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Comparison of the Ni-CODH-I-ECH gene clusters in three Carboxydothermus species. Vertical blocks between sequences indicate regions of shared similarity shaded according to BLASTn. Cpe, C. pertinax; Chy, C. hydrogenoformans; Cis, C. islandicus. Red, cooS; green, cooA; purple, Ni-CODH-I-related gene; blue, ECH-related gene; white, hypothetical gene; gray, unrelated neighboring gene.
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Carboxydothermus 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 Carboxydothermus to show variations in the CO dehydrogenase-energ...
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... Although for some hydrogenogenic carboxydotrophs the addition of an external electron acceptor such as sulfur, thiosulfate, or sulfate led to a partial redirection of CO-derived electrons from protons to a more favorable acceptor, this did not completely stop the H 2 formation [14][15][16][17]. The same seems to be true for those carboxydotrophic acetogens that can utilize electron acceptors other than CO 2 . ...
Anaerobic carboxydotrophy is a widespread catabolic trait in bacteria, with two dominant pathways: hydrogenogenic and acetogenic. The marginal mode by direct oxidation to CO2 using an external e-acceptor has only a few examples. Use of sulfidic sediments from two types of hypersaline lakes in anaerobic enrichments with CO as an e-donor and elemental sulfur as an e-acceptor led to isolation of two pure cultures of anaerobic carboxydotrophs belonging to two genera of sulfur-reducing haloarchaea: Halanaeroarchaeum sp. HSR-CO from salt lakes and Halalkaliarchaeum sp. AArc-CO from soda lakes. Anaerobic growth of extremely halophilic archaea with CO was obligatory depended on the presence of elemental sulfur as the electron acceptor and yeast extract as the carbon source. CO served as a direct electron donor and H2 was not generated from CO when cells were incubated with or without sulfur. The genomes of the isolates encode a catalytic Ni,Fe-CODH subunit CooS (distantly related to bacterial homologs) and its Ni-incorporating chaperone CooC (related to methanogenic homologs) within a single genomic locus. Similar loci were also present in a genome of the type species of Halalkaliarchaeum closely related to AArc-CO, and the ability for anaerobic sulfur-dependent carboxydotrophy was confirmed for three different strains of this genus. Moreover, similar proteins are encoded in three of the four genomes of recently described carbohydrate-utilizing sulfur-reducing haloarchaea belonging to the genus Halapricum and in two yet undescribed haloarchaeal species. Overall, this work demonstrated for the first time the potential for anaerobic sulfur-dependent carboxydotrophy in extremely halophilic archaea.
... The cells of C. hydrogenoformans Z-2901 and Carboxydocella sp. ULO1 were grown in a modified DSM 507 medium (pH 7.0) (Fukuyama et al. 2018), and the cells of C. maritimus KKC1 were grown in a NBRC 1251 medium (pH 7.5). These were grown under 100% CO gas at 65 °C by using 100 ml butyl rubber-stoppered bottles containing 50 ml of medium. ...
The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.
... 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). ...
Calderihabitans maritimus KKC1 is a thermophilic, carbon monoxide (CO)-utilizing, hydrogen-evolving bacterium that harbors seven cooS genes for anaerobic CO dehydrogenases and six hyd genes for [NiFe] hydrogenases and capable of using a variety of electron acceptors coupled to CO oxidation. To understand the relationships among these unique features and the transcriptional adaptation of the organism to CO, we performed a transcriptome analysis of C. maritimus KKC1 grown under 100% CO and N2 conditions. Of its 3114 genes, 58 and 32 genes were significantly upregulated and downregulated in the presence of CO, respectively. A cooS–ech gene cluster, an “orphan” cooS gene, and bidirectional hyd genes were upregulated under CO, whereas hydrogen-uptake hyd genes were downregulated. Transcriptional changes in anaerobic respiratory genes supported the broad usage of electron acceptors in C. maritimus KKC1 under CO metabolism. Overall, the majority of the differentially expressed genes were oxidoreductase-like genes, suggesting metabolic adaptation to the cellular redox change upon CO oxidation. Moreover, our results suggest a transcriptional response mechanism to CO that involves multiple transcription factors, as well as a CO-responsive transcriptional activator (CooA). Our findings shed light on the diverse mechanisms for transcriptional and metabolic adaptations to CO in CO-utilizing and hydrogen-evolving bacteria.
