A novel hexameric NADP+ -reducing [FeFe] hydrogenase from Moorella thermoacetica.

Acetogenic bacteria such as the thermophilic anaerobic model organism Moorella thermoacetica reduce CO2 with H2 as a reductant via the Wood-Ljungdahl pathway (WLP). The enzymes of the WLP of M. thermoacetica require NADH, NADPH, and reduced ferredoxin as reductants. Whereas an electron-bifurcating ferredoxin- and NAD+ -reducing hydrogenase HydABC had been described, the enzyme that reduces NADP+ remained to be identified. A likely candidate is the HydABCDEF hydrogenase from M. thermoacetica. Genes encoding for the HydABCDEF hydrogenase are expressed during growth on glucose and dimethyl sulfoxide (DMSO), an alternative electron acceptor in M. thermoacetica, whereas expression of the genes hydABC encoding for the electron-bifurcating hydrogenase is downregulated. Therefore, we have purified the hydrogenase from cells grown on glucose and DMSO to apparent homogeneity. The enzyme had six subunits encoded by hydABCDEF and contained 58 mol of iron and 1 mol of FMN. The enzyme reduced methyl viologen with H2 as reductant and of the physiological acceptors tested, only NADP+ was reduced. Electron bifurcation with pyridine nucleotides and ferredoxin was not observed. H2 -dependent NADP+ reduction was optimal at pH 8 and 60 °C; the specific activity was 8.5 U·mg-1 and the Km for NADP+ was 0.086 mm. Cell suspensions catalyzed H2 -dependent DMSO reduction, which is in line with the hypothesis that the NADP+ -reducing hydrogenase HydABCDEF is involved in electron transfer from H2 to DMSO.

Using directed evolution to improve hydrogen production in chimeric hydrogenases from algal species.

Algae generate hydrogen from sunlight and water utilizing high-energy electrons generated during photosynthesis. The amount of hydrogen produced in heterologous expression of the wild-type hydrogenase is currently insufficient for industrial applications. One approach to improve hydrogen yields is through directed evolution of the DNA of the native hydrogenase. Here, we created 113 chimeric algal hydrogenase gene variants derived from combining segments of three parent hydrogenases, two from Chlamydomonas reinhardtii (CrHydA1 and CrHydA2) and one from Scenedesmus obliquus (HydA1). To generate chimeras, there were seven segments into which each of the parent hydrogenase genes was divided and recombined in a variety of combinations. The chimeric and parental hydrogenase sequences were cloned for heterologous expression in Escherichia coli, and 40 of the resultant enzymes expressed were assayed for H2 production. Chimeric clones that resulted in equal or greater production obtained with the cloned CrHydA1 parent hydrogenase were those comprised of CrHydA1 sequence in segments #1, 2, 3, and/or 4. These best-performing chimeras all contained one common region, segment #2, the part of the sequence known to contain important amino acids involved in proton transfer or hydrogen cluster coordination. The amino acid sequence distances among all chimeric clones to that of the CrHydA1 parent were determined, and the relationship between sequence distances and experimentally-derived H2 production was evaluated. An additional model determined the correlation between electrostatic potential energy surface area ratios and H2 production. The model yielded several algal mutants with predicted hydrogen productions in a range of two to three times that of the wild-type hydrogenase. The mutant data and the model can now be used to predict which specific mutant sequences may result in even higher hydrogen yields. Overall, results provide more precise details in planning future directed evolution to functionally improve algal hydrogenases.

Carbon amendments in soil microcosms induce uneven response on H2 oxidation activity and microbial community composition.

