Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Abstract

An hydrogenase-deficient (Hup−) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup− mutant, strain JP91, selected by its inability to grow autotrophically (Aut− phenotype) together with other Hup− mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup− mutants, span 15–20 kb of DNA.