energy loss resulting from introducing electrons into the electron transport chain at the redox level of quinol instead of NADH, it is difficult to understand why this enzyme is the sole mechanism for malate-oxaloacetate interconversion in T. crunogena. Perhaps an irreversible reaction ensures that oxaloacetate is always reasonably abundant, which might be necessary to ensure that sufficient quantities of this key metabolic intermediate are available to fuel the unusually rapid growth of this organism.

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39 640585747 Pseudomonas putida F1 641549269 Pseudomonas putida GB-1 637144653 Pseudomonas putida KT2440 641623704 Pseudomonas putida W619 638002958 Pseudomonas entomophila L48 638001372 Pseudomonas entomophila L48 641547969 Pseudomonas putida GB-1 637146347 Pseudomonas putida KT2440 640587217 Pseudomonas putida F1 640817146 Janthinobacterium sp. Marse... 637741971 Pseudomonas fluorescens PfO-1 637319766 Pseudomonas fluorescens Pf-5 637488092 Pseudomonas syringae pv. ph... 637652733 Pseudomonas syringae pv. sy... 639290944 Agrobacterium tumefaciens s... 639296300 Agrobacterium tumefaciens s... 641367309 Methylobacterium extorquens... 641626067 Methylobacterium radiotoler... 637240674 Ralstonia solanacearum GMI1... 641683026 Burkholderia ambifaria MC40... 638152943 Burkholderia cepacia AMMD c... 637767798 Burkholderia sp. 383 chromo... 641651413 Burkholderia cenocepacia MC... 639695235 Burkholderia cenocepacia HI... 638018274 Burkholderia cenocepacia AU... 640798595 Klebsiella pneumoniae subsp... 640901963 Enterobacter sakazakii ATCC... 640799670 Klebsiella pneumoniae subsp... 640492976 Enterobacter sp. 638 640914531 Citrobacter koseri ATCC BAA... 638063389 Escherichia coli 536 637356589 Escherichia coli CFT073 637083715 Escherichia coli O157 637934195 Escherichia coli UTI89 640755152 Escherichia coli APEC O1 637064887 Escherichia coli O157 640433264 Shigella dysenteriae Sd197 637807798 Shigella boydii Sb227 641724916 Shigella boydii CDC 3083-94 640430017 Shigella sonnei Ss046 640921475 Escherichia coli HS 641600859 Escherichia coli ATCC 8739 640926167 Escherichia coli E24377A 641617232 Escherichia coli SECEC SMS-3-5 637015555 Escherichia coli K12 641607798 Escherichia coli str. K-12 ... P33940.2 Escherichia coli K-12 637002180 Escherichia coli W3110 DNA 637379546 Erwinia carotovora subsp. a... 640937056 Serratia proteamaculans 568 638002668 Pseudomonas entomophila L48 641546777 Pseudomonas putida GB-1 640504350 Pseudomonas mendocina ymp 640810728 Pseudomonas aeruginosa PA7 637053872 Pseudomonas aeruginosa PAO1 639324107 Pseudomonas aeru g inosa UCBP... 100 100 100 100 98 100 97 60 100 92 80 95 100 64 100 100 100 100 77 100 92 51 83 77 88 76 54 100 55 96 29 62 62 19 25 33 100 77 99 65 71 100 64 42 43 54 38 27 55Figure 10: Phylogeny of malate: quinone oxi doreductase sequences, with FAD dependent oxidoreductase and sarcosine oxidase as an outgroup.

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40 640153099 Acinetobacter baumannii ATC... 641591543 Acinetobacter baumannii AYE 637517558 Acinetobacter sp. ADP1 639822360 Acidovorax avenae subsp. ci... 637043391 Xylella fastidiosa 9a5c 641649872 Xylella fastidiosa M12 637384553 Xylella fastidiosa Temecula1 641703362 Xylella fastidiosa M23 637981361 Baumannia cicadellinicola s... 637853720 Sodalis glossinidius str. m... 637342002 Wigglesworthia glossinidia ... 637672983 Candidatus Blochmannia penn... 637443999 Candidatus Blochmannia flor... 637979761 Ralstonia metallidurans CH3... 637143181 Neisseria gonorrhoeae FA 1090 637197096 Neisseria meningitidis Z2491 641333839 Neisseria meningitidis 053442 637196713 Neisseria meningitidis MC58 639828834 Neisseria meningitidis FAM18 637456023 Wolinella succinogenes DSM ... 641336112 Gluconacetobacter diazotrop... 637128307 Haemophilus ducreyi 35000HP 640123098 Actinobacillus pleuropneumo... 641531466 Actinobacillus pleuropneumo... 637474465 Rhodopseu domonas palustris ... 641630572 Methylobacterium radiotoler... 641721484 Bordetella avium 197N 638128825 Granulobacter bethesdensis ... 641259515 Azorhizobium caulinodans OR... 637367827 Bradyrhizobium japonicum US... 637624819 Gluconobacter oxydans 621H 640471789 Polynucleobacter sp. QLW-P1... 637834282 Hahella c hejuensis KCTC 2396 639813377 Marinobacter aquaeolei VT8 640806382 Marinomonas sp. MWYL1 637936893 Methylobacillus flagellatus KT 639810325 Marinoba cter aquaeolei VT8 637967787 Chromohalobacter salexigens... AAW88350.1 Pseudomonas citronellolis 637055074 Pseudomonas aeruginosa PAO1 639327554 Pseudomonas aeruginosa UCBP... 640814335 Pseudomonas aeruginosa PA7 640487060 Pseudomonas stutzeri A1501 640504932 Pseudomonas mendocina ymp 637486629 Pseudomonas syringae pv. ph... 637392515 Pseudomonas syringae pv. to... 637651319 Pseudomonas syringae pv. sy... 637322469 Pseudomo nas fluorescens Pf-5 637744622 Pseudomonas fluorescens PfO-1 637999983 Pseudomonas entomophila L48 641624233 Pseudomonas putida W619 641545892 Pseudomonas putida GB-1 637144145 Pseudomonas putida KT2440 640585239 Pseudomonas putida F1 O69282.3 Corynebacterium glutamicum 637432768HelicobacterhepaticusATCC 100 100 85 100 100 49 69 76 100 100 90 99 100 61 57 100 100 100 56 100 100 99 100 74 38 30 79 76 55 100 87 99 86 95 100 98 56 55 99 83 93 90 99 82 55 33 31 24 88 26 11 6 5 18 93 72 50Figure 16 (continued):

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41 Figure 16 (continued): 637432768 Helicobacter hepaticus ATCC... 639811350 Marinobacter aquaeolei VT8 639660682 Candidatus Carsonella ruddi... O24913.1 Helicobacter pylori 637017819 Helicobacter pylori 26695 637022093 Helicobacter pylori J99 638019913 Helicobacter pylori HPAG1 638058756 Helicobacter acinonychis st... 637786616 Thiomicrospira crunogena XCL-2 640929999 Campylobacter concisus 13826 640871843 Campylobact... 637291800 Campylobacter jejuni RM1221 640941280 Campylobacter jejuni subsp.... 640862503 Campylobacter jejuni subsp.... 637039942 Campylobacter jejuni subsp.... 639853141 Campylobacter jejuni subsp.... 639746014 Campylobacter fetus subsp. ... 640871304 Campylobact... 640948903 Arcobacter butzleri RM4018 637793936 Thiomicrospira denitrifican... 640821590 Sulfurovum sp. NBC37-1 mqo outgroup 100 85 66 100 100 58 68 44 100 56 99 29 18 74 44 67 95 100 69 0.2 Epsilonproteobacteria Clade

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42 3.3 Conclusion Based on the analyses conducted here, T. crunogena does have a complete oxidative citric acid cycle, which is unexpected for an obligate autotroph. Although some of the genes (citrate synthase, aconi tase) cluster with other proteobacterial autotrophs, no other autotrophic proteobacterium genome sequenced thus far contains only the MQO gene for oxaloacetate formation, and it seems unlikely that reliance on MQO will be widespread among autotrophic proteobacteria, given its irreversibility as well as energy loss. Although complete, this oxidative CAC is quite different from the familiar one present in mitochondria, with two enzyme substitutions (OAOR, MQO) and evidence it was cobbled together via horizontal gene transfers of at least three of the nine steps. Furthermore, the six steps of the CAC catalyzed by enzymes whose amino acid sequences do cluster with those from other proteobacteria, do so with long branch lengths, which is likely to be due to Gammaproteobacteria related to T. crunogena being undersampled in genome sequencing efforts, and therefore, our study. It will be of great interest to determine whether any other members of the Gammaproteobacteria that are more closely related to T. crunogena have a similar cycle, or whether particular steps (NADH-insensitive citrate synthase, OAOR, MQO) are specific adaptations to its habitat or lifestyle.

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49 NADP+ complexes. Biochemistry, 1991. 30 (35): p. 8671-8. 96. Knapp, J.E., et al., Crystal structure of the truncated cubic core component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex. Journal of Molecular Biology, 1998. 280 (4): p. 655-668. 97. Wolodko, W.T., et al., THE CRYSTAL-STRUCTURE OF SUCCINYL-COA SYNTHETASE FROM ESCHERICHIA-COLI AT 2.5-ANGSTROM RESOLUTION. Journal of Biological Chemistry, 1994. 269 (14): p. 10883-10890. 98. Weaver, T., Structure of free fumarase C from Escherichia coli. Acta Crystallogr D Biol Crystallogr, 2005. 61 (Pt 10): p. 1395-401. 99. Madern, D., Molecular evolution within the L-malate and L-lactate dehydrogenase super-family. Journal of Molecular Evolution, 2002. 54 (6): p. 825840. 100. Sawers, G., The aerobic/anaerobic interface. Curr Opin Microbiol, 1999. 2 (2): p. 181-7. 101. Sawers, G. and G. Watson, A glycyl radical solution: oxygen-dependent interconversion of pyruvate formate-lyase. Mol Microbiol, 1998. 29 (4): p. 945-54. 102. Atteia, A., et al., Pyruvate formate-lyase and a novel route of eukaryotic ATP synthesis in Chlamydomonas mitochondria. J Biol Chem, 2006. 281 (15): p. 990918. 103. Hawkins, C.F., A. Borges, and R.N. Perham, A common structural motif in thiamin pyrophosphate-binding enzymes. FEBS Lett, 1989. 255 (1): p. 77-82. 104. Lessard, I.A. and R.N. Perham, Expression in Escherichia coli of genes encoding the E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex of Bacillus stearothermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro. J Biol Chem, 1994. 269 (14): p. 10378-83. 105. Green, J.D., et al., Three-dimensional structure of a lipoyl domain from the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. J Mol Biol, 1995. 248 (2): p. 328-43. 106. Russell, G.C. and J.R. Guest, Site-directed mutagenesis of the lipoate acetyltransferase of Escherichia coli. Proc Biol Sci, 1991. 243 (1307): p. 155-60. 107. Zhu, P.P. and A. Peterkofsky, Sequence and organization of genes encoding enzymes involved in pyruvate metabolism in Mycoplasma capricolum. Protein Sci, 1996. 5 (8): p. 1719-36. 108. Yu, X., et al., Structures of the human pyruvate dehydrogenase complex cores: a highly conserved catalytic center with flexible N-terminal domains. Structure, 2008. 16 (1): p. 104-14. 109. Arjunan, P., et al., Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 A resolution. J Mol Biol, 1996. 256 (3): p. 590-600.

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50 Appendices

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Appendix 1: Biochemically Characterized Genes of the Citric Acid Cycle Used as Query Sequences for BLAST Searches of IMG 51 Enzyme COG ID Species Genbank accession # Si-citrate synthase COG0372 Pyrobaculum aerophilum Q8ZWP2 Escherichia coli 1K3P_A Thermotoga maritima Q9WYC6 Pyrococcus furiosus 1AJ8_A Sulfolobus solfataricus 1O7X_A Sus scrofa P00889 Re-citrate synthase COG0119 Clostridium kluyveri DSM 555 YP_001394363 Aconitase A COG1048 Bacillus subtilis P09339 Mycobacterium avium O08451 Rattus norvegicus NP 077374 Aconitase B COG1049 Escherichia coli 1L5J_B Aconitase X COG1679, COG1786 Aeropyrum pernix NP_148375 Archaeoglobus fulgidus NP_071158 Pseudomonas aeruginosa NP_249950 Monomeric IDH COG0473 Azotobacter vinelandii 1J1W|A Multimeric IDH COG0538 Thermotoga Maritima 1ZOR|A Bacillus subtilis 1HQS|A Aeropyrum Pernix 1XGV|A 2-OGDH -E1 subunit COG0567 Pseudomonas putida AAC23516 Azotobacter vinelandii P20707 Bacillus subtilis P23129 Corynebacterium glutamicum Q8NRC3* -E2 subunit COG0508 Pseudomonas putida AAC23516 Azotobacter vinelandii P20707 Bacillus subtilis P23129 -E3 subunit COG1249 Stenotrophomonas maltophilia K279a CAQ46640 Leishmania major CAJ08475 2-subunit OAOR -subunit alpha COG0674 Hydrogenobacter thermophilus BAB21494 -subunit beta COG1013 Hydrogenobacter thermophilus BAB21495 4-subunit OAOR -subunit alpha COG0674 Helicobacter pylori AAC38211 Methanothermobacter marburgensis AAB85529 -subunit beta COG1013 Helicobacter pylori AAC38212

