Random transposition by Tn916 in Desulfitobacterium dehalogenans allows for isolation and characterization of halorespiration-deficient mutants

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D, dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron accepters nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.