Several ways one goal-methanogenesis from unconventional substrates.

Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are known: in the hydrogenotrophic pathway H2 and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (Methanosphaera, Methanimicrococcus, Methanomassiliicoccus, Methanonatronarchaeum) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching Methanonatronarchaeum revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of Methanosarcinales strains. Moreover, recently it has been shown that the methanogen Methermicoccus shengliensis can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (N,N,N-trimethylethanolamine) or betaine (N,N,N-trimethylglycine) have been described as substrates for methane production in Methanococcoides and Methanolobus strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways. KEY POINTS: • Newly discovered methanogenic substrates and pathways are reviewed for the first time. • The review provides an in-depth analysis of unusual methanogenesis pathways. • The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.

H2 Metabolism revealed by metagenomic analysis of subglacial sediment from East Antarctica.

Subglacial ecosystems harbor diverse chemoautotrophic microbial communities in areas with limited organic carbon, and lithological H2 produced during glacial erosion has been considered an important energy source in these ecosystems. To verify the H2-utilizing potential there and to identify the related energy-converting metabolic mechanisms of these communities, we performed metagenomic analysis on subglacial sediment samples from East Antarctica with and without H2 supplementation. Genes coding for several [NiFe]-hydrogenases were identified in raw sediment and were enriched after H2 incubation. All genes in the dissimilatory nitrate reduction and denitrification pathways were detected in the subglacial community, and the genes coding for these pathways became enriched after H2 was supplied. Similarly, genes transcribing key enzymes in the Calvin cycle were detected in raw sediment and were also enriched. Moreover, key genes involved in H2 oxidization, nitrate reduction, oxidative phosphorylation, and the Calvin cycle were identified within one metagenome-assembled genome belonging to a Polaromonas sp. As suggested by our results, the microbial community in the subglacial environment we investigated consisted of chemoautotrophic populations supported by H2 oxidation. These results further confirm the importance of H2 in the cryosphere.

Improved subtyping affords better discrimination of Trichomonas gallinae strains and suggests hybrid lineages.

Trichomonas gallinae is a protozoan pathogen that causes avian trichomonosis typically associated with columbids (canker) and birds of prey (frounce) that predate on them, and has recently emerged as an important cause of passerine disease. An archived panel of DNA from North American (USA) birds used initially to establish the ITS ribotypes was reanalysed using Iron hydrogenase (FeHyd) gene sequences to provide an alphanumeric subtyping scheme with improved resolution for strain discrimination. Thirteen novel subtypes of T. gallinae using FeHyd gene as the subtyping locus are described. Although the phylogenetic topologies derived from each single marker are complementary, they are not entirely congruent. This may reflect the complex genetic histories of the isolates analysed which appear to contain two major lineages and several that are hybrid. This new analysis consolidates much of the phylogenetic signal generated from the ITS ribotype and provides additional resolution for discrimination of T. gallinae strains. The single copy FeHyd gene provides higher resolution genotyping than ITS ribotype alone. It should be used where possible as an additional, single-marker subtyping tool for cultured isolates.

Identification of the heme acquisition system in Vibrio vulnificus M2799.

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.

Enzymatic and spectroscopic properties of a thermostable [NiFe]­hydrogenase performing H2-driven NAD+-reduction in the presence of O2.

Biocatalysts that mediate the H2-dependent reduction of NAD+ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD+-reducing [NiFe]­hydrogenase that sustains catalytic activity at high temperatures and in the presence of O2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD+-reducing [NiFe]­hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H2-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H2-mediated NAD+ reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]­hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD+-reducing [NiFe]­hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H2-driven cofactor recycling under oxic conditions at elevated temperatures.

Mixotrophy drives niche expansion of verrucomicrobial methanotrophs.