... In the genome of Thermofilum sp. strain 1505, a [Ni,Fe]-CODH-ECH gene cluster was found, which is a phenomenon typical of all the sequenced genomes of hydrogenogenic carboxydotrophs, with the only known exception being Carboxydothermus pertinax Ug1 that has its [Ni,Fe]-CODH genes and ECH gene cluster physically unconnected [22]. ...
The ability to grow by anaerobic CO oxidation with production of H2 from water is known for some thermophilic bacteria, most of which belong to Firmicutes, as well as for a few hyperthermophilic Euryarchaeota isolated from deep-sea hydrothermal habitats. A hyperthermophilic, neutrophilic, anaerobic filamentous archaeon strain 1505=VKM B-3180=KCTC 15798 was isolated from a terrestrial hot spring in Kamchatka (Russia) in the presence of 30% CO in the gas phase. Strain 1505 could grow lithotrophically using carbon monoxide as the energy source with the production of hydrogen according to the equation CO+H2O→CO2+H2; mixotrophically on CO plus glucose; and organotrophically on peptone, yeast extract, glucose, sucrose, or Avicel. The genome of strain 1505 was sequenced and assembled into a single chromosome. Based on 16S rRNA gene sequence analysis and in silico genome-genome hybridization, this organism was shown to be closely related to the Thermofilum adornatum species. In the genome of Thermofilum sp. strain 1505, a gene cluster (TCARB_0867-TCARB_0879) was found that included genes of anaerobic (Ni,Fe-containing) carbon monoxide dehydrogenase and genes of energy-converting hydrogenase ([Ni,Fe]-CODH-ECH gene cluster). Compared to the [Ni,Fe]-CODH-ECH gene clusters occurring in the sequenced genomes of other H2-producing carboxydotrophs, the [Ni,Fe]-CODH-ECH gene cluster of Thermofilum sp. strain 1505 presented a novel type of gene organization. The results of the study provided the first evidence of anaerobic CO oxidation coupled with H2 production performed by a crenarchaeon, as well as the first documented case of lithotrophic growth of a Thermofilaceae representative.
... Thermincola, Carboxydocella, Carboxydothermus, and Caldanaerobacter were classified into the group (1). In most cases, the phylogeny of CODH genes was corresponding to their taxonomic phylogeny in this group (Adam et al. 2018;Fukuyama et al. 2018;Toshchakov et al. 2018), suggesting that the CODH-ECH gene clusters descended from the common ancestors of each genus. The genus Carboxydothermus has been one of the most studied models of thermophilic carboxydotrophy, and the members of this genus possess four or five CODH genes (Fukuyama et al. 2018). ...
... In most cases, the phylogeny of CODH genes was corresponding to their taxonomic phylogeny in this group (Adam et al. 2018;Fukuyama et al. 2018;Toshchakov et al. 2018), suggesting that the CODH-ECH gene clusters descended from the common ancestors of each genus. The genus Carboxydothermus has been one of the most studied models of thermophilic carboxydotrophy, and the members of this genus possess four or five CODH genes (Fukuyama et al. 2018). A comparative genomic analysis in Carboxydothermus revealed that the CODH-ECH gene clusters were conserved in the members except for C. pertinax, which lacked only the CODH (CODH-I) unit of CODH-ECH gene cluster and Carboxydothermus ferrireducens, which lacked the whole CODH-ECH gene cluster (Fukuyama et al. 2018). ...
... The genus Carboxydothermus has been one of the most studied models of thermophilic carboxydotrophy, and the members of this genus possess four or five CODH genes (Fukuyama et al. 2018). A comparative genomic analysis in Carboxydothermus revealed that the CODH-ECH gene clusters were conserved in the members except for C. pertinax, which lacked only the CODH (CODH-I) unit of CODH-ECH gene cluster and Carboxydothermus ferrireducens, which lacked the whole CODH-ECH gene cluster (Fukuyama et al. 2018). C. ferrireducens can grow carboxydotrophically, but is not hydrogenogenic (Slobodkin et al. 2006). ...
