Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest that H2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature.

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SUMMARY The biological production of methane is vital to the global carbon cycle and accounts for ca. 74% of total methane emissions. The organisms that facilitate this process, methanogenic archaea, belong to a large and phylogenetically diverse group that thrives in a wide range of anaerobic environments. Two main subgroups exist within methanogenic archaea: those with and those without cytochromes. Although a variety of metabolisms exist within this group, the reduction of growth substrates to methane using electrons from molecular hydrogen is, in a phylogenetic sense, the most widespread methanogenic pathway. Methanogens without cytochromes typically generate methane by the reduction of CO2 with electrons derived from H2, formate, or secondary alcohols, generating a transmembrane ion gradient for ATP production via an Na+-translocating methyltransferase (Mtr). These organisms also conserve energy with a novel flavin-based electron bifurcation mechanism, wherein the endergonic reduction of ferredoxin is facilitated by the exergonic reduction of a disulfide terminal electron acceptor coupled to either H2 or formate oxidation. Methanogens that utilize cytochromes have a broader substrate range, and can convert acetate and methylated compounds to methane, in addition to the ability to reduce CO2. Cytochrome-containing methanogens are able to supplement the ion motive force generated by Mtr with an H+-translocating electron transport system. In both groups, enzymes known as hydrogenases, which reversibly interconvert protons and electrons to molecular hydrogen, play a central role in the methanogenic process. This review discusses recent insight into methanogen metabolism and energy conservation mechanisms with a particular focus on the genus Methanosarcina.

The methanogenic archaeon encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H-CO, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δ Δ and Δ Δ Δ mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F dehydrogenase operon were significantly higher in all strains lacking , suggesting a mechanism to sense the redox state of F The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed. Methanogenic archaea are key players in the global carbon cycle due to their ability to facilitate the remineralization of organic substrates in many anaerobic environments. The consequences of biological methanogenesis are far-reaching, with impacts on atmospheric methane and CO concentrations, agriculture, energy production, waste treatment, and human health. The data presented here clarify the function of hydrogenases during methanogenesis, which in turn deepens our understanding of this unique form of metabolism. This knowledge is critical for a variety of important issues ranging from atmospheric composition to human health.

Aims: This study analysed the growth and survival of 18 strains of the six serotypes of non-O157 Shiga toxin-producing Escherichia coli (STEC) (O26, O45, O103, O111, O121 and O145) most frequently implicated in human illness and compared them with Escherichia coli O157:H7 strain ATCC43895. Methods and Results:The data from growth in Luria-Bertani broth (LB)-HCl (pH 4Á0, 4Á5, 4Á8), LB-lactate (pH 4Á5 and 4Á8) and LB-NaCl (5%, 7%) were fitted to modified Gompertz equations to enable quantitative comparisons across strains and media conditions. Serogroup O45 strains had growth rates that were equal to or significantly greater than the O157:H7 control strain in all growth conditions tested. The growth rate was independent from the maximum growth achieved, but three strains (103A, 121A and 45B) had significantly faster growth and greater maximum cell densities in LB-NaCl 5% (strain 103A), LB-HCl pH 4Á0 (strain 121A) and LB-NaCl 7% (strain 45B). Survival in LB-HCl pH 3Á0 of four strains (103C, 111B, 26B and 26C) was significantly greater and five strains (26A, 45A, 111A, 121A and 145A) were significantly reduced in comparison with the O157:H7 control strain. None of the STEC strains had greater survival in LB-NaCl 12% than the O157:H7 control strain. A significant association was found between the exponential phase, but not stationary phase, RpoS level and survival of STEC. Conclusions: Some STEC strains had growth or survival properties that exceeded those of the O157:H7 control strain, but none of the non-O157 STEC had both significantly greater growth and survival properties. STEC survival was associated with the exponential-phase RpoS level. Significance and Impact of the Study: Results from this study define the variability in growth and survival of STEC strains that will be useful defining food product formulations and process interventions to control STEC. The presence of exponential phase r s expands the significance of this alternative sigma factor.