Insight into Energy Conservation via Alternative Carbon Monoxide Metabolism in Carboxydothermus pertinax Revealed by Comparative Genome Analysis

Abstract

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.

Transcriptome analysis of a thermophilic and hydrogenogenic carboxydotroph Carboxydothermus pertinax

“…Despite of its hydrogenogenic carboxydotrophy (Yoneda et al 2012 ), as per genome analysis, C. pertinax lacks genes encoding CODH-I catalytic subunit (CooS-I) and its transcriptional factor CooA-1 in their CODH-I−ECH gene cluster (Fukuyama et al 2017 ). Furthermore, gene expression analysis in C. pertinax has shown that genes encoding CODH-II catalytic subunit ( cooS - II ) and distantly encoding the ECH catalytic large and small subunits are remarkably upregulated under 100% CO, suggesting that C. pertinax performs hydrogenogenic CO metabolism in which CODH-II couples with distal ECH (Fukuyama et al 2018 ). Since C. pertinax possesses one CooA homolog (CooA-2) unlike C. hydrogenoformans possessing two CooA homologs, its transcriptional regulation from CO response is expected to be simpler than that of C. hydrogenoformans .…”

Carbon monoxide-dependent transcriptional changes in a thermophilic, carbon monoxide-utilizing, hydrogen-evolving bacterium Calderihabitans maritimus KKC1 revealed by transcriptomic analysis

“…The functions of these cooS genes have been predicted from their genomic contexts such as ECH, WLP, and ferredoxin-NAD(P)H oxidoreductase (Techtmann et al 2012;Inoue et al 2019), which are presumed to be regulated by CO-responsive transcription factors, such as CooA and RcoM (Shelver et al 1995;Komori et al 2007;Kerby et al 2008). However, recent studies of two hydrogenogenic carboxydotrophs, Carboxydothermus pertinax and Thermoanaerobacter kivui, show that the enzymatic coupling of cooS and ech genes that are distantly encoded in their respective genomes enables CO-dependent H 2 production (Fukuyama et al 2018(Fukuyama et al , 2019aSchoelmerich and Müller 2019). Moreover, cooS expression is upregulated in the presence of CO, despite the fact that no sequence motif is recognized by the CO-responsive transcriptional activator CooA in C. pertinax (Fukuyama et al 2018(Fukuyama et al , 2019a.…”

“…However, recent studies of two hydrogenogenic carboxydotrophs, Carboxydothermus pertinax and Thermoanaerobacter kivui, show that the enzymatic coupling of cooS and ech genes that are distantly encoded in their respective genomes enables CO-dependent H 2 production (Fukuyama et al 2018(Fukuyama et al , 2019aSchoelmerich and Müller 2019). Moreover, cooS expression is upregulated in the presence of CO, despite the fact that no sequence motif is recognized by the CO-responsive transcriptional activator CooA in C. pertinax (Fukuyama et al 2018(Fukuyama et al , 2019a. Similarly, ech expression is upregulated in the presence of CO, although the genome does not encode any previously described COresponsive transcription factors in T. kivui (Schoelmerich and Müller 2019).…”

“…The draft genome of C. maritimus KKC1 encodes six cooS genes that are categorized by their genomic contexts, as follows: cooS1 (KKC1_RS04465), WLP; cooS2 (KKC1_RS06675), ECH; cooS3 (KKC1_RS06585), ferredoxin-NAD(P)H oxidoreductase; cooS4 (KKC1_RS12505), a cysteine synthase and ABC transporter; cooS5 (KKC1_RS04925), 2-oxoglutarate:ferredoxin oxidoreductase (Kor); and cooS6 (KKC1_RS10495), CooA (Omae et al 2017). These six cooS genes harbor the complete sequence motifs that form three types of metal clusters for catalysis, although cooS1 is frame-shifted like other hydrogenogenic, carboxydotrophic Moorella and Carboxydothermus species possibly as a result of cultivation at high CO concentrations (Wu et al 2005;Omae et al 2017;Poehlein et al 2018;Fukuyama et al 2018Fukuyama et al , 2019b. To the best of our knowledge, the genome contains the highest number of cooS genes (Omae et al 2017;Toshchakov et al 2018) and encodes six hydrogenase gene clusters that include two ech gene clusters, a cootype gene cluster (ech1, KKC1_RS06640-KKC1_RS06665) with cooS2, and a hyc/hyf-type gene cluster (ech2, KKC1_ RS01155-KKC1_RS01200) with putative formate dehydrogenase genes (Omae et al 2017).…”

“…RNA isolation and cDNA synthesis were performed, as previously described (Fukuyama et al 2018(Fukuyama et al , 2019a. Total RNA was extracted using a mirVana miRNA Extraction Kit (Ambion, Austin, TX, USA), and the remaining genomic DNA was removed using TURBO DNase (Ambion).…”