Targeted gene-replacement mutagenesis of dcrA, encoding an oxygen sensor of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

Abstract

A gene-replacement mutagenesis method has been developed for the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough and used to delete dcrA, encoding a potential oxygen or redox sensor with homology to the methyl-accepting chemotaxis proteins. A suicide plasmid, containing a cat-marked dcrA allele and a counter-selectable sacB marker was transferred from Escherichia coli S17-1 to D. vulgaris by conjugation. Following plasmid integration the desired dcrA deletion mutant (D. vulgaris F100) was obtained in media containing sucrose and chloramphenicol. Southern blot screening was required to distinguish D. vulgaris F100 from strain in which the sacB marker was inactivated by transposition of an endogenous IS element. No anaerotactic deficiency has so far been detected in D. vulgaris F100, which was found to be more resistant to inactivation by oxygen that the wild-type. Increased transcription of the rbo-rub operon, located immediately downstream from dcrA, was demonstrated by Northern blotting and may be the cause of this unusual phenotype, in view of the recent discovery that Rbo can complement the deleterious effects of superoxide dismutase deficiency in E. coli.