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Appendix 1 (continued): 52 Methanothermobacter marburgensis AAB85530 -subunit Gamma COG1014 Helicobacter pylori AAC38213 Methanothermobacter marburgensis AAB85531 -subunit delta COG1144 Helicobacter pylori AAC38210 Methanothermobacter marburgensis AAB85528 5-subunit OAOR -subunit alpha COG0674 Hydrogenobacter thermophilus BAB62133 -subunit beta COG1013 Hydrogenobacter thermophilus BAB62134 -subunit gamma COG1014 Hydrogenobacter thermophilus BAB62135 -subunit delta COG1144 Hydrogenobacter thermophilus BAB62136 -subunit epsilon Hydrogenobacter thermophilus BAB62132 Succinyl-coA -subunit alpha COG0074 Escherichia coli P0AGE9 Sus scrofa 1EUDA Trichomonas vaginalis P53401 -subunit beta COG0045 Escherichia coli P07460 Sus scrofa 2FP4_B Trichomonas vaginalis AAA30326 SDH-FRD Type A -subunit alpha COG1053 Halobacterium sp. NRC-1 NP_280171 Mycobacterium tuberculosis H37Rv NP_217835 Thermoplasma acidophilum DSM 1728 NP_394461 Archaeoglobus fulgidus DSM 4304 NP_069515 -subunit beta COG0479 Halobacterium sp. NRC-1 NP_280172 Mycobacterium tuberculosis H37Rv NP_217836 Thermoplasma acidophilum DSM 1728 NP_394462 Archaeoglobus fulgidus DSM 4304 NP_069516 -subunit gamma COG2009, COG2048 Halobacterium sp. NRC-1 NP_280174 Mycobacterium tuberculosis H37Rv NP_217833 Thermoplasma acidophilum DSM 1728 NP_394463 Archaeoglobus fulgidus DSM 4304 NP_069517 -subunit delta COG2142 Halobacterium sp. NRC-1 NP_280173 Mycobacterium tuberculosis H37Rv NP_217834 Thermoplasma acidophilum DSM 1728 NP_394464

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Appendix 1 (continued): 53 Archaeoglobus fulgidus DSM 4304 NP_069518 SDH-FRD Type B -subunit alpha COG1053 Campylobacter jejuni subsp. jejuni CG8486 ZP_01810183 Bacillus subtilis subsp. subtilis str. 168 NP_390722 Chlamydophila pneumoniae CWL029 NP_224984 -subunit beta COG0479 Campylobacter jejuni subsp. jejuni CG8486 ZP_01810184 Bacillus subtilis subsp. subtilis str. 168 NP_390721 Chlamydophila pneumoniae CWL029 NP_224985 -subunit gamma COG2009, COG2048 Campylobacter jejuni subsp. jejuni CG8486 ZP_01810182 Bacillus subtilis subsp. subtilis str. 168 NP_390723 Chlamydophila pneumoniae CWL029 NP_224983 SDH-FRD Type C -subunit alpha COG1053 Rhodoferax fermentans BAA31215 Escherichia coli O157:H7 str. Sakai NP_308775 Rickettsia prowazekii str. Madrid E NP_220520 -subunit beta COG0479 Rhodoferax fermentans BAA31216 Escherichia coli O157:H7 str. Sakai NP_308776 Rickettsia prowazekii str. Madrid E NP_220521 -subunit gamma COG2009, COG2048 Rhodoferax fermentans BAA31213 Escherichia coli O157:H7 str. Sakai NP_308773 Rickettsia prowazekii str. Madrid E NP_220518 -subunit delta COG2142 Rhodoferax fermentans BAA31214 Escherichia coli O157:H7 str. Sakai NP_308774 Rickettsia prowazekii str. Madrid E NP_220519 SDH-FRD Type D -subunit alpha COG1053 Escherichia coli str. K-12 substr. MG1655 NP_418578 Haemophilus influenzae Rd KW20 NP_438995 -subunit beta COG0479 Escherichia coli str. K-12 substr. MG1655 NP_418577 Haemophilus influenzae Rd KW20 NP_438994 -subunit gamma COG2009, COG2048 Escherichia coli str. K-12 substr. MG1655 NP_418576 Haemophilus influenzae Rd KW20 NP_438993

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Appendix 1 (continued): 54 -subunit delta COG2142 Escherichia coli str. K-12 substr. MG1655 NP_418575 Haemophilus influenzae Rd KW20 NP_438992 SDH-FRD Type E -subunit alpha COG1053 Campylobacter jejuni subsp. jejuni CF93-6 ZP_01067418 Sulfolobus acidocaldarius CAA70249 Synechocystis sp. PCC 6803 NP_440839 -subunit beta COG0479 Campylobacter jejuni subsp. jejuni 84-25 ZP_01100272 Sulfolobus acidocaldarius CAA70250 Synechocystis sp. PCC 6803 NP_442463 -subunit gamma COG2009, COG2048 Sulfolobus acidocaldarius CAA70251 -subunit delta COG2142 Sulfolobus acidocaldarius CAA70252 Fumarase -Class I COG1951, COG1838 Endoriftia persephone ABG77122 Bradyrhizobium sp. ORS278 CAL78714 Escherichia coli AAA23827 Escherichia coli CAA25204 -Class II COG0114 Bacillus subtilis CAA25849 Escherichia coli 2FUS_A Malate Dehydrogenase COG0039 Sus Scrofa 4MDH Prosthecochloris vibrioformis 1GV1 Aeropyrum Pernix 2D4A Cryptosporidium Parvum 2HJR Malate Quinone Oxidoreductase COG0579 Pseudomonas citronellolis AAW88350 Corynebacterium glutamicum O69282 Helicobacter pylori O24913 Escherichia coli K-12 P33940 PDH (homodimeric) -E1 subunit COG2609 Escherichia coli NP 285810 Pseudomonas aeruginosa AAC45353 Azotobacter vinelandii CAA75394 Ralstonia eutropha AAA21598 -E2 subunit COG0508 Escherichia coli NP 285811 Haemophilus influenzae 39388 Azotobacter vinelandii CAA30987 Pseudomonas aeruginosa AAC45354

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Appendix 1 (continued): 55 -E3 subunit COG1249 Escherichia coli NP 285812 Haemophilus influenzae NP 439387 Ralstonia eutropha AAA21600 PDH (heterodimeric) -E1 alpha COG1071 Zymomonas mobilis CAA73384 Homo sapiens NP 000275 Acidithiobacillus ferrooxidans AAB41626 -E1 beta COG0022 Zymomonas mobilis CAA73385 Homo sapiens NP 000916 Acidithiobacillus ferrooxidans AAB41627 -E2 COG0508 Zymomonas mobilis CAA63808 Homo sapiens NP 001922 Geobacillus stearothermophilus CAA37630 -E3 COG1249 Homo sapiens 1ZY8 Geobacillus stearothermophilus CAA37631 Pyruvate Formate Lyase COG1882 Escherichia coli 1MZO Shewanella oneidensis AAN55926 Thermoanaerobacterium saccharolyticum ACA51671 Streptococcus mutans BAA09085 Lactococcus lactis O32799 1-subunit PAOR COG0674 Desulfovibrio africanus CAA70873 Pantoea agglomerans CAA55302 2-subunit PAOR -subunit alpha COG0674, COG1014 Thermoanaerobacterium saccharolyticum ACA51672 Halobacterium salinarum CAA45825 -subunit beta COG1013 Thermoanaerobacterium saccharolyticum ACA51673 Halobacterium salinarum CAA45826 4-subunit PAOR -subunit alpha COG0674 Pyrococcus furiosus CAA59505 Helicobacter pylori AAC38206 -subunit beta COG1013 Pyrococcus furiosus CAA59506 Helicobacter pylori AAC38207 -subunit gamma COG1014 Pyrococcus furiosus CAA59500 Helicobacter pylori AAC38204 -subunit delta COG1144 Pyrococcus furiosus CAA59504 Helicobacter pylori AAC38205

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Appendix 1 (continued): 56 5-subunit PAOR -subunit alpha COG0674 Hydrogenobacter thermophilus BAA95605 -subunit beta COG1013 Hydrogenobacter thermophilus BAA95606 -subunit Gamma COG1014 Hydrogenobacter thermophilus BAA95607 -subunit delta COG1144 Hydrogenobacter thermophilus BAA95604 -subunit epsilon Hydrogenobacter thermophilus BAA95603 *This species has a fused E1 and E2 subunits.

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Appendix 2: Paralogous Genes with an Alternative Function 57 Enzyme Paralogs with Alternative Function Si-citrate synthase (both types) homocitrate synthase 2-methylcitrate synthase Re-citrate synthase 2-isopropylmalate synthase Aconitase A 3-isopropylmalate synthase large subunit Aconitase B 3-isopropylmalate synthase large subunit Aconitase X 3-isopropylmalate synthase large subunit Isocitrate Dehydrogenase 3-isopropylmalate dehydrogenase tartrate dehydrogenase 2-OGDH -E1 heterodimeric PDH E1 alpha Branched chain dehydrogenase Acetoin dehydrogenase -E2 alphaketobutyrate dehydrogenase Branched chain dehydrogenase PDH homodimeric E2 PDH heterodimeric E2 Acetoin dehydrogenase -E3 methionine degrading enzyme glutathione reductase tryptathione reductase thioredoxin reductase 2-subunit OAOR -subunit alpha 2-subunit PAOR, alpha subunit 2-oxoisovalerate oxidoreductase, beta subunit phenylglyoxylate:acceptor oxidoreductase 4-subunit OAOR, alpha subunit 4-subunit PAOR, alpha subunit -subunit beta 4-subunit IOR, alpha subunit

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Appendix 2 (continued): 58 4-subunit OAOR -all subunits 4-subunit IOR 4-subunit PAOR 4-subunit VOR 4-subunit PGOR -subunit alpha 2-subunit OAOR, alpha subunit 2-subunit PAOR, alpha subunit 1-subunit PAOR Succinyl-coA Synthetase Succinate Dehydrogenase -subunit alpha L-aspartate oxidase -subunit beta Fumarase Class 1 tartrate dehydratase Class 2 argininosuccinate lyase Malate dehydrogenase inosine-5'-monophosphate dehydrogenase Malate Quinone Oxidoreductase FAD dependent oxidoreductase sarcosine oxidase, beta subunit PDH (homodimeric) -E1 alphaketobutyrate dehydrogenase methionine degrading enzyme -E2 alphaketobutyrate dehydrogenase Branched chain dehydrogenase 2-OGDH E2 Acetoin dehydrogenase -E3 methionine degrading enzyme glutathione reductase tryptathione reductase thioredoxin reductase PDH (heterodimeric)

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Appendix 2 (continued): 59 -E1 alpha 2-OGDH E1 Branched chain dehydrogenase Acetoin dehydrogenase -E1 beta Branched chain dehydrogenase Acetoin dehydrogenase -E2 alphaketobutyrate dehydrogenase Branched chain dehydrogenase 2-OGDH E2 Acetoin dehydrogenase -E3 methionine degrading enzyme glutathione reductase tryptathione reductase thioredoxin reductase 1-subunit PAOR 4-subunit PAOR, alpha subunit 4-subunit OAOR, alpha subunit 4-subunit VOR, alpha subunit 4-subunit PGOR, alpha subunit 2-subunit PAOR -subunit alpha 2-subunit OAOR, alpha subunit 4-subunit OAOR, alpha subunit 4-subunit VOR, alpha subunit 4-subunit PGOR, alpha subunit 4-subunit PAOR, alpha subunit -subunit beta 4-subunit IOR, alpha subunit 4-subunit PAOR -all subunits 4-subunit IOR 4-subunit OAOR 4-subunit VOR 4-subunit PGOR -subunit alpha 2-subunit OAOR, alpha subunit 2-subunit PAOR, alpha subunit 1-subunit PAOR, alpha subunit

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Appendix 3: Active Site Residues for Alignment Verification 60 Enzyme Subunit Organism Conserved Residue(s) Role Reference Si-citrate synthase N/A Sus scrofa his174, his 238, his 320, arg 329, arg 401, arg 421, phe 397 stabilize citrateenzyme interaction, substrate binding and catalysis [42] Escherichia coli His264, His229, His-306, Arg-314, Arg-387 and Arg-407 stabilize citrateenzyme interaction, substrate binding and catalysis [93] Re-citrate synthase N/A no info Aconitase A + B N/A Escherichia coli D100, H101, H147, D165, S166, H167, N170, N258, E262, C434, N446, R447, R452, C518, C521, S642, S643 and R644 coordination of the [4Fe-4S] centre, substrate recognition and catalysis [51, 94] Aconitase X N/A Pseudomonas aeruginosa C276, P279, Y280, W11, E13, S14, A16, P60, C19, S29, G32, G15, G25, S26, C27 active center, structurally conserved regions [55] Monomeric IDH N/A Archaeglobus fulgidus I43, S119, R125, R135, R159, Y166, K236, C263, D290, D314, D318, L327, G328, H346, A349, V358, N359, D400 substrate binding and catalysis [57] Multimeric IDH N/A Escherichia coli I43, S119, R125, C133, R135, R159, Y166, C200, C207, K236, D290, C308, D314, D318, I327, G328, C339, H346, A349, V358, N359, D400, C413 substrate binding and catalysis [95] 2-OGDH E1 no info E2 Escherichia coli threonine 323, histidine 375, Asp379 substrate binding and catalysis [96] OAOR 2-subunit no info 4-subunit no info Succinyl-coA Synthetase alpha Escherichia coli K or R242, G248, L or P276 dimerization [67] alpha D103, Y158, E159, T237, K242, G248, L276 binding CoA