Aerobic methanotrophic bacteria have evolved a specialist lifestyle dependent on consumption of methane and other short-chain carbon compounds. However, their apparent substrate specialism runs contrary to the high relative abundance of these microorganisms in dynamic environments, where the availability of methane and oxygen fluctuates. In this work, we provide in situ and ex situ evidence that verrucomicrobial methanotrophs are mixotrophs. Verrucomicrobia-dominated soil communities from an acidic geothermal field in Rotokawa, New Zealand rapidly oxidised methane and hydrogen simultaneously. We isolated and characterised a verrucomicrobial strain from these soils, Methylacidiphilum sp. RTK17.1, and showed that it constitutively oxidises molecular hydrogen. Genomic analysis confirmed that this strain encoded two [NiFe]-hydrogenases (group 1d and 3b), and biochemical assays revealed that it used hydrogen as an electron donor for aerobic respiration and carbon fixation. While the strain could grow heterotrophically on methane or autotrophically on hydrogen, it grew optimally by combining these metabolic strategies. Hydrogen oxidation was particularly important for adaptation to methane and oxygen limitation. Complementary to recent findings of hydrogenotrophic growth by Methylacidiphilum fumariolicum SolV, our findings illustrate that verrucomicrobial methanotrophs have evolved to simultaneously utilise hydrogen and methane from geothermal sources to meet energy and carbon demands where nutrient flux is dynamic. This mixotrophic lifestyle is likely to have facilitated expansion of the niche space occupied by these microorganisms, allowing them to become dominant in geothermally influenced surface soils. Genes encoding putative oxygen-tolerant uptake [NiFe]-hydrogenases were identified in all publicly available methanotroph genomes, suggesting hydrogen oxidation is a general metabolic strategy in this guild.

Enhanced Hydrogen Production by Co-cultures of Hydrogenase and Nitrogenase in Escherichia coli.

Rhodobacter sphaeroides is a bacterium that can produce hydrogen by interaction with hydrogenase and nitrogenase. We report a hydrogen production system using co-cultivation of hydrogenase in liquid medium and immobilized nitrogenase in Escherichia coli. The recombinant plasmid has been constructed to analyze the effect of hydrogen production on the expression of hupSL hydrogenase and nifHDK nitrogenase isolated from R. sphaeroides. All recombinant E. coli strains were cultured anaerobically, and cells for nitrogenase were immobilized in agar gel, whereas cells for hydrogenase were supplemented on the nitrogenase agar gel. The hupSL hydrogenase has been observed to enhance hydrogen production and hydrogenase activity under co-culture with nifHDK nitrogenase. The maximum hydrogen production has been obtained at an agar gel concentration and a cell concentration for co-culture of 2 % and 6.4 × 10(8) CFU. Thus, co-culture of hupSL hydrogenase and nifHDK nitrogenase provides a promising route for enhancing the hydrogen production and hydrogenase activity.

Three different [NiFe] hydrogenases confer metabolic flexibility in the obligate aerobe Mycobacterium smegmatis.

Mycobacterium smegmatis is an obligate aerobe that harbours three predicted [NiFe] hydrogenases, Hyd1 (MSMEG_2262­2263), Hyd2 (MSMEG_2720-2719) and Hyd3 (MSMEG_3931-3928). We show here that these three enzymes differ in their phylogeny, regulation and catalytic activity. Phylogenetic analysis revealed that Hyd1 groups with hydrogenases that oxidize H2 produced by metabolic processes, and Hyd2 is homologous to a novel group of putative high-affinity hydrogenases. Hyd1 and Hyd2 respond to carbon and oxygen limitation, and, in the case of Hyd1, hydrogen supplementation. Hydrogen consumption measurements confirmed that both enzymes can oxidize hydrogen. In contrast, the phylogenetic analysis and activity measurements of Hyd3 are consistent with the enzyme evolving hydrogen. Hyd3 is controlled by DosR, a regulator that responds to hypoxic conditions. The strict dependence of hydrogen oxidation of Hyd1 and Hyd2 on oxygen suggests that the enzymes are oxygen tolerant and linked to the respiratory chain. This unique combination of hydrogenases allows M. smegmatis to oxidize hydrogen at high (Hyd1) and potentially tropospheric (Hyd2) concentrations, as well as recycle reduced equivalents by evolving hydrogen (Hyd3). The distribution of these hydrogenases throughout numerous soil and marine species of actinomycetes suggests that oxic hydrogen metabolism provides metabolic flexibility in environments with changing nutrient fluxes.

Circadian yin-yang regulation and its manipulation to globally reprogram gene expression.