High-affinity H2-oxidizing bacteria (HA-HOB) thriving in soil are responsible for the most important sink of atmospheric H2. Their activity increases with soil organic carbon content, but the incidence of different carbohydrate fractions on the process has received little attention. Here we tested the hypothesis that carbon amendments impact HA-HOB activity and diversity differentially depending on their recalcitrance and their concentration. Carbon sources (sucrose, starch, cellulose) and application doses (0, 0.1, 1, 3, 5% Ceq soildw-1) were manipulated in soil microcosms. Only 0.1% Ceq soildw-1 cellulose treatment stimulated the HA-HOB activity. Sucrose amendments induced the most significant changes, with an abatement of 50% activity at 1% Ceq soildw-1. This was accompanied with a loss of bacterial and fungal alpha diversity and a reduction of high-affinity group 1 h/5 [NiFe]-hydrogenase gene (hhyL) abundance. A quantitative classification framework was elaborated to assign carbon preference traits to 16S rRNA gene, ITS and hhyL genotypes. The response was uneven at the taxonomic level, making carbon preference a difficult trait to predict. Overall, the results suggest that HA-HOB activity is more susceptible to be stimulated by low doses of recalcitrant carbon, while labile carbon-rich environment is an unfavorable niche for HA-HOB, inducing catabolic repression of hydrogenase.

NADP+ or CO2 reduction by frhAGB-encoded hydrogenase through interaction with formate dehydrogenase 3 in the hyperthermophilic archaeon Thermococcus onnurineus NA1.

IMPORTANCE: The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the frhAGB-encoded hydrogenase of Thermococcus onnurineus NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P)+ is significant in that the enzyme can function to supply reducing equivalents, just as F420-reducing hydrogenases in methanogens use coenzyme F420 as an electron carrier. Additionally, it was identified that T. onnurineus NA1 could produce formate from H2 and CO2 by the concerted action of frhAGB-encoded hydrogenase and formate dehydrogenase Fdh3.

Improved preculture management for Cupriavidus necator cultivations.

OBJECTIVES: Research on hydrogenases from Cupriavidus necator has been ongoing for more than two decades and still today the common methods for culture inoculation are used. These methods were never adapted to the requirements of modified bacterial strains, resulting in different physiological states of the bacteria in the precultures, which in turn lead prolonged and different lag-phases. RESULTS: In order to obtain uniform and always equally fit precultures for inoculation, we have established in this study an optimized protocol for precultures of the derivative of C. necator HF210 (C. necator HP80) which is used for homologous overexpression of the genes for the NAD+-reducing soluble hydrogenase (SH). We compared different media for preculture growth and determined the optimal time point for harvest. The protocol obtained in this study is based on two subsequent precultures, the first one in complex nutrient broth medium (NB) and a second one in fructose -nitrogen mineral salt medium (FN). CONCLUSION: Despite having two subsequent precultures our protocol reduces the preculture time to less than 30 h and provides reproducible precultures for cultivation of C. necator HP80.

Proteomic and Mutant Analysis of Hydrogenase Maturation Protein Gene hypE in Symbiotic Nitrogen Fixation of Mesorhizobium huakuii.

Hydrogenases catalyze the simple yet important redox reaction between protons and electrons and H2, thus mediating symbiotic interactions. The contribution of hydrogenase to this symbiosis and anti-oxidative damage was investigated using the M. huakuii hypE (encoding hydrogenase maturation protein) mutant. The hypE mutant grew a little faster than its parental 7653R and displayed decreased antioxidative capacity under H2O2-induced oxidative damage. Real-time quantitative PCR showed that hypE gene expression is significantly up-regulated in all the detected stages of nodule development. Although the hypE mutant can form nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 47% reduction in nitrogen fixation capacity. This phenotype was linked to the formation of smaller abnormal nodules containing disintegrating and prematurely senescent bacteroids. Proteomics analysis allowed a total of ninety differentially expressed proteins (fold change > 1.5 or <0.67, p < 0.05) to be identified. Of these proteins, 21 are related to stress response and virulence, 21 are involved in transporter activity, and 18 are involved in energy and nitrogen metabolism. Overall, the HypE protein is essential for symbiotic nitrogen fixation, playing independent roles in supplying energy and electrons, in bacterial detoxification, and in the control of bacteroid differentiation and senescence.