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Appendix 3 (continued): 61 alpha G14, F15, T16, G17, S18, Q19, G20, P40, G41, K42, V72, P73, F76, S or A80 accepting phosphoryl alpha H246 site of phosphorylation alpha H142 assisting H246 beta Escherichia coli N199, D213 coordinate the Mg necessary to bind NDPs [97] beta Y6, Q7, R or G70, N94, R116, R225, A or E231, E or D249, F319 dimerization beta R348, D274, E242, L374 conserved residues SDH-FRD (all forms) Escherichia coli H242, R286, H354 and R399 (FAD-binding domain); 3 clusters of 4 cysteines (FeS cluster) substrate binding and catalysis [71] Fumarase Class I no info Class II Escherichia coli Asn326, Lys324, Ser98, Thr100, Asn141, His188, Glu331 and His188 substrate binding and catalysis [75, 98] Malate Dehydrogenase Escherichia coli R102, R109, D168, R171, and H195 substrate binding and catalysis [99] Malate Quinone Oxidoreductase no info PAOR [31, 34, 36-38] 1-subunit N/A D. africanus 812, 815, 840, 1071 (4 cysteines) iron-sulfur cluster 689-699 (4 cysteines) iron-sulfur cluster 745-755 (4 cysteines) iron-sulfur cluster 2-subunit alpha Halobacterium salinarum 183-189 (GDILEQN) aldehyde-binding motif--can be variable beta Halobacterium salinarum 30, 33, 64, 212 (4 cysteines) iron-sulfur cluster beta Halobacterium salinarum 105-108 (GDG) TPP-binding 4-subunit beta Pyrococcus furiosus 21, 24, 69, 224 (4 cysteines) iron-sulfur cluster beta Pyrococcus furiosus 109-112 (GDG) TPP-binding gamma Pyrococcus furiosus 161-170 (GELGEKNA) aldehyde-binding motif--can be variable delta Pyrococcus furiosus 53-63 (4 cysteines) iron-sulfur cluster delta Pyrococcus furiosus 83-93 (4 cysteines) iron-sulfur cluster

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Appendix 3 (continued): 62 PFL N/A E. coli 734 (glycine) glycine radical [39, 41, 100-102] 418 and 419 (2 cysteines) form S-C bond with C2 of pyruvate PDH Heterodimeric E1 alpha G. stearothermophilus 173 (GDG) binds TPP [26, 103, 104] Homodimeric E1 E. coli 229-231 (GDG) binds TPP [25, 26, 105-109] E1 E. coli 258-260 (NCN) binds TPP E2 G. stearothermophilus 398-400 (DHR) H for transferring acetyl group to CoA E. coli 602-604 (DHR) H for transferring acetyl group to CoA E. coli 39; 141; 242 [GDKASME; G(D/E)X13(D/S)K X10 (G/C)] lipoyl prosthetic group is covalently bonded to the lysine (K) residues here; some E2s have more than one location that is lipoylated, others do not. This sequence can vary; the key bit is the lysine residue. G. stearothermophilus 27-54 [G(D/E)X13(D/S)K X10 (G/C)] lipoyl binding domain E3 E. coli 13-18; 182-187 (GXGXXG) binds ADP of FAD G. stearothermophilus 16-21; 183-188 (GXGXXG) binds ADP of FAD E. coli 45-50 (CXXXXC) redox-sensitive disulfide center G. stearothermophilus 47-52 (CXXXXC) redox-sensitive disulfide center

PAGE 71

Appendix 4: Pfams Whose HMM Logos Were Used to Verify Alignments 63 Enzyme Pfam Si-citrate synthase pfam00285 Re-citrate synthase pfam00682 Aconitase A pfam00330, pfam00694 Aconitase B pfam00330, pfam06434 Aconitase X pfam01989, pfam04412 Monomeric IDH pfam03971 Multimeric IDH pfam00180 2-OGDH -E1 subunit pfam00676, pfam02779 -E2 subunit pfam00198, pfam00364, pfam02817 -E3 subunit pfam02852, pfam07992 2-subunit OAOR -subunit alpha pfam01558, pfam01855, pfam02780 -subunit beta pfam02775 4-subunit OAOR -subunit alpha pfam01855, pfam02780 -subunit beta pfam02775 -subunit gamma pfam01558 -subunit delta pfam00037 5-subunit OAOR subunit epsilon None available Succinyl-coA -subunit alpha pfam02629

PAGE 72

Appendix 4 (continued) 64 -subunit beta pfam08442, pfam00549 SDH-FRD (all forms) -subunit alpha pfam01266, pfam02910, pfam00890 -subunit beta pfam00037, pfam00111 -subunit gamma pfam02300 -subunit delta pfam02313 Fumarase -Class 1 pfam05681, pfam05683 -Class 2 pfam00206, pfam10415 Malate Dehydrogenase pfam00056, pfam02866 Malate Quinone Oxidoreductase pfam01266, pfam06039 PDH (homodimeric) -E1 subunit pfam00456 -E2 subunit pfam00198, pfam00364, pfam02817 -E3 subunit pfam02852, pfam07992 PDH (heterodimeric) -E1 alpha pfam00676 -E1 beta pfam02779, pfam02780 -E2 pfam00198, pfam00364, pfam02817 -E3 pfam02852, pfam07992 Pyruvate Formate Lyase pfam02901, pfam01228 1-subunit PAOR pfam01855, pfam01558, pfam00037, pfam02775

PAGE 73

Appendix 4 (continued) 65 2-subunit PAOR -subunit alpha pfam01558, pfam01855 -subunit beta pfam02775 4-subunit PAOR -subunit alpha pfam01855 -subunit beta pfam02775 -subunit gamma pfam01558 -subunit delta pfam00037 5-subunit PAOR subunit epsilon None available

PAGE 74

66 Appendix 5: The citric acid cycle of of T. crunogena

PAGE 75

67 Appendix 6: The canonical citric acid cycle of Gammaproteobacteria

PAGE 76

68 Appendix 7: The canonical citric acid cycle of Betaproteobacteria

PAGE 77

69 Appendix 8: The canonical citric acid cycle of Alphaproteobacteria.

PAGE 78

70 Appendix 9: The canonical citric acid cycle of Epsilonproteobacteria.

PAGE 79

71 Appendix 10: The canonical citric acid cycle of Delta and Acido-proteobacteria.

PAGE 80

72 Appendix 11: Citric acid cycle genes (pyruvate to 2-oxoglutarate) present for each species in the Gammaproteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class

PAGE 81

73 Appendix 11 (continued):

PAGE 82

74 Appendix 11 (continued):

PAGE 83

75 Appendix 12: Citric acid cycle genes (pyruvate to 2-oxoglutarate) present for each species in the Betaproteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class.

PAGE 84

76 Appendix 12 (continued):

PAGE 85

77 Appendix 13: Citric acid cycle genes (pyruvate to 2-oxoglutarate) present for each species in the Alphaproteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class.

PAGE 86

78 Appendix 13 (continued):

PAGE 87

79 Appendix 14: Citric acid cycle genes (pyruvate to 2-oxoglutarate) present for each species in the Epsilon-, Delta-, and Acido-proteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class.

PAGE 88

80 Appendix 15: Citric acid cycle genes (2-oxoglutarate to oxaloacetate) present for each species in the Gammaproteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class.

PAGE 89

81 Appendix 15 (continued):

PAGE 90

82 Appendix 15 (continued):

PAGE 91

83 Appendix 16: Citric acid cycle genes (2-oxoglutarate to oxaloacetate) present for each species in the Betaproteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class.

PAGE 92

84 Appendix 16 (continued):

PAGE 93

85 Appendix 17: Citric acid cycle genes (2-oxoglutarate to oxaloacetate) present for each species in the Alphaproteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class.

PAGE 94

86 Appendix 17 (continued):

PAGE 95

87 Appendix 18: Citric acid cycle genes (2-oxoglutarate to oxaloacetate) present for each species in the Epsilon-, Delta-, and Acido-proteobacteria The phylogenetic trees adjacent to these tables were made using 16S RNA sequences and show the evolutionary relatedness and distance between the species in each class.

PAGE 96

88 637805918 Shigella boydii Sb227 641724363 Shigella boydii CDC 3083-94 641605663 Escherichia coli str. K-12 ... 641615025 Escherichia coli SECEC SMS-3-5 640427973 Shigella sonnei Ss046 640919305 Escherichia coli HS 637000110 Escherichia coli W3110 DNA 640923845 Escherichia coli E24377A 637332397 Shigella flexneri 2a str. 301 637422296 Shigella flexneri 2a str. 2... 638073932 Shigella flexneri 5 str. 8401 NP 285810 Escherichia coli 637080769 Escherichia coli O157 637061853 Escherichia coli O157 641602962 Escherichia coli ATCC 8739 637013489 Escherichia coli K12 640432574 Shigella dysenteriae Sd197 638061264 Escherichia coli 536 637354003 Escherichia coli CFT073 637931832 Escherichia coli UTI89 640753108 Escherichia coli APEC O1 641305994 Salmonella enterica subsp. ... 641320630 Salmonella enterica subsp. ... 637404004 Salmonella enterica subsp. ... 637215444 Salmonella enterica subsp. ... 637638998 Salmonella enterica subsp. ... 637600461 Salmonella enterica subsp. ... 637210905 Salmonella typhimurium LT2 640917224 Citrobacter koseri ATCC BAA... 640490831 Enterobacter sp. 638 640797162 Klebsiella pneumoniae subsp... 640901374 Enterobacter sakazakii ATCC... 637380277 Erwinia carotovora subsp. a... 637465377 Photorhabdus luminescens su... 640074271 Yersinia enterocolitica sub... 640939571 Serratia proteamaculans 568 640866378 Yersinia pseudotuberculosis... 641598575 Yersinia pseudotuberculosis... 637532318 Yersinia pseudotuberculosis... 638045032 Yersinia pestis Antiqua 640479797 Yersinia pestis Pestoides F 637309209 Yersinia pestis KIM 641339022 Yersinia pestis Angola 637202391 Yersinia pestis CO92 638038680 Yersinia pestis Nepal516 637490959 Yersinia pestis biovar Micr... 637852399 Sodalis glossinidius str. m... 637672595 Candidatus Blochmannia penn... 637443626 Candidatus Blochmannia flor... 637981863 Baumannia cicadellinicola s... 637056223 Buchnera aphidicola str. AP... 637306089 Buchnera aphidicola str. Sg... 639633185 Buchnera aphidicola str. Bp... 637341702 Wigglesworthia glossinidia ... 639660895 Buchnera aphidicola str. Cc... 638104657 Haemophilus somnus 129PT 641657972 Haemophilus somnus 2336 637070502 Pasteurella multocida subsp... 100 99 100 100 85 94 59 37 100 76 99 66 66 100 56 99 99 80 50 98 77 100 54 48 100 48 78 84 27 62 62 64 100 51 58Appendix 19: Phylogeny of homodimeric pyruvate dehydrogenase sequences, using genes encoding methionine degradation enzyme as an outgroup.

PAGE 97

89 p 637550855 Mannheimia succiniciproduce... 640807863 Actinobac illus succinogenes... 637129587 Haemophilus ducreyi 35000HP 640122441 Actinobacillus pleuropneumo... 641530778 Actinobacillus pleuropneumo... 637663653 Haemophilus influenzae 86-0... 640610710 Haemophilus influenzae PittGG 640609466 Haemophilus influenzae PittEE NP 439389 Haemophilus influenzae 637008419 Haemophilus influenzae Rd KW20 639716437 Aeromonas hydrophila subsp.... 641523029 Aeromonas salmonicida subsp... 639798045 Psychromonas ingrahamii 37 637733271 Pseudoalteromonas haloplank... 639779712 Shewanella amazonensis SB2B 641234956 Shewanella pealeana ATCC 70... 641631803 Shewanella woodyi ATCC 51908 637957436 Shewanella denitrificans OS217 637636892 3 Vibrio fischeri ES114 chr... 640908944 Vibrio harveyi ATCC BAA-111... 637587896 Photobacterium profundum SS... 637048902 Vibrio cholerae O1 biovar e... 640533447 Vibrio cholerae O395 chromo... 637399506 Vibrio parahaemolyticus RIM... 637361989 Vibrio vulnificus CMCP6 chr... 637469537 Vibrio vulnificus YJ016 chr... 640949855 Arcobacter butzleri RM4018 637287247 Colwellia psychrerythraea 34H 638056381 Pseudoalteromonas atlantica... 637604972 Idiomarina loihiensis L2TR 640931276 Shewanella sediminis HAW-EB3 641551233 Shewanella halifaxensis HAW... 640165043 Shewanella loihica PV-4 638136590 Shewanella frigidimarina NC... 640832197 Shewanella baltica OS185 641290404 Shewanella baltica OS195 640121179 Shewanella baltica OS155 639814564 Shewanella sp. W3-18-1 640500616 Shewanella putrefaciens CN-32 637342426 Shewanella oneidensis MR-1 639724962 Shewanella sp. ANA-3 chromo... 638116987 Shewanella sp. MR-4 638124283 Shewanella sp. MR-7 641556610 Francisella philomiragia su... 641732854 Francisella tularensis subs... 639753531 Francisella tularensis subs... 638146389 Francisella tularensis subs... 640890373 Francisella tularensis subs... 637906898 Francisella tularensis subs... 640189795 Francisella tularensis subs... 638060866 Francisella tularensis subs... 637615361 Francisella tularensis subs... 641267892 Dinoroseobacter shibae DFL 12 640525121 Dichelobacter nodosus VCS1703A 637451285 Chromobacterium violaceum A... 637141841 Neisseria gonorrhoeae FA 1090 637195964 Neisseria meningitidis MC58 639828132 Neisseria meningitidis FAM18 637198226 Neisseria meningitidis Z2491 641333040 Neisseria menin g itidis 053442 98 86 58 34 39 100 100 100 71 41 83 100 82 82 58 100 91 100 100 100 48 50 48 100 97 82 80 100 100 100 100 100 100 99 93 64 88 89 98 100 82 73 93 24 83 72 63 73 90 45 43 85 100 98 99 100 82 81Appendix 19 (continued):

