Methanogens utilize an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane (CH4) gas. Previous biochemical studies suggest that the proton pumping F420H2 dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri ∆fpo mutants investigated in Chapter 2 display no measurable phenotype on this substrate. In contrast, ∆frh mutants lacking the cytoplasmic F420-dependent hydrogenase (Frh) are severely affected in their ability to grow and make methane from methanol, while double ∆fpo ∆frh mutants are completely unable to utilize this substrate. These data suggest that while M. barkeri has the flexibility to use the Fpo-dependent electron transport chain when needed, the preferred energy conservation pathway involves production of H2 gas by Frh hydrogenase within the cytoplasm. The H2 can then diffuse out of the cell, where it can be oxidized by the periplasmic methanophenazine-dependent hydrogenase (Vht), with transfer of electrons into the electron transport chain. Consistent with this “H2-cycling” proposal, a conditional vht mutant isolated in Chapter 3, is unable to grow using any of the methanogenic substrates tested under non-permissive conditions, suggesting that Vht is essential for growth of M. barkeri. Moreover, repression of vht expression results in a rapid increase in H2 partial pressure, which supports the hypothesis that Vht is required for H2 uptake. H2 accumulation in the culture headspace of conditional vht mutant is accompanied with cessation of methanogenesis and growth, implying that H2 uptake is essential for anaerobic respiration and viability. In contrast, Vht is not essential in mutants lacking the H2-producing Frh hydrogenase. This is consistent with the hypothesis that Vht is required for H2 uptake, only when Frh produces H2, that is, Frh and Vht hydrogenases are functionally coupled in a “H2-cycling” energy conservation mechanism. Because production of H2 by Frh consumes protons within the cytoplasm and oxidation of H2 by Vht releases protons outside the cell, this electron transport chain is capable of establishing a trans-membrane proton gradient that can be used to make ATP by the ATP synthase. Our study provides the first direct experimental evidence for H2-cycling, since it was proposed to be involved in energy conservation in sulfate-reducing bacteria in 1981. To further dissect the roles of these hydrogenases in M. barkeri physiology, I constructed a series of hydrogenase deletion mutants in various combinations in Chapter 4, including a mutant that is devoid of all three types of hydrogenases, ferredoxin-dependent Ech, Frh and Vht. My data show that each of the three types of hydrogenases is needed for growth via the CO2 reduction pathway. In contrast, none of the hydrogenases is essential during methylotrophic growth, indicating the presence of H2-independent electron transport chains, which are able to support wild-type growth yields. Either Vht or Ech hydrogenase alone can support growth using the methyl respiration pathway. However, both Ech and Vht hydrogenases are required for acetate utilization. The data presented in chapter 4 also suggest that Ech and/or Frh hydrogenases block the methyl oxidative pathway by catalyzing conversion of H2 to Fdred and F420H2, respectively. In addition, evidence is provided for involvement of Hyp proteins in maturation of Ech hydrogenase. This work highlights the similarities and differences between H2-independent electron transport chains of the hydrogenotroph M. barkeri and the non-hydrogenotroph Methanosarcina acetivorans.
【 预 览 】
附件列表
Files
Size
Format
View
Analysis of hydrogen metabolism in methanosarcina barkeri fusaro
PDF
download
878987797
028-85220240