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 H2 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.
... 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 . 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. ...
... To prepare RNA for transcriptome analysis, C. pertinax was grown at 65 °C in modified DSM medium 507 under a headspace of 100% CO or 100% N 2 gas according to methods previously described (Fukuyama et al. 2018). We added sodium thiosulfate (final concentration, 1 g/L) as the terminal electron acceptor and sodium pyruvate (final concentration, 2 g/L) as the electron donor and carbon source to the medium in both the gas phase conditions to collect enough cells for analysis. ...
... When C. pertinax reached the late exponential phase as indicated by arrows (Fig. S1), 10 mL of the culture in both the conditions was collected and total RNA was extracted according to the methods previously described (Fukuyama et al. 2018). After removal of contaminating DNA using TURBO DNase (Invitrogen, Carlsbad, CA, USA), total RNA was purified using Agencourt RNAClean XP (Beckman Coulter, Brea, CA, USA) according to the manufacturer's instructions. ...
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% N2 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% N2, 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% N2, 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.
Waste biomass is considered a promising renewable energy feedstock that can be converted by anaerobic digestion. However, anaerobic digestion application can be challenging due to the structural complexity of several waste biomass kinds. Therefore, coupling anaerobic digestion with thermochemical processes can offset the limitations and convert the hardly biodegradable waste biomass, including digestate residue, into value-added products: syngas and pyrogas (gaseous mixtures consisting mainly of H2, CO, CO2), bio-oil, and biochar for further valorisation. In this review, the utilisation boundaries and benefits of the aforementioned products by anaerobic culture are discussed. First, thermochemical process parameters for an enhanced yield of desired products are summarised. Particularly, the microbiology of CO and H2 mixture biomethanation and fermentation in anaerobic digestion is presented. Finally, the state-of-the-art biological conversion of syngas and pyrogas to CH4 mediated by anaerobic culture is adequately described. Extensive research shows the successful selective biological conversion of CO and H2 to CH4, acetic acid, and alcohols. The main bottleneck is the gas-liquid mass transfer which can be enhanced appropriately by bioreactors' configurations. A few research groups focus on bio-oil and biochar addition into anaerobic digesters. However, according to the literature review, there has been no research for utilising all value-added products at once in anaerobic digestion published so far. Although synergic effects of such can be expected. In summary, the combination of anaerobic digestion and thermochemical processes is a promising alternative for wide-scale waste biomass utilisation in practice.
Carbon monoxide (CO) is a gas that is toxic to various organisms including humans and even microbes; however, it has low redox potential, which can fuel certain microbes, namely, CO oxidizers. Hydrogenogenic CO oxidizers utilize an energy conservation system via a CO dehydrogenase/energy-converting hydrogenase complex to produce hydrogen gas, a zero emission fuel, by CO oxidation coupled with proton reduction. Biochemical and molecular biological studies using a few model organisms have revealed their enzymatic reactions and transcriptional response mechanisms using CO. Biotechnological studies for CO-dependent hydrogen production have also been carried out with these model organisms. In this chapter, we review recent advances in the studies of these microbes, which reveal their unique and versatile metabolic profiles and provides future perspectives on ecological roles and biotechnological applications. Over the past decade, the number of isolates has doubled (37 isolates in 5 phyla, 20 genera, and 32 species). Some of the recently isolated ones show broad specificity to electron acceptors. Moreover, accumulating genomic information predicts their unique physiologies and reveals their phylogenomic relationships with novel potential hydrogenogenic CO oxidizers. Combined with genomic database surveys, a molecular ecological study has unveiled the wide distribution and low abundance of these microbes. Finally, recent biotechnological applications of hydrogenogenic CO oxidizers have been achieved via diverse approaches (e.g., metabolic engineering and co-cultivation), and the identification of thermophilic facultative anaerobic CO oxidizers will promote industrial applications as oxygen-tolerant biocatalysts for efficient hydrogen production by genomic engineering.
