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In hydrothermal environments, carbon monoxide (CO) utilisation by thermophilic hydrogenogenic carboxydotrophs may play an important role in microbial ecology by reducing toxic levels of CO and providing H 2 for fuelling microbial communities. We evaluated thermophilic hydrogenogenic carboxydotrophs by microbial community analysis. First, we analysed the correlation between carbon monoxide dehydrogenase (CODH)–energy-converting hydrogenase (ECH) gene cluster and taxonomic affiliation by surveying an increasing genomic database. We identified 71 genome-encoded CODH–ECH gene clusters, including 46 whose owners were not reported as hydrogenogenic carboxydotrophs. We identified 13 phylotypes showing > 98.7% identity with these taxa as potential hydrogenogenic carboxydotrophs in hot springs. Of these, Firmicutes phylotypes such as Parageobacillus , Carboxydocella , Caldanaerobacter, and Carboxydothermus were found in different environmental conditions and distinct microbial communities. The relative abundance of the potential thermophilic hydrogenogenic carboxydotrophs was low. Most of them did not show any symbiotic networks with other microbes, implying that their metabolic activities might be low. Electronic supplementary material The online version of this article (10.1007/s00203-019-01661-9) contains supplementary material, which is available to authorized users.

Carboxydotrophic anaerobic thermophiles have been isolated from various hydrothermal environments and are considered to be important carbon monoxide (CO) scavengers or primary producers. However, the ecological factors that influence the distribution, abundance and CO-oxidizing activities of these bacteria are poorly understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus spp. in a hot spring sample and found that they constituted up to 10% of the total bacterial cells. In this study, we investigated environmental features, potential microbial CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot springs to determine environmental factors that affect CO oxidizers and to see whether Carboxydothermus spp. are common in these environments. We detected potential microbial CO-oxidation activities in samples that showed relatively high values of total organic carbon, total nitrogen, oxidation-reduction potential and soil-water content. The abundance of Carboxydothermus spp. did not correlate with the presence of potential microbial CO-oxidation activities; however, Carboxydothermus spp. were detected in a wide range of environments, suggesting that these bacteria are widely distributed in spite of the relatively low population size. This study implies that thermophilic CO oxidizers occur in a wide range of environments and oxidize CO in somewhat oxidative environments rich in organic matter.

species are some of the most studied thermophilic carboxydotrophs. Their varied carboxydotrophic growth properties suggest distinct strategies for energy conservation via carbon monoxide (CO) metabolism. In this study, we used comparative genome analysis of the genus to show variations in the CO dehydrogenase-energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H production (hydrogenogenic CO metabolism). Indeed, the ability or inability to produce H with CO oxidation is explained by the presence or absence of this gene cluster in ,, and Interestingly, despite its hydrogenogenic CO metabolism, lacks the Ni-CO dehydrogenase catalytic subunit (CooS-I) and its transcriptional regulator-encoding genes in this gene cluster, probably due to inversion. Transcriptional analysis in showed that the Ni-CO dehydrogenase gene () and distantly encoded energy-converting-hydrogenase-related genes were remarkably upregulated with 100% CO. In addition, when thiosulfate was available as a terminal electron acceptor in 100% CO, the maximum cell density and maximum specific growth rate of were 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H produced was only 62% of the amount of CO consumed, less than expected according to hydrogenogenic CO oxidation (CO + HO → CO + H). Accordingly, would couple CO oxidation by Ni-CO dehydrogenase II with simultaneous reduction of not only HO but also thiosulfate when grown in 100% CO. Anaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche by scavenging potentially toxic CO and producing H as an available energy source for thermophilic microbes. This hydrogenogenic carboxydotrophy relies on a Ni-CO dehydrogenase-energy-converting hydrogenase gene cluster. This feature is thought to be common to these organisms. However, the hydrogenogenic carboxydotroph lacks the gene for the Ni-CO dehydrogenase catalytic subunit encoded in the gene cluster. Here, we performed a comparative genome analysis of the genus, a transcriptional analysis, and a cultivation study in 100% CO to prove the hydrogenogenic CO metabolism. Results revealed that could couple Ni-CO dehydrogenase II alternatively to the distal energy-converting hydrogenase. Furthermore, represents an example of the functioning of Ni-CO dehydrogenase that does not always correspond to its genomic context, owing to the versatility of CO metabolism and the low redox potential of CO.