Rubredoxin Protein Scaffolds Sourced from Diverse Environmental Niches as an Artificial Hydrogenase Platform.

Nickel-substituted rubredoxin (NiRd) from Desulfovibrio desulfuricans has previously been shown to act as both a structural and functional mimic of the [NiFe] hydrogenase. However, improvements both in turnover frequency and overpotential are needed to rival the native [NiFe] hydrogenase enzymes. Characterization of a library of NiRd mutants with variations in the secondary coordination sphere suggested that protein dynamics played a substantial role in modulating activity. In this work, rubredoxin scaffolds were selected from diverse organisms to study the effects of distal sequence variation on catalytic activity. It was found that though electrochemical catalytic activity was only slightly impacted across the series, the Rd sequence from a psychrophilic organism exhibited substantially higher levels of solution-phase hydrogen production. Additionally, Eyring analyses suggest that catalytic activation properties relate to the growth temperature of the parent organism, implying that the general correlation between the parent organism environment and catalytic activity often seen in naturally occurring enzymes may also be observed in artificial enzymes. Selecting protein scaffolds from hosts that inhabit diverse environments, particularly low-temperature environments, represents an alternative approach for engineering artificial metalloenzymes.

Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site.

BACKGROUND: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. RESULTS: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. CONCLUSION: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

Helicobacter hepaticus promotes liver fibrosis through oxidative stress induced by hydrogenase in BALB/c mice.

BACKGROUND: It has been documented that Helicobacter hepaticus produces a nickel-containing hydrogen-oxidizing hydrogenase enzyme, which is necessary for hydrogen-supported amino acid uptake. Although H. hepaticus infection has been shown to promote liver inflammation and fibrosis in BALB/c mice, the impact of hydrogenase on the progression of liver fibrosis induced by H. hepaticus has not been explored. MATERIALS AND METHODS: BALB/c mice were inoculated with hydrogenase mutant (ΔHyaB) or wild type (WT) H. hepaticus 3B1 for 12 and 24 weeks. H. hepaticus colonization, hepatic histopathology, serum biochemistry, expression of inflammatory cytokines, and oxidative stress signaling pathways were detected. RESULTS: We found that ΔHyaB had no influence on the colonization of H. hepaticus in the liver of mice at 12 and 24 weeks post infection (WPI). However, mice infected by ΔHyaB strains developed significantly alleviated liver inflammation and fibrosis compared with WT infection. Moreover, ΔHyaB infection remarkably increased the expression of hepatic GSH, SOD, and GSH-Px, and decreased the liver levels of MDA, ALT, and AST compared to WT H. hepaticus infected group from 12 to 24 WPI. Furthermore, mRNA levels of Il-6, Tnf-α, iNos, Hmox-1, and α-SMA were significantly decreased with an increase of Nfe2l2 in the liver of mice infected by ΔHyaB strains. In addition, ΔHyaB H. hepaticus restored the activation of the Nrf2/HO-1 signaling pathway, which is inhibited by H. hepaticus infection. CONCLUSIONS: These data demonstrated that H. hepaticus hydrogenase promoted liver inflammation and fibrosis development mediated by oxidative stress in male BALB/c mice.

Valorization of whey-based side streams for microbial biomass, molecular hydrogen, and hydrogenase production.

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O2-tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H2), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation-reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37 °C, pH 7.5 and 30 °C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD600 4.0) and H2-oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H2 yields of ~ 5 mmol/L and cumulative H2 ~ 94 mL g/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable ß-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production. KEY POINTS: • Archaeal thermostable ß-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey. • Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey. • Enhanced biomass and H2 production was shown in a genetically modified strain of Escherichia coli.

Microorganisms Involved in Hydrogen Sink in the Gastrointestinal Tract of Chickens.

