Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1 µl of 100 nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

Diversity analysis of thermophilic hydrogenogenic carboxydotrophs by carbon monoxide dehydrogenase amplicon sequencing using new primers.

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.

Draft Genome Sequence of Thermanaeromonas sp. Strain C210, Isolated in the Presence of Carbon Monoxide.

The genus Thermanaeromonas comprises two species of thermophilic, strictly anaerobic, spore-forming bacteria. Here, we report the draft genome sequence of Thermanaeromonas sp. strain C210, which was first isolated in the presence of carbon monoxide. The genome sequence provides insight into carbon monoxide-dependent metabolism for members of the genus Thermanaeromonas.

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

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.

Anaerobic and hydrogenogenic carbon monoxide-oxidizing prokaryotes: Versatile microbial conversion of a toxic gas into an available energy.

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.

Draft Genome Sequences of Two Thermophilic Moorella sp. Strains, Isolated from an Acidic Hot Spring in Japan.

The thermophilic Moorella sp. strains E308F and E306M were isolated from an acidic hot spring in Japan. Here, we report the draft genome sequences of E308F (3.06 Mbp; G+C content, 54.0%) and E306M (2.99 Mbp; G+C content, 54.4%), to advance the genomic information available on the genus Moorella.

Diversity and distribution of thermophilic hydrogenogenic carboxydotrophs revealed by microbial community analysis in sediments from multiple hydrothermal environments in Japan.

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.

Transcriptome analysis of a thermophilic and hydrogenogenic carboxydotroph Carboxydothermus pertinax.

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.

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

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-energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H2 production (hydrogenogenic CO metabolism). Indeed, the ability or inability to produce H2 with CO oxidation is explained by the presence or absence of this gene cluster in Carboxydothermus hydrogenoformans, Carboxydothermus islandicus, and Carboxydothermus ferrireducens Interestingly, despite its hydrogenogenic CO metabolism, Carboxydothermus pertinax 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 C. pertinax showed that the Ni-CO dehydrogenase gene (cooS-II) 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 C. pertinax were 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H2 produced was only 62% of the amount of CO consumed, less than expected according to hydrogenogenic CO oxidation (CO + H2O → CO2 + H2). Accordingly, C. pertinax would couple CO oxidation by Ni-CO dehydrogenase II with simultaneous reduction of not only H2O but also thiosulfate when grown in 100% CO.IMPORTANCE Anaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche by scavenging potentially toxic CO and producing H2 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 Carboxydothermus pertinax 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 Carboxydothermus, a transcriptional analysis, and a cultivation study in 100% CO to prove the hydrogenogenic CO metabolism. Results revealed that C. pertinax could couple Ni-CO dehydrogenase II alternatively to the distal energy-converting hydrogenase. Furthermore, C. pertinax 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.

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

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. Genome-based 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 CO2-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 (acetyl-CoA) 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.IMPORTANCECalderihabitans 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 H2 production, whereas the latter include acetogenic bacteria that grow on H2 plus CO2 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 high-energy 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.

Draft Genome Sequences of Two Hydrogenogenic Carboxydotrophic Bacteria, Carboxydocella sp. Strains JDF658 and ULO1, Isolated from Two Distinct Volcanic Fronts in Japan.

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%).

Draft Genome Sequences of Carboxydothermus pertinax and C. islandicus, Hydrogenogenic Carboxydotrophic Bacteria.

Carboxydothermus spp. are some of the most studied carbon monoxide-oxidizing anaerobic thermophiles. For further investigation into the carbon monoxide metabolism of Carboxydothermus spp., we report here the draft genome sequences of the hydrogenogenic carboxydotrophs Carboxydothermus pertinax (2.47 Mb; G+C content, 40.7%) and C. islandicus (2.39 Mb; G+C content, 42.0%).

Detection of anaerobic carbon monoxide-oxidizing thermophiles in hydrothermal environments.

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.

Over-expression of carbon monoxide dehydrogenase-I with an accessory protein co-expression: a key enzyme for carbon dioxide reduction.

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.