BACKGROUND: The cyanobacterial circadian program exerts genome-wide control of gene expression. KaiC undergoes rhythms of phosphorylation that are regulated by interactions with KaiA and KaiB. The phosphorylation status of KaiC is thought to mediate global transcription via output factors SasA, CikA, LabA, RpaA, and RpaB. Overexpression of kaiC has been reported to globally repress gene expression. RESULTS: Here, we show that the positive circadian component KaiA upregulates "subjective dusk" genes and that its overexpression deactivates rhythmic gene expression without significantly affecting growth rates in constant light. We analyze the global patterns of expression that are regulated by KaiA versus KaiC and find in contrast to the previous report of KaiC repression that there is a "yin-yang" regulation of gene expression whereby kaiA overexpression activates "dusk genes" and represses "dawn genes," whereas kaiC overexpression complementarily activates dawn genes and represses dusk genes. Moreover, continuous induction of kaiA latched KaiABC-regulated gene expression to provide constitutively increased transcript levels of diverse endogenous and heterologous genes that are expressed in the predominant subjective dusk phase. In addition to analyzing KaiA regulation of endogenous gene expression, we apply these insights to the expression of heterologous proteins whose products are of potential value, namely human proinsulin, foreign luciferase, and exogenous hydrogenase. CONCLUSIONS: Both KaiC and KaiA complementarily contribute to the regulation of circadian gene expression via yin-yang switching. Circadian patterns can be reprogrammed by overexpression of kaiA or kaiC to constitutively enhance gene expression, and this reprogramming can improve 24/7 production of heterologous proteins that are useful as pharmaceuticals or biofuels.

Identification and structure of a novel archaeal HypB for [NiFe] hydrogenase maturation.

HypB (metal-binding GTPase) and HypA (nickel metallochaperone) are required for nickel insertion into [NiFe] hydrogenase. However, the HypB homolog proteins are not found in some archaeal species including Thermococcales. In this article, we identify a novel archaeal Mrp/MinD family ATPase-type HypB from Thermococcus kodakarensis (Tk-mmHypB) and determine its crystal structure. The mmhypB gene is conserved among species lacking the hypB gene and is located adjacent to the hypA gene on their genome. Deletion of the mmhypB gene leads to a significant reduction in hydrogen-dependent growth of T. kodakarensis, which is restored by nickel supplementation. The monomer structure of Tk-mmHypB is similar to those of the Mrp/MinD family ATPases. The ADP molecules are tightly bound to the protein. Isothermal titration calorimetry shows that Tk-mmHypB binds ATP with a K(d) value of 84 nM. ADP binds more tightly than does ATP, with a K(d) value of 15 nM. The closed Tk-mmHypB dimer in the crystallographic asymmetric unit is consistent with the ATP-hydrolysis-deficient dimer of the Mrp/MinD family Soj/MinD proteins. Structural comparisons with these proteins suggest the ATP-binding dependent conformational change and rearrangement of the Tk-mmHypB dimer. These observations imply that the nickel insertion process during the [NiFe] hydrogenase maturation is performed by HypA, mmHypB, and a nucleotide exchange factor in these archaea.

Redirecting the electron flow towards the nitrogenase and bidirectional Hox-hydrogenase by using specific inhibitors results in enhanced H2 production in the cyanobacterium Anabaena siamensis TISTR 8012.

The inhibition of competitive metabolic pathways by various inhibitors in order to redirect electron flow towards nitrogenase and bidirectional Hox-hydrogenase was investigated in Anabaena siamensis TISTR 8012. Cells grown in BG11(0) supplemented with KCN, rotenone, DCMU, and DL-glyceraldehyde under light condition for 24 h showed enhanced H(2) production. Cells grown in BG11 medium showed only marginal H(2) production and its production was hardly increased by the inhibitors tested. H(2) production with either 20mM KCN or 50 µM DCMU in BG11(0) medium was 22 µmol H(2) mg chl a(-1) h(-1), threefold higher than the control. The increased H(2) production caused by inhibitors was consistent with the increase in the respective Hox-hydrogenase activities and nifD transcript levels, as well as the decrease in hupL transcript levels. The results suggested that interruption of metabolic pathways essential for growth could redirect electrons flow towards nitrogenase and bidirectional Hox-hydrogenase resulting in increased H(2) production.