PAGE 98

90 638003995 Pseudomonas entomophila L48 641545447 Pseudomonas putida GB-1 641624676 Pseudomonas putida W619 637143718 Pseudomonas putida KT2440 640584810 Pseudomonas putida F1 637317852 Pseudomonas fluorescens Pf-5 637740304 Pseudomonas fluorescens PfO-1 637650858 Pseudomonas syringae pv. sy... 637486124 Pseudomonas syringae pv. ph... 637396326 Pseudomonas syringae pv. to... 640501789 Pseudomonas mendocina ymp AAC45353 Pseudomonas aeruginosa 639327948 Pseudomo nas aeruginosa UCBP... 637055458 Pseudomo nas aeruginosa PAO1 640814795 Pseudomonas aeruginosa PA7 CAA75394 Azotobacter vinelandii 640489794 Pseudomonas stutzeri A1501 640804667 Marinomonas sp. MWYL1 638078705 Alcanivorax borkumensis SK2 637966024 Chromohalobacter salexigens... 637921671 Saccharophagus degradans 2-40 637831665 Hahella chejuensis KCTC 2396 639813306 Marinobacter aquaeolei VT8 639758116 Candidatus Ruthia magnifica... 640537890 Candidatus Vesicomyosocius ... 640569246 Legionella pneumophila str.... 637131444 Legionella pneumophila subs... 637580156 Legionella pneumophila str.... 637583293 Legionella pneumophila str.... 637671295 Candidatus Pelagibacter ubi... 637785722 Thiomicrospira crunogena XCL-2 640889471 Coxiella burnetii Dugway 7E... 637162145 Coxiella burnetii RSA 493 641330074 Coxiella burnetii RSA 331 637942334 Polaromonas sp. JS666 639834787 Polaromonas naphthalenivora... 637917033 Rhodoferax ferrireducens T118 641664400 Leptothrix cholodnii SP-6 639823667 Acidovorax avenae subsp. ci... 641299078 Delftia acidovorans SPH-1 639839196 Acidovorax sp. JS42 639849686 Verminephrobacter eiseniae ... 640090696 Methylibium petroleiphilum PM1 640816105 Janthinobacterium sp. Marse... 641722419 Bordetella avium 197N 641363749 Bordetella petrii 637112302 Bordetella pertussis Tohama I 637104678 Bordetella bronchiseptica RB50 637108588 Bordetella parapertussis 12822 637043114 Xylella fastidiosa 9a5c 641649632 Xylella fastidiosa M12 637384310 Xylella fastidiosa Temecula1 641703102 Xylella fastidiosa M23 637756295 Xanthomonas campestris pv. ... 637293827 Xanthomonas axonopodis pv. ... 637851545 Xanthomonas oryzae pv. oryz... 637656090 Xanthomonas campestris pv. ... 637275729 Xanthomonas campestris pv. ... 637765856 Burkholderia sp. 383 chromo... 640817334 Janthinobacterium sp. Marse... 76 100 100 37 100 100 92 91 100 100 32 42 100 88 100 100 98 45 74 40 100 23 60 100 72 100 87 100 86 99 100 86 100 77 98 90 93 80 92 99 74 100 99 100 74 100 24 21 29 30 45 15 36 12 48 18 13 9 2 59 5Appendix 19 (continued):

PAGE 99

91 p 638124994 Alkalilimnicola ehrlichei M... 639855508 Halorhodospira halophila SL1 637172126 Methylococcus capsulatus st... 637938994 Methylobacillus flagellatus KT 641641526 Methylobacterium sp. 4-46 641646397 Methylobacterium sp. 4-46 637371285 6 Bradyrhizobium japonicum ... 637694111 Ralstonia eutropha JMP134 c... 637979685 Ralstonia metallidurans CH3... 640447861 Ralstonia eutropha H16 chro... 637863668 Anaeromyxobacter dehalogena... 640845655 Anaeromyxobacter sp. Fw109-5 637607280 Azoarcus sp. EbN1 639786512 Azoarcus sp. BH72 637678883 Dechloromonas aromatica RCB 640470891 Polynucleobacter sp. QLW-P1... 641671699 Polynucleobacter necessariu... AAA21598 Ralstonia eutropha 640447494 Ralstonia eutropha H16 chro... 637690691 Ralstonia eutropha JMP134 c... 637976582 Ralstonia metallidurans CH3... 637237987 Ralstonia solanacearum GMI1000 637948091 Burkholderia xenovorans LB4... 638019220 Burkholderia cenocepacia AU... 639694192 Burkholderia cenocepacia HI... 641638735 Burkholderia cenocepacia MC... 638149999 Burkholderia cepacia AMMD c... 641681813 Burkholderia ambifaria MC40... 637763750 Burkholderia sp. 383 chromo... 640187991 Burkholderia vietnamiensis ... 641313554 Burkholderia multivorans AT... 637841533 Burkholderia thailandensis ... 640128540 Burkholderia pseudomallei 6... 639846494 Burkholderia mallei SAVP1 c... 640150160 Burkholderia mallei NCTC 10... 640098327 Burkholderia mallei NCTC 10... 640135894 Burkholderia pseudomallei 1... 637729202 Burkholderia pseudomallei 1... 637562395 Burkholderia mallei ATCC 23... 637568282 Burkholderia pseudomallei K... 637670016 Psychrobacter arcticus 273-4 637973515 Psychrobacter cryohalolenti... 640599622 Psychrobacter sp. PRwf-1 637519867 Acinetobacter sp. ADP1 640155504 Acinetobacter baumannii ATC... 641588967 Acinetobacter baumannii AYE 641585534 Acinetobacter baumannii 639684914 Solibacter usitatus Ellin6076 637984810 Acidobacteria bacterium Ell... 637737756 Nitrosococcus oceani ATCC 1... mde outgroup pp mde outgroup 100 100 96 100 100 100 100 100 100 73 100 99 58 63 100 49 68 78 98 75 100 97 100 100 100 100 100 98 100 100 100 99 100 62 99 33 99 33 23 10 93 96 66 77 44 75 30 24 0.2Appendix 19 (continued):

PAGE 100

92 Appendix 20: Phylogeny of isocitrate dehydrogenase sequences, using genes encoding 3isopropylmalate and tartrate dehydrogenases as an outgroup. multimeric IDH multimeric IDH 95 58

PAGE 101

93 tar Saccharophagus degradans 2-40 640929591 mono Campylobacter concisus... 637291999 mono Campylobacter jejuni R... 637040082 mono Campylobacter jejuni s... 639853279 mono Campylobacter jejuni s... 640941415 mono Campylobacter jejuni s... 640862367 mono Campylobacter jejuni s... 637432241 mono Helicobacter hepaticus... 639746294 mono Campylobacter fetus su... 640870818 mono Campylobacter hominis ... 640820120 mono Nitratiruptor sp. SB155-2 637793472 mono Thiomicrospira denitri... 640821733 mono Sulfurovum sp. NBC37-1 641583506 mono Acinetobacter baumannii 641589782 mono Acinetobacter baumanni... 640154653 mono Acinetobacter baumanni... 637517729 mono Acinetobacter sp. ADP1 639839368 mono Acidovorax sp. JS42 641298744 mono Delftia acidovorans SPH-1 637454509 mono Chromobacterium violac... 639786278 mono Azoarcus sp. BH72 637681591 mono Dechloromonas aromatic... 637693633 mono Ralstonia eutropha JMP134 640451754 mono Ralstonia eutroph... 637979152 mono Ralstonia metalliduran... 640470517 mono Polynucleobacter sp. Q... 641671069 mono Polynucleobacter neces... 637607651 mono Azoarcus sp. EbN1 639848401 mono Verminephrobacter eise... 639836698 mono Polaromonas naphthalen... 641667159 mono Leptothrix cholodnii SP-6 640600326 mono P sychr obacter sp. PRwf-1 637668974 mono Psychrobacter arcticus... 637972764 mono P sychr obacter cryohalo... 637283818 mono Colwellia p sychrerythr... 638079378 mono Alcanivorax borkumensi... 638055388 mono Pseudoalteromonas atla... 639810046 mono Marinobacter aquaeolei... 640805641 mono Marinomonas sp. MWYL1 640503641 mono Pseudomonas mendocina ymp 640488246 mono Pseudomonas stutzeri A... 637053026 mono Pseudomonas aeruginosa... 639324998 mono Pseudomonas aeruginosa... 640811634 mono Pseudomonas aeruginosa... 637833532 mono Hahella chejuensis KCT... 638125872 mono Alkalilimnicola ehrlic... 639815850 mono Shewanella sp. W3-18-1 640499392 mono Shewanella putrefacien... 641288882 mono Shewanella baltica OS195 640830681 mono Shewanella baltica OS185 640119698 mono Shewanella baltica OS155 637344456 mono Shewanella oneidensis ... 639726333 mono Shewanella sp. ANA-3 638118219 mono Shewanella sp. MR-4 638122340 mono Shewanella sp. MR-7 639781436 mono Shewanella amazonensis... 637955848 mono Shewanella denitrifica... 638135054 mono Shewanella frigidimari... 68 54 94 99 62 99 99 99 62 99 71 85 99 99 99 64 96 99 99 79 18 90 62 34 98 99 99 54 99 98 25 42 11 20 32 43 85 38 64 33 26 96 51 77 22 26 83 59 11 16 91 79 58 14 62 99Appendix 20 (continued):

PAGE 102

94 Appendix 20 (continued): 640163125 mono Shewanella loihica PV-4 640932769 mono Shewanella sediminis H... 641634233 mono Shewanella woodyi ATCC... 641237161 mono Shewanella pealeana AT... 641552518 mono Shewanella halifaxensi... 637785822 mono Thiomicrospira crunogena XL-2 641351186 mono Sorangium cellulosum S... 637920765 mono Saccharophagus degrada... 637734636 mono Pseudoalteromonas halo... 637636473 mono Vibrio fischeri E... 637047617 mono Vibrio cholerae O1 bio... 640532169 mono Vibrio cholerae O395 637397987 mono Vibrio parahaemolyticu... 640907054 mono Vibrio harveyi ATCC BA... 637362448 mono Vibrio vulnificus CMCP6 637469086 mono Vibrio vulnificus YJ016 637605973 mono Idiomarina loihiensis ... 637285350 mono Colwellia p sychrerythr... 639796200 mono P sychromonas ingrahami... 641556667 mono Francisella philomirag... 639753471 mono Francisella tularensis... 637907154 mono Francisella tularensis... 638146631 mono Francisella tularensis... 640890676 mono Francisella tularensis... 641732822 mono Francisella tularensis... 640189427 mono Francisella tularensis... 637615399 mono Francisella tularensis... 638060904 mono Francisella tularensis... 638105227 mono Haemophilus somnus 129PT 641658764 mono Haemophilus somnus 2336 637863765 mono Anaeromyxobacter dehal... 640843097 mono Anaeromyxobacter sp. F... 640549691 mono Geobacter uraniumreduc... 637778641 mono Geobacter metallireduc... 637126141 mono Geobacter sulfurreduce... 637819215 mono Magnetospirillum magne... 640949697 mono Arcobacter butzleri RM... 637613857 mono Silicibacter pomeroyi ... 638005773 mono Silicibacter sp. TM1040 641267909 mono Dinoroseobacter shibae... 639635617 mono Roseobacter denitrific... 637904729 mono Jannaschia sp. CCS1 639722345 mono Magnetococcus sp. MC-1 640600327 mono Psychrobacter sp. PRwf-1 641332651 mono Neisseria meningitidis... 637142297 mono Neisseria gonorrhoeae ... 637197838 mono Neisseria meningitidis... 637195564 mono Neisseria meningitidis... 639827807 mono Neisseria meningitidis... 637634210 mono Xanthomonas oryzae pv .... 637851478 mono Xanthomonas oryzae pv .... 637297084 mono Xanthomonas axonopodis... 637275886 mono Xanthomonas campestris... 637659360 mono Xanthomonas campestris... 637759689 mono Xanthomonas campestris... 637045161 mono Xylella fastidiosa 9a5c 641650189 mono Xylella fastidiosa M12 637384856 mono X y lella fastidiosa Tem... monomeric IDH 99 67 60 99 99 90 72 95 81 43 75 38 99 65 54 99 99 99 99 99 99 99 71 48 99 87 76 95 94 46 81 98 99 47 27 11 96 8 76 31 4 40 72 2 18 28 2 45 39 6 12 0 2 0 0 8 11

PAGE 103

95 Appendix 20 (continued): 641703673 mono Xylella fastidiosa M23 641722616 mono Bordetella avium 197N 638001227 mono Pseudomonas entomophil... 641548739 mono Pseudomonas putida GB-1 637147423 mono Pseudomonas putida KT2440 640586289 mono Pseudomonas putida F1 641623199 mono Pseudomonas putida W619 637852641 mono Sodalis glossinidius s... 637488625 mono Pseudomonas syringae p... 637653532 mono Pseudomonas syringae p... 637394707 mono Pseudomonas syringae p... 637321226 mono Pseudomonas fluorescen... 637743470 mono Pseudomonas fluorescen... 637965692 mono Chromohalobacter salex... 1J1W|A Azotobacter vinelandii 640170742 mono Herminiimonas arsenico... 640819160 mono Janthinobacterium sp. ... 637952145 mono Burkholderia xenovoran... 641313186 mono Burkholderia multivora... 640188381 mono Burkholderia vietnamie... 638150409 mono Burkholderia cepacia AMMD 641682218 mono Burkholderia ambifaria... 637764174 mono Burkholderia sp. 383 641639141 mono Burkholderia cenocepac... 638015193 mono Burkholderia cenocepac... 639694589 mono Burkholderia cenocepac... 83 54 99 96 99 98 79 91 99 80 99 72 99 83 40 69 99 38 49 81 26 13 14 18 30 62 p 3-isopropylmalate dehydrogenase & tartrate dehydrogenase outgroup 96