Hydrogen sink is a beneficial process, which has never been properly examined in chickens. Therefore, the aim of this study was to assess the quantity and quality of microbiota involved in hydrogen uptake with the use of real-time PCR and metagenome sequencing. Analyses were carried out in 50 free-range chickens, 50 commercial broilers, and 54 experimental chickens isolated from external factors. The median values of acetogens, methanogens, sulfate-reducing bacteria (SRB), and [NiFe]-hydrogenase utilizers measured in the cecum were approx. 7.6, 0, 0, and 3.2 log10/gram of wet weight, respectively. For the excreta samples, these values were 5.9, 4.8, 4, and 3 log10/gram of wet weight, respectively. Our results showed that the acetogens were dominant over the other tested groups of hydrogen consumers. The quantities of methanogens, SRB, and the [NiFe]-hydrogenase utilizers were dependent on the overall rearing conditions, being the result of diet, environment, agrotechnical measures, and other factors combined. By sequencing of the 16S rRNA gene, archaea of the genus Methanomassiliicoccus (Candidatus Methanomassiliicoccus) were discovered in chickens for the first time. This study provides some indication that in chickens, acetogenesis may be the main metabolic pathway responsible for hydrogen sink.

Synthetic engineering of a new biocatalyst encapsulating [NiFe]-hydrogenases for enhanced hydrogen production.

Hydrogenases are microbial metalloenzymes capable of catalyzing the reversible interconversion between molecular hydrogen and protons with high efficiency, and have great potential in the development of new electrocatalysts for renewable fuel production. Here, we engineered the intact proteinaceous shell of the carboxysome, a self-assembling protein organelle for CO2 fixation in cyanobacteria and proteobacteria, and sequestered heterologously produced [NiFe]-hydrogenases into the carboxysome shell. The protein-based hybrid catalyst produced in E. coli shows substantially improved hydrogen production under both aerobic and anaerobic conditions and enhanced material and functional robustness, compared to unencapsulated [NiFe]-hydrogenases. The catalytically functional nanoreactor as well as the self-assembling and encapsulation strategies provide a framework for engineering new bioinspired electrocatalysts to improve the sustainable production of fuels and chemicals in biotechnological and chemical applications.

H2 Is a Major Intermediate in Desulfovibrio vulgaris Corrosion of Iron.

Desulfovibrio vulgaris has been a primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe0 have been proposed. We investigated Fe0 oxidation with a mutant of D. vulgaris in which hydrogenase genes were deleted. The hydrogenase mutant grew as well as the parental strain with lactate as the electron donor, but unlike the parental strain, it was not able to grow on H2. The parental strain reduced sulfate with Fe0 as the sole electron donor, but the hydrogenase mutant did not. H2 accumulated over time in Fe0 cultures of the hydrogenase mutant and sterile controls but not in parental strain cultures. Sulfide stimulated H2 production in uninoculated controls apparently by both reacting with Fe0 to generate H2 and facilitating electron transfer from Fe0 to H+. Parental strain supernatants did not accelerate H2 production from Fe0, ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe0 and D. vulgaris via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe0 and either riboflavin or anthraquinone-2,6-disulfonate, and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe0 as the electron donor. The results demonstrate that D. vulgaris primarily accepts electrons from Fe0 via H2 as an intermediary electron carrier. These findings clarify the interpretation of previous D. vulgaris corrosion studies and suggest that H2-mediated electron transfer is an important mechanism for iron corrosion under sulfate-reducing conditions. IMPORTANCE Microbial corrosion of iron in the presence of sulfate-reducing microorganisms is economically significant. There is substantial debate over how microbes accelerate iron corrosion. Tools for genetic manipulation have only been developed for a few Fe(III)-reducing and methanogenic microorganisms known to corrode iron and in each case those microbes were found to accept electrons from Fe0 via direct electron transfer. However, iron corrosion is often most intense in the presence of sulfate-reducing microbes. The finding that Desulfovibrio vulgaris relies on H2 to shuttle electrons between Fe0 and cells revives the concept, developed in some of the earliest studies on microbial corrosion, that sulfate reducers consumption of H2 is a major microbial corrosion mechanism. The results further emphasize that direct Fe0-to-microbe electron transfer has yet to be rigorously demonstrated in sulfate-reducing microbes.

