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.

Gammaproteobacterium Thiomicrospira crunogena thrives at deep-sea vents despite extreme oscillations in the environmental supply of dissolved inorganic carbon (DIC; =CO2 + HCO3- + CO3-2). Survival in this habitat is likely aided by the presence of a carbon concentrating mechanism (CCM). Though CCMs are well-documented in cyanobacteria, based on this study T. crunogena is the first chemolithoautotroph to have a physiologically characterized CCM. T. crunogena is capable of rapid growth in the presence of 20 micrometers DIC, has the ability to use both extracellular HCO3- and CO2, and generates intracellular DIC concentrations 100-fold greater than extracellular, all of which are consistent with a CCM analogous to those present in cyanobacteria. Interestingly, however, the T.crunogena genome lacks apparent orthologs of many of the components of the cyanobacteria CCM (e.g., HCO3- transporters). However, despite this lack, several candidate genes were identified during genome annotation as likely to play a role in DIC uptake and fixation (three carbonic anhydrase genes: alpha-CA, beta-CA, and csoSCA, as well as genes encoding three RubisCO enzymes: cbbLS, CScbbLS, and cbbM, which encode a cytoplasmic form I RubisCO, a carboxysomal form I RubisCO, and a form II RubisCO, respectively). In order to clarify their possible roles in DIC uptake and fixation, alpha-CA, beta-CA and csoSCA transcription by low-DIC and high-DIC T. crunogena were assayed by qRT PCR, heterologous expression in E. coli, and potentiometric assays of low-DIC and high-DIC T. crunogena. Transcription of alpha-CA and beta-CA were not sensitive to the DIC concentration available during growth. When overexpressed in E.coli, carbonic anhydrase activity was detectable, and it was possible to measure the effects of the classical carbonic anhydrase inhibitors ethoxyzolamide and acetazolamide, as well as dithiothreitol (DTT; recently determined to be a carboxysomal CA inhibitor). The alpha-CA was sensitive to both of the classical inhibitors, but not DTT. Beta-CA was insensitive to all inhibitors tested, and the carboxysomal carbonic anhydrase was sensitive to both ethoxyzolamide and DTT. The observation that the CA activity measureable potentiometrically with intact T. crunogena cells is sensitive to classical inhibitors, but not DTT, strongly suggests the alpha-CA is extracellular. The presence of carbonic anhydrase activity in crude extracts of high-DIC cells that was resistant to classical inhibitors suggests that beta-CA may be more active in high-DIC cells. Incubating cells with ethoxyzolamide (which permeates cells rapidly) resulted in inhibition of carbon fixation, but not DIC uptake, while incubation with acetazolamide (which does not permeate cells rapidly) had no apparent effect on either carbon fixation or DIC uptake. The observations that inhibition of alpha-CA has no effect on DIC uptake and fixation, and that the beta-CA is not transcribed more frequently under low-DIC conditions, make it unlikely that either play a role in DIC uptake and fixation in low-DIC cells. Further studies are underway to determine the roles of alpha-CA and beta-CA in T. crunogena. To assay the entire genome for genes transcribed more frequently under low-DIC conditions, and therefore likely to play a role in the T. crunogena CCM, oligonucleotide arrays were fabricated using the T. crunogena genome sequence. RNA was isolated from cultures grown in the presence of both high (50 mM) and low (0.05 mM) concentrations of DIC, directly labeled with cy5 fluorophore, and hybridized to microarrays. Genes encoding the three RubisCO enzymes present in this organism demonstrated differential patterns of transcription consistent with what had been observed previously in Hydrogenovibrio marinus. Genes encoding two conserved hypothetical proteins were also found to be transcribed more frequently under low-DIC conditions, and this transcription pattern was verified by qRT-PCR. Knockout mutants are currently being generated to determine whether either gene is necessary for growth under low-DIC conditions. Identifying CCM genes and function in autotrophs beyond cyanobacteria will serve as a window into the physiology required to flourish in microbiallydominated ecosystems where noncyanobacterial primary producers dominate.