Iron restriction induces preferential down-regulation of H(2)-consuming over H(2)-evolving reactions during fermentative growth of Escherichia coli.

BACKGROUND: Escherichia coli synthesizes three anaerobically inducible [NiFe]-hydrogenases (Hyd). All three enzymes have a [NiFe]-cofactor in the large subunit and each enzyme also has an iron-sulfur-containing small subunit that is required for electron transfer. In order to synthesize functionally active Hyd enzymes iron must be supplied to the maturation pathways for both the large and small subunits. The focus of this study was the analysis of the iron uptake systems required for synthesis of active Hyd-1, Hyd-2 and Hyd-3 during fermentative growth. RESULTS: A transposon-insertion mutant impaired in hydrogenase enzyme activity was isolated. The mutation was in the feoB gene encoding the ferrous iron transport system. The levels of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 as determined by specific in-gel activity staining were reduced at least 10-fold in the mutant after anaerobic fermentative growth in minimal medium, while the hydrogen-evolving Hyd-3 activity was less severely affected. Supplementation of the growth medium with ferric iron, which is taken up by e.g. the siderophore enterobactin, resulted in phenotypic complementation of the feoB mutant. Growth in rich medium demonstrated that a mutant lacking both the ferrous iron transport system and enterobactin biosynthesis (entC) was devoid of Hyd-1 and Hyd-2 activity but retained some hydrogen-evolving Hyd-3 activity. Analysis of crude extracts derived from the feoB entC double null mutant revealed that the large subunits of the hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 were absent. Analysis of lacZ fusions demonstrated, however, that expression of the hya, hyb and hyc operons was reduced only by maximally 50% in the mutants compared with the wild type. CONCLUSIONS: Our findings demonstrate that the ferrous iron transport system is the principal route of iron uptake for anaerobic hydrogenase biosynthesis, with a contribution from the ferric-enterobactin system. Hydrogen-oxidizing enzyme function was abolished in a feoB entC double mutant and this appears to be due to post-translational effects. The retention of residual hydrogen-evolving activity, even in the feoB entC double null mutant suggests that sufficient iron can be scavenged to synthesize this key fermentative enzyme complex in preference to the hydrogen-uptake enzymes.

Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3).

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²âº (Ni²âº-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO4²â» ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO4²â» were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO4²â» and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

Transcription of fdh and hyd in Syntrophobacter spp. and Methanospirillum spp. as a diagnostic tool for monitoring anaerobic sludge deprived of molybdenum, tungsten and selenium.

Formate dehydrogenases and hydrogenases contain molybdenum or tungsten and/or selenium. These enzymes are crucial for interspecies formate and hydrogen transfer between propionate degrading Syntrophobacter spp. and methanogenic Methanospirillum spp. Here we used reverse transcription of total RNA followed by quantitative PCR (RT-qPCR) with specific primers to get insight into interspecies formate and hydrogen transfer. Transcriptional regulation of formate dehydrogenases and hydrogenases in Syntrophobacter and Methanospirillum spp. in a propionate-fed up-flow anaerobic sludge bed (UASB) reactor was examined. In both microorganisms formate dehydrogenase and hydrogenase coding genes (fdh and hyd respectively) were transcribed simultaneously. During 249 days in which molybdenum, tungsten and selenium were not supplied to the reactor feed, the microbial activity and transcription of fdh and hyd in Syntrophobacter spp. decreased. Transcription of fdh and hyd in Methanospirillum spp. did not decrease, but transcription of fdh increased when after 249 days molybdenum, tungsten and selenium were supplied to the reactor feed. The developed RT-qPCR is a technique that can give rapid information about active processes in methanogenic granular sludge and may contribute to predict metal limitation and failure in UASB reactors.

High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

BACKGROUND: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. PRINCIPAL FINDINGS: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms. SIGNIFICANCE: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli.

