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.

Abstract The gammaproteobacterium Thiomicrospira crunogena XCL–2 is a hydrothermal vent chemolithoautotroph that has a carbon concentrating mechanism (CCM), which is functionally similar to that of cyanobacteria. At hydrothermal vents, dissolved inorganic carbon (DIC) concentrations and pH values fluctuate over time, with CO2 concentrations ranging from 20 μM to greater than 1 mM, therefore having a CCM would provide an advantage when CO2 availability is very low as CCMs generate intracellular DIC concentrations much higher than extracellular, thereby providing sufficient substrate for carbon fixation. The CCM in T. crunogena includes α–carboxysomes (intracellular inclusions containing form IA RubisCO and carbonic anhydrase), and also presumably requires at least one active HCO3 µ transporter to generate the elevated intracellular concentrations of DIC. To determine whether RubisCO itself might be adapted to low CO2 concentrations, the KCO2 for purified carboxysomal RubisCO was measured (250 μM SD ±; 40) and was much greater than that of whole cells (1.03 μM). This finding suggests that the primary adaptation by T. crunogena to low–DIC conditions has been to enhance DIC uptake, presumably by energy–dependent membrane transport systems that are either ATP–dependent and/or dependent on membrane potential (δ ψ). To determine the mechanism for active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis andδ ψ. After separate incubations with the ATP synthase inhibitor DCCD and the protonophore CCCP, intracellular ATP was diminished, as was the concentration of intracellular DIC and fixed carbon, despite the absence of an inhibitory effect on δ ψ in the DCCD–incubated cells. In some organisms, DCCD inhibits the NDH–1 and bc1 complexes so it was necessary to verify that ATP synthase was the primary target of DCCD in T. crunogena. Both electron transport complex activities were assayed in the presence and absence of DCCD and there was no significant difference between inhibited (309.0 μmol/s for NDH–1 and 3.4 μmol/s for bc1) and uninhibited treatments (271.7 μmol/s for NDH–1 and 3.6 μmol/s for bc1). These data support the hypothesis that an ATP–dependent transporter is primarily responsible for HCO3 µ transport in T. crunogena. The ATP–dependent transporter solute–binding protein gene (cmpA) from Synechococcus elongatus PCC 7942, was used to perform a BLAST query. Tcr_1153 was the closest match in the T. crunogena genome. However, the gene neighborhood and the result of a maximum likelihood tree suggest that Tcr_1153 is a nitrate transporter protein. Work is underway to find the genes responsible for this ATP–dependent transporter.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24679号 / 農博第2562号 / 新制||農||1100(附属図書館) / 学位論文||R5||N5460(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 澤山 茂樹, 教授 吉田 天士, 准教授 中川 聡 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS). / Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).