Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans

Abstract

A strain of Desulfovibrio fructosovorans deleted from the hydN [NiFe]hydrogenase structural gene was constructed. A plasmid carrying a 7 kb DNA fragment on which the hydN gene had been replaced by the npt reporter gene (kanamycin-resistant, KnR) was introduced into D. fructosovorans by electroporation. Southern analysis of one KnR clone demonstrated that the hydN gene had been eliminated by marker exchange. This mutant, which was devoid of the [NiFe]hydrogenase gene, still showed a 10% residual hydrogenase activity. Its ability to grow efficiently with H2 as sole energy source is discussed. This is the first report, in a member of the sulphate-reducing bacteria, of a successful transformation and concomitant homologous recombination leading to a fully controlled genotype.

Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans

“…D. fructosovorans ⌬hndC is a null mutant which was obtained by performing marker exchange mutagenesis as described by Rousset et al. (18).…”

“…This property might make it possible to obtain Hyd-null mutations that are not lethal to the organism even if Hyds are involved in the mechanism underlying oxidative phosphorylation, as has been previously suggested (13). D. fructosovorans MR400, from which the [NiFe] Hyd structural genes (hynAB) had been deleted, was constructed in order to correlate the functional deficit with the genotype of the mutant (18). Significant levels of residual Hyd activity have been observed in MR400, which were attributable to additional enzymes.…”

Evidence for a Fourth Hydrogenase in Desulfovibrio fructosovorans

“…Methyl viologen reduction activity of the triple mutant. Hydrogenase activity in D. fructosovorans is usually determined by measurement of hydrogen-dependent methyl viologen reduction activity in soluble cellular extract obtained from cultures grown on a 30 mM fructose-50 mM sulfate medium (4,5,12,19). The presence of the hydrogenase activities in the D. fructosovorans strains was tested in native polyacrylamide gel electrophoresis (Fig.…”

“…In this species, three hydrogenases have been already characterized: a periplasmic [NiFe] hydrogenase which represents about 1% of the total proteins (8,20), a cytoplasmic NADPreducing hydrogenase (13), and a periplasmic [Fe] hydrogenase (4). In order to elucidate the relative importance of these various hydrogenases in the energy-generating metabolism of D. fructosovorans, deletions were first made by marker exchange mutagenesis of the genes encoding the [NiFe] hydrogenase (19) and the NADP-reducing hydrogenase (12). All mutants (single or double) showed significant growth on organic substrates as well as on medium containing H 2 as the sole energy source.…”

“…1). This recombinant suicide plasmid was introduced into the DM4 strain (⌬hynABC Kan r ; ⌬hndD Cm r ) (12) depleted of the [NiFe] and NADPreducing hydrogenases, using an electrotransformation procedure as previously described (19). The electrotransformed cells were first grown for 6 h without any antibiotics and then subcultured in the presence of antibiotics (50 g of kanamycin ml Ϫ1 , 34 g of thiamphenicol ml Ϫ1 , and 20 g of gentamicin ml Ϫ1 ) in liquid medium.…”