NMR-based metabolomic analysis of the physiological role of the electron-bifurcating FeFe-hydrogenase Hnd in Solidesulfovibrio fructosivorans under pyruvate fermentation.

Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans), an anaerobic sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of hydrogen gas (H2) into protons and electrons. One of these, named Hnd, was demonstrated to be an electron-bifurcating hydrogenase Hnd (Kpebe et al., 2018). It couples the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 and whose function has been recently shown to be involved in ethanol production under pyruvate fermentation (Payne 2022). To understand further the physiological role of Hnd in S. fructosivorans, we compared the mutant deleted of part of the hnd gene with the wild-type strain grown on pyruvate without sulfate using NMR-based metabolomics. Our results confirm that Hnd is profoundly involved in ethanol metabolism, but also indirectly intervenes in global carbon metabolism and additional metabolic processes such as the biosynthesis of branched-chain amino acids. We also highlight the metabolic reprogramming induced by the deletion of hndD that leads to the upregulation of several NADP-dependent pathways.

Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes.

Here, we outline how to identify hydrogenase enzymes from metagenomic fosmid libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis MR-1 (ΔhyaB) via triparental mating. If a fosmid clone exhibits hydrogen-uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. The screen enables screening of 48 metagenomic fosmid clones in parallel.

A regulatory hydrogenase gene cluster observed in the thioautotrophic symbiont of Bathymodiolus mussel in the East Pacific Rise.

The mytilid mussel Bathymodiolus thermophilus lives in the deep-sea hydrothermal vent regions due to its relationship with chemosynthetic symbiotic bacteria. It is well established that symbionts reside in the gill bacteriocytes of the mussel and can utilize hydrogen sulfide, methane, and hydrogen from the surrounding environment. However, it is observed that some mussel symbionts either possess or lack genes for hydrogen metabolism within the single-ribotype population and host mussel species level. Here, we found a hydrogenase cluster consisting of additional H2-sensing hydrogenase subunits in a complete genome of B. thermophilus symbiont sampled from an individual mussel from the East Pacific Rise (EPR9N). Also, we found methylated regions sparsely distributed throughout the EPR9N genome, mainly in the transposase regions and densely present in the rRNA gene regions. CRISPR diversity analysis confirmed that this genome originated from a single symbiont strain. Furthermore, from the comparative analysis, we observed variation in genome size, gene content, and genome re-arrangements across individual hosts suggesting multiple symbiont strains can associate with B. thermophilus. The ability to acquire locally adaptive various symbiotic strains may serve as an effective mechanism for successfully colonizing different chemosynthetic environments across the global oceans by host mussels.

Integration of QTL Mapping and Whole Genome Sequencing Identifies Candidate Genes for Alkalinity Tolerance in Rice (Oryza sativa).

Soil alkalinity is an important stressor that impairs crop growth and development, resulting in reduced crop productivity. Unlike salinity stress, research efforts to understand the mechanism of plant adaptation to alkaline stress is limited in rice, a major staple food for the world population. We evaluated a population of 193 recombinant inbred lines (RIL) developed from a cross between Cocodrie and N22 under alkaline stress at the seedling stage. Using a linkage map consisting of 4849 SNP markers, 42 additive QTLs were identified. There were seven genomic regions where two or more QTLs for multiple traits colocalized. Three important QTL clusters were targeted, and several candidate genes were identified based on high impact variants using whole genome sequences (WGS) of both parents and differential expression in response to alkalinity stress. These genes included two expressed protein genes, the glucan endo-1,3-beta-glucosidase precursor, F-box domain-containing proteins, double-stranded RNA-binding motif-containing protein, aquaporin protein, receptor kinase-like protein, semialdehyde hydrogenase, and NAD-binding domain-containing protein genes. Tolerance to alkaline stress in Cocodrie was most likely due to the low Na+/K+ ratio resulting from reduced accumulation of Na+ ions and higher accumulation of K+ in roots and shoots. Our study demonstrated the utility of integrating QTL mapping with WGS to identify the candidate genes in the QTL regions. The QTLs and candidate genes originating from the tolerant parent Cocodrie should be targeted for introgression to improve alkalinity tolerance in rice and to elucidate the molecular basis of alkali tolerance.