Calderihabitans maritimus KKC1 is a thermophilic, hydrogenogenic carboxydotroph isolated from a submerged marine caldera. Here, we describe the de novo sequencing and feature analysis of the C. maritimus KKC1 genome. Genomebased phylogenetic analysis confirmed that C. maritimus KKC1 was most closely related to the genus Moorella, which includes well-studied acetogenic members. Comparative genomic analysis revealed that, like Moorella, C. maritimus KKC1 retained both the CO 2 -reducing Wood-Ljungdahl pathway and energy-converting hydrogenase-based module activated by reduced ferredoxin, but it lacked the HydABC and NfnAB electron-bifurcating enzymes and pyruvate:ferredoxin oxidoreductase required for ferredoxin reduction for acetogenic growth. Furthermore, C. maritimus KKC1 harbored six genes encoding CooS, a catalytic subunit of the anaerobic CO dehydrogenase that can reduce ferredoxin via CO oxidation, whereas Moorella possessed only two CooS genes. Our analysis revealed that three cooS genes formed known gene clusters in other microorganisms, i.e., cooS-acetyl coenzyme A (acetylCoA) synthase (which contained a frameshift mutation), cooS-energy-converting hydrogenase, and cooF-cooS-FAD-NAD oxidoreductase, while the other three had novel genomic contexts. Sequence composition analysis indicated that these cooS genes likely evolved from a common ancestor. Collectively, these data suggest that C. maritimus KKC1 may be highly dependent on CO as a low-potential electron donor to directly reduce ferredoxin and may be more suited to carboxydotrophic growth compared to the acetogenic growth observed in Moorella, which show adaptation at a thermodynamic limit.IMPORTANCE Calderihabitans maritimus KKC1 and members of the genus Moorella are phylogenetically related but physiologically distinct. The former is a hydrogenogenic carboxydotroph that can grow on carbon monoxide (CO) with H 2 production, whereas the latter include acetogenic bacteria that grow on H 2 plus CO 2 with acetate production. Both species may require reduced ferredoxin as an actual "energy equivalent," but ferredoxin is a low-potential electron carrier and requires a highenergy substrate as an electron donor for reduction. Comparative genomic analysis revealed that C. maritimus KKC1 lacked specific electron-bifurcating enzymes and possessed six CO dehydrogenases, unlike Moorella species. This suggests that C. maritimus KKC1 may be more dependent on CO, a strong electron donor that can directly reduce ferredoxin via CO dehydrogenase, and may exhibit a survival strategy different from that of acetogenic Moorella, which solves the energetic barrier associated with endergonic reduction of ferredoxin with hydrogen.

A thermophilic and hydrogenogenic carboxydotroph, Carboxydothermus pertinax, performs hydrogenogenic CO metabolism in which CODH-II couples with distally encoded ECH. To enhance our knowledge of its hydrogenogenic CO metabolism, we performed whole transcriptome analysis of C. pertinax grown under 100% CO or 100% N 2 using RNA sequencing. Of the 2577 genes, 36 and 64 genes were differentially expressed genes (DEGs) with false discovery rate adjusted P value < 0.05 when grown under 100% CO or 100% N 2 , respectively. Most of the DEGs were components of 23 gene clusters, suggesting switch between metabolisms via intensive expression changes in a relatively low number of gene clusters. Of the 9 significantly expressed gene clusters under 100% CO, CODH-II and ECH gene clusters were found. Only the ECH gene cluster was regulated by the CO-responsive transcriptional factor CooA, suggesting that others were separately regulated in the same transcriptional cascade as the ECH gene cluster. Of the 14 significantly expressed gene clusters under 100% N 2 , ferrous iron transport gene cluster involved in anaerobic respiration and prophage region were found. Considering that the expression of the temperate phage was strictly repressed under 100% CO, hydrogenogenic CO metabolism might be stable for C. pertinax . Electronic supplementary material The online version of this article (10.1007/s00792-019-01091-x) contains supplementary material, which is available to authorized users.

Carbon monoxide dehydrogenase-I (CODH-I) from the CO-utilizing bacterium Carboxydothermus hydrogenoformans are expected to be utilized as a part of reproducible carbon dioxide photoreduction system. However, the over-expression system for CODH-I remains to be constructed. CODH-I constitutes a hydrogenase/CODH gene cluster including a gene encoding a Ni-insertion accessory protein, CooC (cooC3). Through co-expression of CooC3, we found an over-expression system with higher activity. The Rec-CODH-I with the co-expression exhibits 8060 U/mg which was approximately threefold than that without co-expression (2270 U/mg). In addition, co-expression resulted in Ni(2+) content increase; the amount of Ni atoms of Rec-CODH-I was approximately thrice than that without co-expression.

Hydrogenogenic carboxydotrophs may provide hydrogen as primary energy for the microbial community via carbon monoxide oxidation. To investigate the genetics of carbon monoxide metabolism, we report here the draft genome sequences of the hydrogenogenic carboxydotrophs Carboxydocella sp. strains JDF658 (2.60 Mbp; G+C content, 49.2%) and ULO1 (2.70 Mbp; G+C content, 48.8%).