Identification and partial characterization of an Escherichia coli mutant with altered hydrogenase activity

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Abstract
An E. coli mutant strain with altered hydrogenase activity was isolated using a filter paper assay. This assay depends on the ability of hydrogenase-containing microorganisms to reduce methyl viologen impregnated in filter paper, producing purple-colored colonies in the presence of H2. Membrane-bound and cytoplasmic hydrogenase activities of wild-type and mutant strains were compared by amperometric measurement of H2 production. The cytoplasmic activities of mutant and wild type were comparable. The membrane-bound activity was lower in the mutant than in the wild type. Upon addition of detergent to the membrane fraction the specific activity of the enzyme from the mutant strain increased so that it equaled that of the wild type. The mutant requires an exogenous electron acceptor for anaerobic growth providing evidence for the function of the hydrogenase in anaerobic growth.