Comparative Physiology and Genomics of Hydrogen-Producing Vibrios.

The Hyf-type formate hydrogen lyase (FHL) complex was first proposed based on sequence comparisons in Escherichia coli in 1997 (Andrews et al. in Microbiology 143:3633-3647, 1997). The hydrogenase in the Hyf-type FHL was estimated to be a proton-translocating energy-conserving [NiFe]-hydrogenase. Although the structure of FHL is similar to that of complex I, silent gene expression in E. coli has caused delays in unveiling the genetic and biochemical features of the FHL. The entire set of genes required for Hyf-type FHL synthesis has also been found in the genome sequences of Vibrio tritonius in 2015 (Matsumura et al. in Int J Hydrog Energy 40:9137-9146, 2015), which produces more hydrogen (H2) than E. coli. Here we investigate the physiological characteristics, genome comparisons, and gene expressions to elucidate the genetic backgrounds of Hyf-type FHL, and how Hyf-type FHL correlates with the higher H2 production of V. tritonius. Physiological comparisons among the seven H2-producing vibrios reveal that V. porteresiae and V. tritonius, grouped in the Porteresiae clade, show greater capacity for H2 production than the other species. The structures of FHL-Hyp gene clusters were closely related in both Porteresiae species, but differed from those of the other species with the presence of hupE, a possible nickel permease gene. Interestingly, deeper genome comparisons revealed the co-presence of nickel ABC transporter genes (nik) with the Hyf-type FHL gene only on the genome of the Porteresiae clade species. Therefore, active primary Ni transport might be one of the key factors characterizing higher H2 production in V. tritonius. Furthermore, the expression of FHL gene cluster was significantly up-regulated in V. tritonius cells stimulated with formate, indicating that formate is likely to be a control factor for the gene expression of V. tritonius FHL in a similar way to the formate regulon encoding the E. coli FHL.

An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Impaired glucose metabolism by deleting the operon of hydrogenase 2 in Escherichia coli.

Although Escherichia coli has four hydrogenases, their definite roles in fermentation are still not clear. In this study, all the operon deletion mutants of E.coli hydrogenases (∆hya, ∆hyb, ∆hyc, or ∆hyf) were constructed to evaluate the hydrogen metabolism in comparison to their respective single-gene deletion mutants of large subunits (∆hyaB, ∆hybC, ∆hycE, and ∆hyfG). Besides the hyc operon mutant that expectedly showed no hydrogen synthesis, the hyb operon mutant showed low hydrogen production and demonstrated significantly reduced growth under anaerobic conditions. The present work also provided first-hand data where deleterious effects of operon deletion were compared with single-gene deletion mutations and the results showed that the former type of deletion was found to cause more prominent phenotypic effects than the latter one. Interestingly, hyb operon mutant was remarkably distinct from other operon mutants, specifically in its inability to utilize glucose under both aerobic and anaerobic conditions. Further studies on this mutant revealed a significant reduction of the total intracellular ATP and NADH concentrations, which could explain its impaired glucose metabolism. In this way, Hyd-2 was verified as crucial not only in glucose metabolism but also in energy balance and redox homeostasis of the cells. Furthermore, a decreased expression of glucose metabolism-associated genes, particularly ppc and pykA, indicated their regulation by hyb operon, and thereby, glucose consumption. Moreover, the transcriptional changes in this mutant indicated the wide genomic connectivity of hyb operon to other metabolisms.