PAGE 104

96 638118250 Shewanella sp. MR-4 639726296 Shewanella sp. ANA-3 638122371 Shewanella sp. MR-7 637343818 Shewanella oneidensis MR-1 639815813 Shewanella sp. W3-18-1 640499428 Shewanella putrefaciens CN-32 640830718 Shewanella baltica OS185 640119735 Shewanella baltica OS155 641288919 Shewanella baltica OS195 637956204 Shewanella denitrificans OS217 638135137 Shewanella frigidimarina NC... 640163220 Shewanella loihica PV-4 640933730 Shewanella sediminis HAW-EB3 641633343 Shewanella woodyi ATCC 51908 641236381 Shewanella pealeana ATCC 70... 641553313 Shewanella halifaxensis HAW... 639780780 Shewanella amazonensis SB2B 637284679 Colwellia psychrerythraea 34H 637734552 Pseudoalteromonas haloplank... 640805170 Marinomonas sp. MWYL1 637835008 Hahella chejuensis KCTC 2396 637921192 Saccharophagus degradans 2-40 640487831 Pseudomonas stutzeri A1501 637051983 Pseudomonas aeruginosa PAO1 639326097 Pseudomonas aeruginosa UCBP... 640812735 Pseudomonas aeruginosa PA7 640503743 Pseudomonas mendocina ymp 637147584 Pseudom onas putida KT2440 640586139 Pseudomonas putida F1 641623295 Pseudomonas putida W619 638002570 Pseudomonas entomophila L48 641548878 Pseudomonas putida GB-1 637319061 Pseudomonas fluorescens Pf-5 637741472 Pseudomonas fluorescens PfO-1 637652362 Pseudomonas syringae pv. sy... 637393584 Pseudomonas syringae pv. to... 637487556 Pseudomonas syringae pv. ph... 639811124 Marinoba cter aquaeolei VT8 637606038 Idiomarina loihiensis L2TR 638054812 Pseudoalteromonas atlantica... 637966399 Chromohalo bacter salexigens... 638079594 Alcanivo rax borkumensis SK2 639714507 Aeromonas hydrophila subsp.... 641524901 Aeromonas salmonicida subsp... 639797389 Psychromonas ingrahamii 37 637585760 Photobacterium profundum SS9 637635499 Vibrio fischeri ES114 637048557 Vibrio cholerae O1 biovar e... 640533127 Vibrio cholerae O395 637360571 Vibrio vulnificus CMCP6 637467785 Vibrio vulnificus YJ016 637397819 Vibrio parahaemolyticus RIM... 640906850 Vibrio harveyi ATCC BAA-1116 637463246 Photorhabdus luminescens su... 640479436 Yersinia pestis Pestoides F 59 100 100 100 98 74 62 96 59 72 82 59 99 100 100 100 91 99 99 67 65 96 53 95 83 88 97 84 99 89 89 100 92 100 99 100 100 69 70 25 38 47 59 92 18 2 11 45 15 54 54Appendix 21: Phylogeny of succinyl-coA synthetase sequences, using sequences of succinyl-coA synthetase from Epsilonproteobacteria as the outgroup.

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97 640479436 Yersinia pestis Pestoides F 640865878 Yersinia pseudotuberculosis... 638042675 Yersinia pestis Antiqua 637200199 Yersinia pestis CO92 638040929 Yersinia pestis Nepal516 641339346 Yersinia pestis Angola 637311512 Yersinia pestis KIM 641598040 Yersinia pseudotuberculosis... 637491728 Yersinia pestis biovar Micr... 637532766 Yersinia pseudotuberculosis... 640076429 Yersinia enterocolitica sub... 637377827 Erwinia carotovora subsp. a... 640936797 Serratia proteamaculans 568 637852827 Sodalis glossinidius str. m... 640491411 Enterobacter sp. 638 640900770 Enterobacter sakazakii ATCC... 637215988 Salmonella enterica subsp. ... 637405884 Salmonella enterica subsp. ... 637602213 Salmonella enterica subsp. ... 641323225 Salmonella enterica subsp. ... 637639608 Salmonella enterica subsp. ... 637211484 Salmonella typhimurium LT2 641305367 Salmonella enterica subsp. ... 640797778 Klebsiella pneumoniae subsp... 640916394 Citrobacter koseri ATCC BAA... 637806373 Shigella boydii Sb227 641724845 Shigella boydii CDC 3083-94 637014092 Escherichia coli K12 637062505 Escherichia coli O157 640924468 Escherichia coli E24377A 638061882 Escherichia coli 536 640919952 Escherichia coli HS 640428510 Shigella sonnei Ss046 641615630 Escherichia coli SECEC SMS-3-5 637000717 Escherichia coli W3110 DNA 640433070 Shigella dysenteriae Sd197 641606326 Escherichia coli str. K-12 ... P0AGE9.2 E. coli 641602339 Escherichia coli ATCC 8739 637354661 Escherichia coli CFT073 637081405 Escherichia coli O157 640753642 Escherichia coli APEC O1 637932429 Escherichia coli UTI89 637332847 Shigella flexneri 2a str. 301 637422738 Shigella flexneri 2a str. 2... 638074412 Shigella flexneri 5 str. 8401 638104512 Haemophilus somnus 129PT 641658386 Haemophilus somnus 2336 640808499 Actinobacillus succinogenes... 637550870 Mannheimia succiniciproduce... 637069863 Pasteurella multocida subsp... 640122120 Actinobacillus pleuropneumo... 641530493 Actinobacillus pleuropneumo... 640609836 Haemophilus influenzae PittEE 637008384 Haemophilus influenzae Rd KW20 637663154 Haemophilus influenzae 86-0... 637981457 Baumannia cicadellinicola s... 100 85 100 100 85 85 60 34 100 38 39 62 100 100 33 34 98 18 24 64 66 96 76 60 41 31 100 23 96 64 100Appendix 21 (continued):

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98 637341798 Wigglesworthia glossinidia ... 637443805 Candidatus Blochmannia flor... 637672779 Candidatus Blochmannia penn... 640888503 Coxiella burnetii Dugway 7E... 641330945 Coxiella burnetii RSA 331 637163055 Coxiella burnetii RSA 493 637130477 Legionella pneumophila subs... 637579297 Legionella pneumophila str.... 637582356 Legionella pneumophila str.... 640571048 Legionella pneumophila str.... 637936340 Escherichia coli UTI89 640757136 Escherichia coli APEC O1 637358899 Escherichia coli CFT073 638065426 Escherichia coli 536 640917815 Citrobacter koseri ATCC BAA... 637614452 Francisella tularensis subs... 638059957 Francisella tularensis subs... 639752638 Francisella tularensis subs... 640190394 Francisella tularensis subs... 637908025 Francisella tularensis subs... 638147408 Francisella tularensis subs... 640891712 Francisella tularensis subs... 641733673 Francisella tularensis subs... 641555636 Francisella philomiragia su... 637668790 Psychrobacter arcticus 273-4 637972557 Psychrobacter cryohalolenti... 640598480 Psychrobacter sp. PRwf-1 637451849 Chromobacterium violaceum A... 641583345 Acinetobacter baumannii 641589592 Acinetobacter baumannii AYE 640154895 Acinetobacter baumannii ATC... 637519286 Acinetobacter sp. ADP1 637142153 Neisseria gonorrhoeae FA 1090 641332693 Neisseria meningitidis 053442 637197875 Neisseria meningitidis Z2491 637195602 Neisseria meningitidis MC58 639827844 Neisseria meningitidis FAM18 638127307 Alkalilimnicola ehrlichei M... 639856715 Halorhodospira halophila SL1 637384731 Xylella fastidiosa Temecula1 641703548 Xylella fastidiosa M23 641650061 Xylella fastidiosa M12 637045003 Xylella fastidiosa 9a5c 637275197 Xanthomonas campestris pv. ... 637656556 Xanthomonas campestris pv. ... 637296484 Xanthomonas axonopodis pv. ... 637759075 Xanthomonas campestris pv. ... 637631889 Xanthomonas oryzae pv. oryz... 637848977 Xanthomonas oryzae pv. oryz... 637786107 Thiomicrospira crunogena XCL-2 637709484 Thiobacillus denitrificans ... 637938804 Methylobacillus flagellatus KT 637170156 Methylococcus capsulatus st... 637737217 Nitrosococcus oceani ATCC 1... 640525883 Dichelobacter nodosus VCS1703A 639757826 Candidatus Ruthia magnifica... 76 56 100 100 100 98 93 61 100 81 100 100 62 100 49 39 40 100 96 73 22 24 99 100 100 71 100 100 100 98 72 88 100 100 99 91 100 100 78 63 99 66 40 59 29 49 99 7 10 7 39 38 77Appendix 21 (continued):

PAGE 107

99 640537617 Candidatus Vesicomyosocius ... 637727433 Burkholderia pseudomallei 1... 639846934 Burkholderia mallei SAVP1 640097642 Burkholderia mallei NCTC 10229 640148684 Burkholderia mallei NCTC 10247 637561134 Burkholderia mallei ATCC 23344 637566765 Burkholderia pseudomallei K... 640126742 Burkholderia pseudomallei 668 640134048 Burkholderia pseudomallei 1... 637840320 Burkholderia thailandensis ... 641313069 Burkholderia multivorans AT... 640188535 Burkholderia vietnamiensis G4 637764294 Burkholderia sp. 383 638150533 Burkhol deria cepacia AMMD 641682344 Burkholde ria ambifaria MC40-6 639694714 Burkholderia cenocepacia HI... 638015316 Burkholderia cenocepacia AU... 641639269 Burkholderia cenocepacia MC0-3 637948955 Burkholderia xenovorans LB400 637236914 Ralstonia solanacearum GMI1000 637975845 Ralstonia metallidurans CH34 641669674 Cupriavidus taiwanensis 637689922 Ralstonia eutropha JMP134 640446671 Ralstonia eutropha H16 637104225 Bordetella bronchiseptica RB50 637109717 Bordetella parapertussis 12822 637113706 Bordetella pertussis Tohama I 641362783 Bordetella petrii 641723067 Bordetella avium 197N 637607906 Azoarcus sp. EbN1 639788494 Azoarcus sp. BH72 637681835 Dechloromonas aromatica RCB 640472003 Polynucleobacter sp. QLW-P1... 641672070 Polynucleobacter necessariu... 639850494 Verminephrobacter eiseniae ... 637918769 Rhodoferax ferrireducens T118 639825879 Acidovorax avenae subsp. ci... 639841087 Acidovorax sp. JS42 641295948 Delftia acidovorans SPH-1 640088618 Methylibium petroleiphilum PM1 637944330 Polaromonas sp. JS666 639836969 Polaromonas naphthalenivora... 641663243 Leptothrix cholodnii SP-6 641665477 Leptothrix cholodnii SP-6 640167701 Herminiim onas arsenicoxydans 640815813 Janthinobacterium sp. Marse... 637812084 Nitrosospira multiformis AT... 637426430 Nitrosomonas europaea ATCC ... 638130468 Nitrosomonas eutropha C71 641353592 Sorangium cellulosum So ce 56 637779362 Geobacter metallireducens G... 637779551 Geobacter metallireducens G... 640551830 Geobacter uraniumreducens Rf4 640547655 Geobacter uraniumreducens Rf4 640550971 Geobacter uraniumreducens Rf4 637484236 Bdellovibrio bacteriovorus ... 100 100 100 68 70 94 86 85 90 69 83 100 99 99 37 83 93 89 97 100 100 100 68 89 91 100 58 98 78 98 100 70 100 100 80 100 48 82 61 98 100 99 56 98 100 100 44 21 17 36 63 2Appendix 21 (continued):

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100 637484236 Bdellovibrio bacteriovorus ... 638024899 Myxococcus xanthus DK 1622 640845244 Anaeromyxobacter sp. Fw109-5 637863164 Anaeromyx obacter dehalogena... 640843695 Anaeromyxobacter sp. Fw109-5 639722490 Magnetococcus sp. MC-1 640953755 Rickettsia rickettsii str. ... 641529257 Rickettsia rickettsii str. ... 637193838 Rickettsia conorii str. Mal... 641233956 Rickettsia massiliae MTU5 637661032 Rickettsia felis URRWXCal2 640952447 Rickettsia akari str. Hartford 637029633 Rickettsia prowazekii str. ... 637530985 Rickettsia typhi str. Wilmi... 640951356 Rickettsia canadensis str. ... 637931354 Rickettsia bellii RML369-C 640954724 Rickettsia bellii OSU 85-389 640567195 Orientia tsutsugamushi Boryong 637671100 Candidatus Pelagibacter ubi... 637900406 Neorickettsia sennetsu str.... 637429918 Anaplasma marginale str. St... 637899857 Anaplasma phagocytophilum HZ 637173357 Wolbachia endosymbiont of D... 637630171 Wolbachia endosymbiont stra... 637698087 Ehrlichi a canis str. Jake 637902096 Ehrlichia chaffeensis str. ... 637627805 Ehrlichia ruminantium str. ... 639304190 Ehrlichia ruminantium str. ... 639313645 Ehrlichia ruminantium str. ... 638129876 Granulobact er bethesdensis ... 640553539 Acidiphilium cryptum JF-5 641336900 Gluconacetobacter diazotrop... 637979779 Ralstonia metallidurans CH34 640877026 Parvibaculum lavamentivoran... 637822571 Magnetospirillum magneticum... 637825301 Rhodospirillum rubrum ATCC ... 638005027 Silicibacter sp. TM1040 meg... 639635052 Roseobacter denitrificans O... 637287838 Silicibacter pomeroyi DSS-3 641268808 Dinoroseobacter shibae DFL 12 637903174 Jannaschia sp. CCS1 639767876 Paracoccus denitrificans PD... 640481632 Rhodobacter sphaeroides ATC... 640072193 Rhodobacter sphaeroides 2.4.1 640113226 Rhodobacter sphaeroides ATC... 637611984 Zymomonas mobilis subsp. mo... 640443917 Novosphingobium aromaticivo... 640580846 Sphingomonas wittichii RW1 638010607 Sphingopyxis alaskensis RB2256 637856869 Erythrobacter litoralis HTC... 640518187 Bradyrhizobium sp. ORS278 640555413 Bradyrhizobium sp. BTAi1 637367998 Bradyrhizobium japonicum US... 637711961 Nitrobacter winogradskyi Nb... 637969073 Nitrobacter hamburgensis X14 639317921 Rhodopseudomonas palustris ... 637923305 Rhodo p seudomonas p alustris ... 92 100 98 100 100 100 74 94 100 99 74 99 100 89 90 100 100 100 100 100 100 99 61 78 38 79 30 50 100 90 95 75 100 100 59 96 96 83 68 73 99 60 95 86 51 44 41 49 48 65 58 84 15 6 7Appendix 21 (continued):

