Genomic Analysis of Calderihabitans maritimus KKC1, a Thermophilic, Hydrogenogenic, Carboxydotrophic Bacterium Isolated from Marine Sediment

Abstract

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.

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

“…A CooA-binding consensus sequence (5′-TGTC-N 8 -GACA) was found in the upstream regions of the cooS2-ech1 gene cluster and cooS6, as previously reported ( Fig. 3a and b) (Omae et al 2017), whereas Rex-binding sequence (5′-TTG TGA -N 6 -TCA CAA )-like motifs were found in the upstream regions of the transcriptional units containing the cooS2-ech1, hyd-3b, kor, fdh, and nap gene clusters and three mhc genes (Fig. 3a and c).…”

“…C. maritimus KKC1 requires yeast extract for CO-dependent growth unlike chemolithoautotrophic, hydrogenogenic carboxydotrophs, such as Moorella stamsii and Carboxydothermus hydrogenoformans, and is unable to grow with H 2 and CO 2 unlike chemolithoautotrophic acetogens, such as Moorella thermoacetica (Svetlichny et al 1991;Drake and Daniel 2004;Alves et al 2013;Yoneda et al 2013). 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.…”

“…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).…”

“…The multiplicity of Ni-CODHs and hydrogenases and the broad usage of electron acceptors in C. maritimus KKC1 suggest genomic adaptation for its carboxydotrophic growth (Yoneda et al 2013;Omae et al 2017); however, its metabolic and transcriptional responses to CO remain largely unknown. In this study, we performed a transcriptome analysis of C. maritimus KKC1 grown in the presence or absence of CO using RNA sequencing (RNA-seq).…”

“…1c). The C. maritimus KKC1genome also contains two partial cooS-like gene fragments (KKC1_ RS14835 and KKC1_RS01100) (Omae et al 2017) sharing 65 bp and 16 aa perfect matches encoding one protein (hereafter designated as CooS7). The expression level of cooS7 judged as non-DEGs was also tested.…”