BACKGROUND: Hydrogenases catalyze reversible reaction between hydrogen (H2) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H2 producing ability. RESULTS: Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H2 (12.5 mL H2/(h.L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H2 even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (approximately 65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his6-tag displayed oxygen-tolerant activity of approximately 12 nmol H2/(min.mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition. CONCLUSIONS: This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H2 production. We found that [NiFe]-hydrogenase 1 has H2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect the H2 production process from oxygen. Therefore, we propose that [NiFe]-hydrogenase can be successfully applied as an efficient biohydrogen production tool under micro-aerobic conditions.

First molecular characterisation of hydrogenosomes in the protozoan parasite Histomonas meleagridis.

Histomonas meleagridis is a trichomonad species that undergoes a flagellate-to-amoeba transformation during tissue invasion and causes a serious disease in gallinaceous birds (blackhead disease or histomoniasis). Living in the avian cecum, the flagellated form can be grown in vitro in the presence of an ill-defined bacterial flora. Its cytoplasm harbours numerous spherical bodies which structurally resemble hydrogenosomes. To test whether these organelles may be involved in anaerobic metabolism, we undertook the identification of H. meleagridis genes encoding some potentially conserved hydrogenosomal enzymes. The strategy was based on several PCR amplification steps using primers designed from available sequences of the phylogenetically-related human parasite Trichomonas vaginalis. We first obtained a C-terminal sequence of an iron-hydrogenase homologue (Hm_HYD) with typical active site signatures (H-cluster domain). Immunoelectron microscopy with anti-Hm_HYD polyclonal antibodies showed specific gold labelling of electron-dense organelles, thus confirming their hydrogenosomal nature. The whole genes encoding a malic enzyme (Hm_ME) and the alpha-subunit of a succinyl coenzyme A synthetase (Hm_alpha-SCS) were then identified. Short N-terminal presequences for hydrogenosomal targeting were predicted in both proteins. Anti-Hm_ME and anti-Hm_alpha-SCS antisera provided immunofluorescence staining patterns of H. meleagridis cytoplasmic granules similar to those observed with anti-Hm_HYD antiserum or mAb F5.2 known to react with T. vaginalis hydrogenosomes. Hm_ME, Hm_alpha-SCS and Hm_HYD were also detected as reactive bands on immunoblots of proteins from purified hydrogenosomes. Interestingly, anti-Hm_alpha-SCS staining of the cell surface in non-permeabilised parasites suggests a supplementary role for SCS in cytoadherence, as previously demonstrated in T. vaginalis.

Hydrogenosomal activity of Trichomonas vaginalis cultivated under different iron conditions.

To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.

Regulation of uptake hydrogenase and effects of hydrogen utilization on gene expression in Rhodopseudomonas palustris.

Rhodopseudomonas palustris is a purple, facultatively phototrophic bacterium that uses hydrogen gas as an electron donor for carbon dioxide fixation during photoautotrophic growth or for ammonia synthesis during nitrogen fixation. It also uses hydrogen as an electron supplement to enable the complete assimilation of oxidized carbon compounds, such as malate, into cell material during photoheterotrophic growth. The R. palustris genome predicts a membrane-bound nickel-iron uptake hydrogenase and several regulatory proteins to control hydrogenase synthesis. There is also a novel sensor kinase gene (RPA0981) directly adjacent to the hydrogenase gene cluster. Here we show that the R. palustris regulatory sensor hydrogenase HupUV acts in conjunction with the sensor kinase-response regulator protein pair HoxJ-HoxA to activate hydrogenase expression in response to hydrogen gas. Transcriptome analysis indicated that the HupUV-HoxJA regulatory system also controls the expression of genes encoding a predicted dicarboxylic acid transport system, a putative formate transporter, and a glutamine synthetase. RPA0981 had a small effect in repressing hydrogenase synthesis. We also determined that the two-component system RegS-RegR repressed expression of the uptake hydrogenase, probably in response to changes in intracellular redox status. Transcriptome analysis indicated that about 30 genes were differentially expressed in R. palustris cells that utilized hydrogen when growing photoheterotrophically on malate under nitrogen-fixing conditions compared to a mutant strain that lacked uptake hydrogenase. From this it appears that the recycling of reductant in the form of hydrogen does not have extensive nonspecific effects on gene expression in R. palustris.