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101 pp 637473315 Rhodopseudomonas palustris ... 637882162 Rhodopseudo monas palustris ... 637960726 Rhodopseudom onas palustris ... 640879400 Xanthobact er autotrophicus Py2 641261652 Azorhizobium caulinodans OR... 640879738 Xanthobacter autotrophicus Py2 641642391 Methylobacterium sp. 4-46 641367443 Methylobacterium extorquens... 641626630 Methylobacterium radiotoler... 641707560 Beijerinckia indica subsp. ... 637086371 Caulobacter crescentus CB15 641560714 Caulobacter sp. K31 638142884 Hyphomonas neptunium ATCC 1... 638142313 Maricaulis maris MCS10 637076672 Mesorhizobium loti MAFF303099 638069291 Mesorhizobium sp. BNC1 637183886 Sinorhizobium meliloti 1021 640791893 Sinorhizobium medicae WSM419 639292710 Agrobacteri um tumefaciens s... 639298123 Agrobacteri um tumefaciens s... 639650318 Rhizobium leguminosarum bv.... 640440897 Rhizobium etli CFN 42 637511331 Bartonella henselae str. Ho... 641344102 Bartonella tribocorum CIP 1... 637509792 Bartonella quintana str. To... 639841224 Bartonella bacilliformis KC583 640833577 Ochrobactrum anthropi ATCC ... 640578809 Brucella ovis ATCC 25840 641328098 Brucella canis ATCC 23365 637330863 Brucella suis 1330 641360441 Brucella suis ATCC 23445 637814721 Brucella melitensis biovar ... 637247150 Brucella melitensis 16M 637645891 Brucella abortus biovar 1 s... 1EUD|A S. scrofa P53401.1 T. vaginalis 639702471 Syntrophoba cter fumaroxidan... 641262650 Candidatus Desulfococcus ol... 637527535 Desulfotalea psychrophila L... 637985898 Acidobacteria bacterium Ell... 639682915 Solibacter usitatus Ellin6076 637858319 Syntrophus aciditrophicus SB 639320831 Rhodopseudomonas palustris ... 639660670 Candidatus Carso n 637860763 Syntrophus aciditrophicus SB 637749827 Pelobacter carbinolicus DSM... 639755763 Pelobacter propionicus DSM ... 640551478 Geobacter uraniumreducens Rf4 637125728 Geobacter sulfurreducens PCA 637778008 Geobacter metallireducens G... 637170902 Methylococcus capsulatus st... 641367599 Methylobac terium extorquens... 641629376 Methylobacterium radiotoler... 641646646 Methylobacterium sp. 4-46 638127806 Granulobacter bethesdensis ... 640091829 Meth y libium p etrolei p hilum PM1 100 100 100 100 88 100 45 55 51 100 97 99 100 100 100 100 95 99 100 73 96 54 61 100 89 99 28 99 58 100 74 100 87 78 100 69 83 100 100 100 76 58 33 87 98 77 38 99 94 19 77 21 6 52Appendix 21:

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102 ypp 637428148 Nitrosomonas europaea ATCC ... 638130849 Nitrosomonas eutropha C71 637811164 Nitrosospira multiformis AT... 641268416 Dinoroseobacter shibae DFL 12 637074382 Mesorhizobium loti MAFF303099 639634625 Roseobacter denitrificans O... 637289051 Silicibacter pomeroyi DSS-3 639637521 Roseobacter denitrificans O... epsilon outgroup 100 100 100 100 35 47 53 100 98 0.1Appendix 21:

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103 637062499 Escherichia coli O157 637932424 Escherichia coli UTI89 640753635 Escherichia coli APEC O1 640924463 Escherichia coli E24377A 638061877 Escherichia coli 536 637422744 Shigella flexneri 2a str. 2457 637332853 Shigella flexneri 2a str. 301 638074417 Shigella flexneri 5 str. 8401 637354656 Escherichia coli CFT073 E. coli sdhA (type C) 641606321 Escherichia coli str. K-12 sub 637000712 Escherichia coli W3110 DNA 637014087 Escherichia coli K12 640433064 Shigella dysenteriae Sd197 640428504 Shigella sonnei Ss046 641724839 Shigella boydii CDC 3 083-94 641615625 Escherichia coli SECEC SMS-3-5 640919947 Escherichia coli HS 641602344 Escherichia coli ATCC 8739 637806367 Shigella boydii Sb227 637081399 Escherichia coli O157 640797773 Klebsiella pneumoniae subsp. p 637215983 Salmonella enterica subsp. ent 637405889 Salmonella enterica subsp. ent 637639603 Salmonella enterica subsp. ent 637602218 Salmonella enterica subsp. ent 641305372 Salmonella enterica subsp. ari 637211479 Salmonella typhimurium LT2 641323231 Salmonella enterica subsp. ent 640916401 Citrobacter koseri ATCC BAA-89 640491406 Enterobacter sp. 638 640900775 Enterobacter sakazakii ATCC BA 640936792 Serratia proteamaculans 568 637377822 Erwinia carotovora subsp. atro 640076434 Yersinia enterocolitica subsp. 637311517 Yersinia pestis KIM 637532761 Yersinia pseudotuberculosis IP 641339341 Yersinia pestis Angola 640865883 Yersinia pseudotuberculosis IP 637200194 Yersinia pestis CO92 641598045 Yersinia pseudotuberculosis YP 638042670 Yersinia pestis Antiqua 638040934 Yersinia pestis Nepal516 637491733 Yersinia pestis biovar Microtu 640479441 Yersinia pestis Pestoides F 637463241 Photorhabdus luminescens subsp 637852822 Sodalis glossinidius str. mors 637360576 Vibrio vulnificus CMCP6 637467780 Vibrio vulnificus YJ016 637397814 Vibrio parahaemolyticus RIMD 2 640906845 Vibrio harveyi ATCC BAA1116 637048562 Vibrio cholerae O1 biovar elto 640533132 Vibrio cholerae O395 637635494 Vibrio fischeri ES114 637585755 Photobacterium profundum SS9 639714502 Aeromonas hydrophila subsp. hy 641524906 Aeromonas salmonicida subsp. s 100 100 99 100 75 100 88 100 78 99 100 100 100 47 77 46 52 95 50 68 52 99 100 71 88 53 86 64 65 100Appendix 22: Phylogeny of succinate dehydrogenase/fumarate reductase sequences, using Type D succinate dehydrogenase/fumarate reductase sequences as an outgroup.

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104 p 637606043 Idiomarina loihiensis L2TR 638054807 Pseudoalteromonas atlantica T6 639797394 Psychromonas ingrahamii 37 637284674 Colwellia psychrerythraea 34H 637734557 Pseudoalteromonas haloplanktis 639780775 Shewanella amazonensis SB2B 640933735 Shewanella sediminis HAW-EB3 641633338 Shewanella woodyi ATCC 51908 640163215 Shewanella loihica PV-4 641236376 Shewanella pealeana ATCC 70034 641553318 Shewanella halifaxensis HAW-EB 637956209 Shewanella denitrificans OS217 638135142 Shewanella frigidimarina NCIMB 640119740 Shewanella baltica OS155 641288924 Shewanella baltica OS195 640830723 Shewanella baltica OS185 637343813 Shewanella oneidensis MR-1 639815808 Shewanella sp. W3-18-1 640499433 Shewanella putrefaciens CN-32 638122366 Shewanella sp. MR-7 638118245 Shewanella sp. MR-4 639726291 Shewanella sp. ANA-3 637341803 Wigglesworthia glossinidia end 637443800 Candidatus Blochmannia florida 637672774 Candidatus Blochmannia pennsyl 637835014 Hahella chejuensis KCTC 2396 639811118 Marinobacter aquaeolei VT8 640805176 Marinomonas sp. MWYL1 638079600 Alcanivorax borkumensis SK2 637966393 Chromohalobacter salexigens DS 637921198 Saccharophagus degradans 2-40 640487825 Pseudomonas stutzeri A1501 637051977 Pseudomonas aeruginosa PAO1 639326103 Pseudomonas aeruginosa UCBPP-P 640812741 Pseudomonas aeruginosa PA7 640503749 Pseudomo nas mendocina ymp 637393578 Pseudomonas syringae pv. tomat 637487550 Pseudomonas syringae pv. phase 637652356 Pseudomonas syringae pv. syrin 637319055 Pseudomonas fluorescens Pf-5 637741466 Pseudomonas fluorescens PfO-1 638002576 Pseudomonas entomophila L48 641548884 Pseudomonas putida GB-1 641623301 Pseudomonas putida W619 637147590 Pseudomonas putida KT2440 640586133 Pseudomonas putida F1 637130472 Legionella pneumophila subsp. 640571053 Legionella pneumophila str. Co 637579292 Legionella pneumophila str. Pa 637582351 Legionella pneumophila str. Le 641556386 Francisella philomiragia subsp 637614055 Francisella tularensis subsp. 640189216 Francisella tularensis subsp. 638059559 Francisella tularensis subsp. 641732636 Francisella tularensis subsp. 639753673 Francisella tularensis subsp. 640891972 Francisella tularensis subsp. 637908243 Francisella tularensis subs p 93 64 100 100 96 100 99 59 91 80 100 100 74 100 84 94 99 89 53 100 99 100 48 58 84 61 99 65 100 92 90 100 100 100 100 77 99 97 41 79 93 80 45 31 93 100 98 95 94 100 40 57 22 50 78 Appendix 22 (continued):

PAGE 113

105 638147594 Francisella tularensis subsp. 640888497 Coxiella burnetii Dugway 7E9-1 637163060 Coxiella burnetii RSA 493 641330950 Coxiella burnetii RSA 331 637738950 Nitrosococcus oceani ATCC 1970 637786778 Thiomicrospira crunogena XCL-2 637709868 Thiobacillus denitrificans ATC 637197866 Neisseria meningitidis Z2491 639827835 Neisseria meningitidis FAM18 641332684 Neisseria meningitidis 053442 637195592 Neisseria meningitidis MC58 637142162 Neisseria gonorrhoeae FA 1090 637451840 Chromobacterium violaceum ATCC 637681332 Dechloromonas aromatica RCB 637611101 Azoarcus sp. EbN1 639786692 Azoarcus sp. BH72 637810943 Nitrosospira multiformis ATCC 637427415 Nitrosomonas europaea ATCC 197 638132695 Nitrosomonas eutropha C71 641583339 Acinetobacter baumannii 641589586 Acinetobacter baumannii AYE 640154889 Acinetobacter baumannii ATCC 1 637519292 Acinetobacter sp. ADP1 640598473 P sychrobacter sp. PRwf-1 637668784 Psychrobacter arcticus 273-4 637972551 Psychrobacter cryohalolentis K 637110252 Bordetella parapertussis 12822 637113534 Bordetella pertussis Tohama I 637105827 Bordetella bronchiseptica RB50 641721940 Bordetella avium 197N 641362540 Bordetella petrii 640169085 Herminiimonas arsenicoxydans 640816956 Janthinobacterium sp. Marseill 637943261 Polaromonas sp. JS666 639836047 Polaromonas naphthalenivorans 640090736 Methylibium petroleiphilum PM1 641665481 Leptothrix cholodnii SP-6 637916622 Rhodoferax ferrireducens T118 R. fermentans frdA (type C) 639852097 Verminephrobacter eiseniae EF0 639823407 Acidovorax avenae subsp. citru 639839852 Acidovorax sp. JS42 641297507 Delftia acidovorans SPH-1 640470917 Polynucleobacter sp. QLW-P1DMW 641671673 Polynucleobacter necessarius S 637691728 Ralstonia eutropha JMP134 640448720 Ralstonia eutropha H16 637977883 Ralstonia meta llidurans CH34 637238388 Ralstonia solanacearum GMI1000 637949816 Burkholderia xenovorans LB400 637731045 Burkholderia pseudomallei 1710 637565624 Burkholderia mallei ATCC 23344 640132407 Burkholderia pseudomallei 668 639844138 Burkholderia mallei SAVP1 640094879 Burkholderia mallei NCTC 10229 640145437 Burkholderia pseudomallei 1106 640146777 Burkholderia mallei NCTC 10247 637571150 Burkholderia pseudomallei K962 type C 100 100 97 100 47 33 46 100 100 72 93 100 100 100 100 98 65 99 100 64 96 100 100 100 92 99 54 100 100 54 99 99 97 97 77 49 95 90 95 55 100 100 37 100 99 87 44 47 90 94 99 90 55 99Appendix 22 (continued):

