Generally, hydrogen is supplied by those heterotrophs with very low concentrations. We are particularly focusing on the effect of substrate (hydrogen) concentrations on the expression of genes responsible for methanogenesis. Genetic and biochemical studies revealed that hydrogenotrophic methanogens harbor two isofunctional methyl-coenzyme M reductases and several other enzymes. Of the enzymes, methyl-coenzyme M reductase is the key enzyme that catalyzes the last step of methanogenesis.
M. thermoautotrophicum genome sequence has allowed us to design various gene probes to detect specific mRNA to determine whether hydrogen concentration affect the expression of those isofunctional genes. We cultivated M. thermoautotrophicum under high hydrogen partial pressures and low hydrogen partial pressures to do Northem analysis. Cultivation at high hydrogen concentrations was simply done in pure culture using serum vials pressurized by hydrogen (1 atm). Cultivation under low hydrogen partial pressures (less than 0.001 atm) was possible when the methanogen was cocultured with acetate- or butyrate-oxidizing syntrophs using acetate and butyrate, respectively, as sole carbon and energy source. Acetate or butyrate was oxidized by the syntroph to constantly produce very low concentrations of hydrogen that could be immediately converted to methane. Northern analysis and 2-D protein electrophoresis showed that only one of the methyl-coenzyme M reductases was expressed at low hydrogen partial pressures whereas both of the genes were expressed at high hydrogen partial pressures.
Our experiment is one of the good examples of how the complete genome sequence information was useful to investigate genetic characteristics of methanogens. The genome analysis will shed light not only on genetic traits of the methanogen but also on physiological and ecological characteristics of this environmentally important microorganism.