PAGE 114

106 637571150 Burkholderia pseudomallei K962 637837967 Burkholderia thailandensis E26 641318944 Burkholderia multivorans ATCC 641320127 Burkholderia multivorans ATCC 637766996 Burkholderia sp. 383 640184721 Burkholderia vietnamiensis G4 638151155 Burkholderia cepacia AMMD 641683600 Burkholderia ambifaria MC40-6 641650699 Burkholderia cenocepacia MC0-3 638017718 Burkholderia cenocepacia AU 10 639695995 Burkholderia cenocepacia HI242 640953322 Rickettsia rickettsii str. She 641528802 Rickettsia rickettsii str. Iow 637193401 Rickettsia conorii str. Malish 641233627 Rickettsia mass iliae MTU5 637661542 Rickettsia felis URRWXCal2 640952031 Rickettsia akari str. Hartford 637530683 Rickettsia typhi str. Wilmingt 637029331 Rickettsia prowazekii str. Mad R. prowazekii sdhA (type C) 637931290 Rickettsia bellii RML369-C 640954791 Rickettsia bellii OSU 85-389 640950845 Rickettsia canadensis str. McK 640568075 Orientia tsutsugamushi Boryong 637671105 Candidatus Pelagibacter ubique 637900532 Neorickettsia sennetsu str. Mi 637172641 Wolbachia endosymbiont of Dros 637630097 Wolbachia endosymbiont strain 637899003 Anaplasma phagocytophilum HZ 637899006 Anaplasma phagocytophilum HZ 637429881 Anaplasma marginale str. St. M 637698634 Ehrlichia canis str. Jake 637901439 Ehrlichia chaffeensis str. Ark 637628395 Ehrlichia ruminantium str. Gar 639304741 Ehrlichia ruminantium str. Wel 639314239 Ehrlichia ruminantium str. Wel 638011418 Sphingopyxis alaskensis RB2256 640444487 Novosphingobium aromaticivoran 637856700 Erythrobacter litoralis HTCC25 640580857 Sphingomonas wittichii RW1 637295326 Xanthomonas axonopodis pv. cit 637757957 Xanthomonas campestris pv. ves 637632538 Xanthomonas oryzae pv. oryzae 637849694 Xanthomonas oryzae pv. oryzae 637274234 Xanthomonas campestris pv. cam 637657482 Xanthomonas campestris pv. cam 637043525 Xylella fastidiosa 9a5c 641648453 Xylella fastidiosa M12 637383178 Xylella fastidiosa Temecula1 641701878 Xylella fastidiosa M23 638126087 Alkal ilimnicola ehrlichei MLHE 639854502 Halorhodospira halophila SL1 637170723 Methylococcus capsulatus str. 637822564 Magnetospir illum magneticum AM 637825293 Rhodospir illum rubrum ATCC 111 641335350 Gluconacetobacter diazotrophic 638129885 Granulobacter bethesdensis CGD 640553803 Acidiphilium cryptum JF-5 637882211 Rhodopseudomonas palustris HaA 100 87 100 100 99 100 81 99 61 100 100 100 100 100 100 91 100 64 97 100 87 92 67 100 61 100 100 98 99 94 97 53 100 100 100 84 77 87 95 91 52 69 100 99 100 89 40 51 92 71 35 26 61 44 100Appendix 22 (continued):

PAGE 115

107 pp 637960666 Rhodopseudomonas palustris Bis 637473342 Rhodopseudomonas palustris CGA 637923186 Rhodopseudomonas palustris Bis 639318138 Rhodopseudomonas palustris Bis 637368059 Bradyrhizobium japonicum USDA 640518032 Bradyrhizobium sp. ORS278 640555255 Bradyrhizobium sp. BTAi1 637714367 Nitrobacter winogradskyi Nb-25 637972030 Nitrobacter hamburgensis X14 640877008 Parvibaculum lavamentivorans D 640881217 Xanthobacter autotrophicus Py2 641261380 Azorhizobium caulinodans ORS 5 641706698 Beijerinckia indica subsp. ind 641646139 Methylobacterium sp. 4-46 641369395 Methylobacterium extorquens PA 641626166 Methylobacterium radiotolerans 637089587 Caulobacter crescentus CB15 641565358 Caulobacter sp. K31 638142341 Maricaulis maris MCS10 638145802 Hyphomonas neptunium ATCC 1544 637287852 Silicibacter pomeroyi DSS-3 638005042 Silicibacter sp. TM1040 mega p 641268791 Dinoroseobacter shibae DFL 12 639635069 Roseobacter denitrificans OCh 639767887 Paracoccus denitrificans PD122 640481642 Rhodobacter sphaeroides ATCC 1 640072203 Rhodobacter sphaeroides 2.4.1 640113236 Rhodobacter sphaeroides ATCC 1 637903156 Jannaschia sp. CCS1 637511258 Bartonella henselae str. Houst 641343984 Bartonella tribocorum CIP 1054 637509731 Bartonella quintana str. Toulo 639841290 Bartonella bac illiformis KC583 638069137 Mesorhizobium sp. BNC1 637183902 Sinorhizobium me liloti 1021 640791908 Sinorhizobium medicae WSM419 639292717 Agrobacterium tumefaciens str. 639298129 Agrobacterium tumefaciens str. 639650326 Rhizobium leguminosarum bv. vi 640440903 Rhizobium etli CFN 42 637076641 Mesorhizobium loti MAFF303099 640833598 Ochrobactrum anthropi ATCC 491 637330841 Brucella suis 1330 641328074 Brucella canis ATCC 23365 637247172 Brucella melitensis 16M 637645870 Brucella abortus biovar 1 str. 637814700 Brucella melitensis biovar Abo 640578786 Brucella ovis ATCC 25840 641360418 Brucella suis ATCC 23445 639702871 Syntrophobacter fumaroxidans M M. tuberculosis sdhA (type A) 639700929 Syntrophobacter fumaroxidans M H. sp. NRC-1 sdhA (type A) 639721178 Magnetococcus sp. MC-1 637793454 Thiomicrospira denitrificans A 640819972 Nitratiruptor sp. SB155-2 640822359 Sulfurovum sp. NBC37-1 639727073 Shewanella s p ANA-3 92 100 100 100 100 100 65 100 88 53 46 100 100 100 100 100 93 100 100 100 95 100 100 99 100 99 35 82 100 80 97 65 81 100 100 54 100 56 100 91 53 75 78 100 47 96 39 80 95 33 96 78 72 60 Appendix 22 (continued):

PAGE 116

108 S. sp. PCC 6803 sdhA (type E) type B type E S. acidocaldarius sdhA (type E) A. fulgidus frdA (type A) T. acidophilum frdA (type A) type D 100 98 80 60 42 41 39 29 19 10 60 0.2Appendix 22 (continued):

PAGE 117

109 637082954 Escherichia coli O157 640925495 Escherichia coli E24377A 640429336 Shigella sonnei Ss046 640920834 Escherichia coli HS 641601431 Escherichia coli ATCC 8739 641725867 Shigella boydii CDC 3083-94 637807256 Shigella boydii Sb227 640434169 Shigella dysenteriae Sd197 637064129 Escherichia coli O157 638075374 Shigella flexneri 5 str. 8401 637423777 Shigella flexneri 2a str. 2... 637333873 Shigella flexneri 2a str. 301 638062692 Escherichia coli 536 637355839 Escherichia coli CFT073 637933504 Escherichia coli UTI89 640754532 Escherichia coli APEC O1 641607223 Escherichia coli str. K-12 ... 637014970 Escherichia coli K12 2FUS|A Escherichia coli 637001595 Escherichia coli W3110 DNA 641616465 Escherichia coli SECEC SMS-3-5 640915586 Citrobacter koseri ATCC BAA... 641304673 Salmonella enterica subsp. ... 637212205 Salmonella typhimurium LT2 641322285 Salmonella enterica subsp. ... 637640363 Salmonella enterica subsp. ... 637601622 Salmonella enterica subsp. ... 637405123 Salmonella enterica subsp. ... 637216762 Salmonella enterica subsp. ... 640492023 Enterobacter sp. 638 640900118 Enterobacter sakazakii ATCC... 640798558 Klebsiella pneumoniae subsp... 637378723 Erwinia carotovora subsp. a... 637853423 Sodalis glossinidius str. m... 637464139 Photorhabdus luminescens su... 640937818 Serratia proteamaculans 568 640075541 Yersinia enterocolitica sub... 641597054 Yersinia pseudotuberculosis... 637533806 Yersinia pseudotuberculosis... 640864857 Yersinia pseudotuberculosis... 640477720 Yersinia pestis Pestoides F 638043720 Yersinia pestis Antiqua 641340167 Yersinia pestis Angola 637310550 Yersinia pestis KIM 637201288 Yersinia pestis CO92 638039769 Yersinia pestis Nepal516 637492729 Yersinia pestis biovar Micr... 637981772 Baumannia cicadellinicola s... 637341721 Wigglesworthia glossinidia ... 637443844 Candidatus Blochmannia flor... 637672819 Candidatus Blochmannia penn... 637812562 Nitrosospira multiformis AT... 637428605 Nitrosomonas europaea ATCC ... 638132347 Nitrosomonas eutropha C71 639693121 Burkholderia cenocepacia HI... 641637619 Burkholderia cenocepacia MC... 638013875 Burkholderia cenocepacia AU... 637762487 Burkholderia sp. 383 chromo... 66 100 100 52 83 99 95 97 35 58 45 48 100 47 98 66 69 97 27 56 66 90 98 54 100 90 49 63 98 90 70 84 65 65 100 54 39 42 53Appendix 23: Phylogeny of class II fumarase sequences, using genes encoding argininosuccinate lyase as an outgroup.

PAGE 118

110 p 640186766 Burkholderia vietnamiensis ... 638148768 Burkholderia cepacia AMMD c... 641680720 Burkholderia ambifaria MC40... 641314656 Burkholderia multivorans AT... 637948599 Burkholderia xenovorans LB4... 637841354 Burkholderia thailandensis ... 640128754 Burkholderia pseudomallei 6... 637568453 Burkholderia pseudomallei K... 640136112 Burkholderia pseudomallei 1... 640096141 Burkholderia mallei NCTC 10... 637561328 Burkholderia mallei ATCC 23... 639846776 Burkholderia mallei SAVP1 c... 640148927 Burkholderia mallei NCTC 10... 637729396 Burkholderia pseudomallei 1... 638027754 Myxococcus xanthus DK 1622 639715869 Aeromonas hydrophila subsp.... 641523613 Aeromonas salmonicida subsp... 639850978 Verminephrobacter eiseniae ... 639838176 Acidovorax sp. JS42 637241142 Ralstonia solanacearum GMI1... 641297750 Delftia acidovorans SPH-1 639822694 Acidovorax avenae subsp. ci... 639835888 Polaromonas naphthalenivora... 637941719 Polaromonas sp. JS666 640091905 Methylibium petroleiphilum PM1 637822262 Magnetospirillum magneticum... 637681404 Dechloromonas aromatica RCB 640169815 Herminiimonas arsenicoxydans 640818071 Janthinobacterium sp. Marse... 637451898 Chromobacterium violaceum A... 641721488 Bordetella avium 197N 641361538 Bordetella petrii 637111643 Bordetella pertussis Tohama I 637106206 Bordetella bronchiseptica RB50 637110640 Bordetella parapertussis 12822 641353581 Sorangium cellulosum So ce 56 CAA25849.1 Bacillus subt ilis 639746344 Campylobacter fetus subsp. ... 640871779 Campylobacter curvus 525.92 640949102 Arcobacter butzleri RM4018 640821738 Sulfurovum sp. NBC37-1 637793474 Thiomicrospira denitrifican... 638021128 Helicobacter pylori HPAG1 637023255 Helicobacter pylori J99 637019081 Helicobacter pylori 26695 638057469 Helicobacter acinonychis st... 640861404 Campylobacter jejuni subsp.... 637292909 Campylobacter jejuni RM1221 640942228 Campylobacter jejuni subsp.... 637040898 Campylobacter jejuni subsp.... 639854092 Campylobacter jejuni subsp.... 637581706 Legionella pneumophila str.... 640571521 Legionella pneumophila str.... 637132869 Legionella pneumophila subs... 637584642 Legionella pneumophila str.... 640889072 Coxiella burnetii Dugway 7E... 637162765 Coxiella burnetii RSA 493 641330673 Coxiella burnetii RSA 331 68 48 83 100 73 85 100 59 100 95 57 100 63 100 78 100 99 90 67 100 100 100 49 12 49 6 47 99 80 44 83 100 97 62 93 100 39 62 25 100 90 79 2 65 39 47 55 80 59 51 42 77 62 16 72 18 17Appendix 23 (continued):

PAGE 119

111 637319479 Pseudomonas fluorescens Pf-5 637609343 Azoarcus sp. EbN1 640525084 Dichelobacter nodosus VCS1703A 640154162 Acinetobacter baumannii ATC... 641590323 Acinetobacter baumannii AYE 637518397 Acinetobacter sp. ADP1 637670353 P sychr obacter arcticus 273-4 637974366 P sychr obacter cryohalolenti... 640600207 Psychrobacter sp. PRwf-1 637142253 Neisseria gonorrhoeae FA 1090 637196076 Neisseria meningitidis MC58 639828227 Neisseria meningitidis FAM18 637198345 Neisseria meningitidis Z2491 641333155 Neisseria meningitidis 053442 637481913 Bdellovibrio bacteriovorus ... 640828721 Shewanella baltica OS185 641286799 Shewanella baltica OS195 640121065 Shewanella baltica OS155 639817661 Shewanella sp. W3-18-1 640497725 Shewanella putrefaciens CN-32 640123465 Actinobac illus pleur opneumo... 641531835 Actinobacillus pleuropneumo... 637129903 Haemophilus ducreyi 35000HP 637550262 Mannheimia succiniciproduce... 640807878 Actinobacillus succinogenes... 638104665 Haemophilus somnus 129PT 641658037 Haemophilus somnus 2336 637070428 Pasteurella multocida subsp... 640609581 Haemophilus influenzae PittEE 637663500 Haemophilus influenzae 86-0... 637008576 Haemophilus influenzae Rd KW20 640610534 Haemophilus influenzae PittGG 637917182 Rhodoferax ferrireducens T118 640444437 Novosphingobium aromaticivo... 640582269 Sphingomonas wittichii RW1 638011537 Sphingopyxis alaskensis RB2256 637051241 Pseudomonas aeruginosa PAO1 639326864 Pseudomonas aeruginosa UCBP... 640813712 Pseudomonas aeruginosa PA7 640487637 Pseudomonas stutzeri A1501 640502918 Pseudomonas mendocina ymp 637489191 Pseudomonas syringae pv. ph... 637654020 Pseudomonas syringae pv. sy... 637393103 Pseudomonas syringae pv. to... 637321665 Pseudomonas fluorescens Pf-5 637743978 Pseudomonas fluorescens PfO-1 637145158 Pseudomonas putida KT2440 640588432 Pseudomonas putida F1 641546449 Pseudomonas putida GB-1 638000529 Pseudomonas entomophila L48 641621136 Pseudomonas putida W619 640954168 Rickettsia rickettsii str. ... 641529678 Rickettsia rickettsii str. ... 637194267 Rickettsia conorii str. Mal... 641234268 Rickettsia massiliae MTU5 637660632 Rickettsia felis URRWXCal2 640952813 Rickettsia akari str. Hartford 640951594 Rickettsia canadensis str. ... 100 84 100 90 94 99 100 83 94 73 100 94 57 100 40 54 100 75 93 96 44 100 100 100 59 100 100 100 69 84 89 100 82 77 67 75 100 100 50 24 35 100 78 100 94 82 100 100 78 100 77 35 70 18 12 0 8 10Appendix 23 (continued):

PAGE 120

112 640951594 Rickettsia canadensis str. ... 637029862 Rickettsia prowazekii str. ... 637531222 Rickettsia typhi str. Wilmi... 637931190 Rickettsia be llii RM L369-C 640954896 Rickettsia bel lii OSU 85-389 640567525 Orientia tsutsugamushi Boryong 637670875 Candidatus Pelagibacter ubi... 637900212 Neorickettsia sennetsu str.... 637430444 Anaplasma marginale str. St... 637899086 Anaplasma phagocytophilum HZ 637630156 Wolbachia endosymbiont stra... 637172690 Wolbachia endosymbiont of D... 637698584 Ehrlichia canis str. Jake 637901500 Ehrlichia chaffeensis str. ... 637628341 Ehrlichia ruminantium str. ... 639304694 Ehrlichia ruminantium str. ... 639314186 Ehrlichia ruminantium str. ... 637529697 Desulfotalea p sychrophila L... 641707191 Beijerinckia indica subsp. ... 637250185 Brucella melitensis 16M chr... 640576243 Brucella ovis ATCC 25840 ch... 637331311 Brucella suis 1330 chromoso... 637817725 Brucella melitensis biovar ... 637646315 Brucella abortus biovar 1 s... 641328530 Brucella canis ATCC 23365 c... 641354005 Brucella suis ATCC 23445 ch... 640835699 Ochrobactrum anthropi ATCC ... 638066237 Mesorhizobium sp. BNC1 639842341 Bartonella baci lliformis KC583 641343779 Bartonella tribocorum CIP 1... 637511051 Bartonella henselae str. Ho... 637509567 Bartonella quintana str. To... 640560820 Bradyrhizobium sp. BTAi1 640523104 Bradyrhizobium sp. ORS278 637963565 Rhodopseudomonas palustris ... 637883924 Rhodopseudomonas palustris ... 637476645 Rhodopseudomonas palustris ... 637926382 Rhodopseudomonas palustris ... 639319767 Rhodopseudomonas palustris ... 637374108 Bradyrhizobium japonicum US... 637971185 Nitrobacter hamburgensis X14 637713917 Nitrobacter winograd skyi Nb... 641368465 Methylobacterium extorquens... 641630235 Methylobacterium radiotoler... 641641385 Methylobacterium sp. 4-46 638141547 Maricaulis maris MCS10 640070306 Rhodobacter sphaeroides 2.4... 640111396 Rhodobacter sphaeroides ATC... 640482279 Rhodobacter sphaeroides ATC... 639769228 Paracoccus denitrificans PD... 637904752 Jannaschia sp. CCS1 639635681 Roseobacter denitrificans O... 637289385 S ilicibacter pomeroyi DSS-3 638006763 S ilicibacter sp. TM 1040 641268258 Dinoroseobacter shibae DFL 12 638145508 Hyphomonas neptunium ATCC 1... 640880913 Xanthobacter autotrophicus Py2 641260187 Azorhizobium caulinodans OR... 640878021 Parvibaculum lavamentivoran... 100 100 90 100 76 100 100 99 94 22 12 71 100 100 100 100 80 100 100 100 99 98 100 99 97 100 99 82 67 77 65 72 37 100 55 45 100 69 100 99 100 86 64 92 80 76 39 60 36 47 18 30 82 43 40 28 12Appendix 23 (continued):

PAGE 121

113 640878021 Parvibaculum lavamentivoran ... 637088153 Caulobacter crescentus CB15 641563873 Caulobacter sp. K31 637182673 Sinorhizobium me liloti 1021 640790520 Sinorhizobium medicae WSM419 637079796 Mesorhizobium loti MAFF303099 639648579 Rhizobium leguminosarum bv.... 640439406 Rhizobium etli CFN 42 639291731 Agrobacterium tumefaciens s... 639297104 Agrobacterium tumefaciens s... 641353565 Sorangium cellulosum So ce 56 641630575 Methylobacterium radiotoler... 638128822 Granulobacter bethesdensis ... 637474463 Rhodopseudomonas palustris ... 641259517 Azorhizobium caulinodans OR... 640553354 Acidiphilium cryptum JF-5 640471791 Polynucleobacter sp. QLW-P1... 639683824 Solibacter usitatus Ellin6076 637367829 Bradyrhizobium japonicum US... 637624392 Gluconobacter oxydans 621H 641367249 Methylobacterium extorquens... 641556006 Francisella philomiragia su... 639752269 Francisella tularensis subs... 638146305 Francisella tularensis subs... 640890274 Francisella tularensis subs... 637906806 Francisella tularensis subs... 641733805 Francisella tularensis subs... 640190573 Francisella tularensis subs... 638059772 Francisella tularensis subs... 637614267 Francisella tularensis subs... 637121281 Desulfovibrio vulgaris subs... 639821142 Desulfovibrio vulgaris subs... 637826231 Rhodospir illum rubrum ATCC ... 637738102 Nitrosococcus oceani ATCC 1... 637781100 Desulfovibrio desulfuricans... 637981175 Ralstonia meta llidurans CH3... 637694942 Ralstonia eutropha JMP134 c... 640449937 Ralstonia eutropha H16 chro... 641670319 Cupriavidus taiwanensis 638069515 Mesorhizobium sp. BNC1 639852341 Verminephrobacter eiseniae ... 641296516 Delftia acidovorans SPH-1 639702869 Syntrophobacter fumaroxidan... 637749904 Pelobacter carbinolicus DSM... 638080866 Alcanivorax borkumensis SK2 639813384 Marinobacter aquaeolei VT8 638132983 Shewanella frigidimarina NC... 637786118 Thiomicrospira crunogena XCL-2 637294791 Xanthomonas axonopodis pv. ... 637757287 Xanthomonas campestris pv. ... 637632306 Xanthomonas oryzae pv. oryz... 637849439 Xanthomonas oryzae pv. oryz... 637658238 Xanthomonas campestris pv. ... 637273598 Xanthomonas campestris pv. ... 641648942 Xylella fastidiosa M12 637044012 Xylella fastidiosa 9a5c 641702339 Xylella fastidiosa M23 637383584 Xylella fastidiosa Temecula1 638126112 Alkalilimnicola ehrlichei M... 98 77 100 100 100 71 100 100 100 87 53 42 57 99 100 99 100 99 95 98 100 100 100 100 6 15 8 98 100 100 87 100 100 84 100 99 92 44 49 46 98 40 99 66 100 65 73 52 98 97 40 51 93 80 50 66 53Appendix 23 (continued):

PAGE 122

114 639854483 Halorhodospira halophila SL1 638055064 Pseudoalteromonas atlantica... 637921080 Saccharophagus degradans 2-40 637048048 Vibrio cholerae O1 biovar e... 640532635 Vibrio cholerae O395 chromo... 637399908 Vibrio parahaemolyticus RIM... 640905659 Vibrio harveyi ATCC BAA-111... 637588714 Photobacterium profundum SS... 637732924 Pseudoalteromonas haloplank... 637605454 Idiomarina loihiensis L2TR 637967309 Chromohalobacter salexigens... 637170716 Methylococcus capsulatus st... 637955660 Shewanella denitrificans OS217 639796919 P sychrom onas ingrahamii 37 639780156 Shewanella amazonensis SB2B 640161580 Shewanella loihica PV-4 640806069 Marinomonas sp. MWYL1 639785810 Azoarcus sp. BH72 640931547 Shewanella sediminis HAW-EB3 641635830 Shewanella woodyi ATCC 51908 637830462 Hahella chejuensis KCTC 2396 637054899 Pseudomonas aeruginosa PAO1 639327262 Pseudomonas aeruginosa UCBP... 640814130 Pseudomonas aeruginosa PA7 637318247 Pseudomonas fluorescens Pf-5 637740699 Pseudomonas fluorescens PfO-1 637489655 Pseudomonas syringae pv. ph... 637395801 Pseudomonas syringae pv. to... 638000168 Pseudomonas entomophila L48 637144339 Pseudomonas putida KT2440 640585442 Pseudomonas putida F1 641546052 Pseudomonas putida GB-1 641624065 Pseudomonas putida W619 637125146 Geobacter sulfurreducens PCA 637780370 Geobacter meta llir educens G... 640548436 Geobacter uraniumreducens Rf4 639754587 Pelobacter propionicus DSM ... 637710822 Thiobacillus denitrificans ... 637864144 Anaeromyxobacter dehalogena... 640842767 Anaeromyxobacter sp. Fw109-5 637984706 Acidobacteria bacterium Ell... 639755538 Pelobacter propionicus DSM ... 639702609 Syntrophobacter fumaroxidan... 639819619 Desulfovibrio vulgaris subs... 640805027 Marinomonas sp. MWYL1 637832216 Hahella chejuensis KCTC 2396 639780123 Shewanella amazonensis SB2B 637490603 Pseudomonas syringae pv. ph... 640804252 Marinomonas sp. MWYL1 639293982 Agrobacterium tumefaciens s... 640471919 Polynucleobacter sp. QLW-P1... 641369749 Methylobacterium extorquens... 637814751 Brucella melitensis biovar ... 637973881 P sychr obacter cryohalolenti... 640599763 P sychr obacter sp. PRwf-1 638058335 Helicobacter acinonychis st... 637431752 Helicobacter hepaticus ATCC... 637918671 Rhodoferax ferrireducens T118 100 100 100 100 99 100 100 100 76 100 65 100 100 99 100 100 100 100 99 99 100 74 75 91 98 99 81 40 48 73 45 62 58 63 99 98 71 77 81 48 46 69 69 32 42 99 12 18 26 41 57 4 6 9 95 91 82 95Appendix 23 (continued):

PAGE 123

115 637918671 Rhodoferax ferrireducens T118 639701345 Syntrophobacter fumaroxidan... 637483356 Bdellovibrio bacteriovorus ... 637341775 Wigglesworthia glossinidia ... 640905650 Vibrio harveyi ATCC BAA-111... 641306453 Salmonella enterica subsp. ... 641325870 Salmonella enterica subsp. ... 640917661 Citrobacter koseri ATCC BAA... 638065528 Escherichia coli 536 641603288 Escherichia coli ATCC 8739 638077929 Shigella flexneri 5 str. 8401 637085784 Escherichia coli O157 640923478 Escherichia coli HS 641728433 Shigella boydii CDC 3083-94 637004076 Escherichia coli W3110 DNA 640928306 Escherichia coli E24377A 637936441 Escherichia coli UTI89 640431978 Shigella sonnei Ss046 637066930 Escherichia coli O157 640436625 Shigella dysenteriae Sd197 637359081 Escherichia coli CFT073 640757293 Escherichia coli APEC O1 637426243 Shigella flexneri 2a str. 2... 637809925 Shigella boydii Sb227 637336447 Shigella flexneri 2a str. 301 640609583 Haemophilus influenzae PittEE 640610536 Haemophilus influenzae PittGG fumarase outgroup fumarase outgroup 100 100 72 99 99 68 11 100 96 100 100 90 98 99 65 83 0.2Appendix 23 (continued):


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Quasem, Ishtiaque.
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The citric acid cycle of _thiomicrospira crunogena_ :
b an oddity amongst the proteobacteria
h [electronic resource] /
by Ishtiaque Quasem.
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[Tampa, Fla] :
University of South Florida,
2009.
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Thesis (M.S.)--University of South Florida, 2009.
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ABSTRACT: Thiomicrospira crunogena, a deep-sea hydrothermal vent chemolithoautotroph, uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately autotrophic Gammaproteobacterium would use a 'wishbone' version of the citric acid cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the fully oxidative version to minimize carbon loss as carbon dioxide. However, upon examination of its complete genome sequence, it became apparent that this organism did not fulfill this expectation. Instead of a wishbone pathway, T. crunogena appears to run a fully oxidative CAC. The cycle is 'locked' in the oxidative direction by replacement of the reversible enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both oxidoreductases was confirmed via assays on T. crunogena cell extracts. To determine whether this peculiar CAC was novel, complete genome sequences of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated Microbial Genome database. Genes catalyzing steps in the CAC were collected from each organism and vetted for paralogs that had adopted an alternative, 'non-CAC' function through genome context and cluster analysis. Alignments were made with the remaining sequences and were verified by comparing them to curated alignments at Pfam database and examination of active site residues. Phylogenetic trees were constructed from these alignments, and instances of horizontal gene transfer were determined by comparison to a 16S tree. These analyses verified that the CAC in T. crunogena is indeed unique, as it does not resemble any of the canonical cycles of the six classes of proteobacteria. Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes encoded by genes that are likely to have been acquired via horizontal gene transfer. The gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with those present in the Cyanobacteria, while those encoding oxoglutarate: acceptor oxidoreductase cluster among the Firmicutes, and malate: quinone oxidoreductase clusters with the Epsilonproteobacteria.
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Advisor: Kathleen M. Scott, Ph.D.
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Thiomicrospira crunogena
Central carbon metabolism
Citric acid cycle
Proteobacteria
Chemolithoautotroph
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Dissertations, Academic
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x Biology
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