Mutational analysis of the Streptomyces lividans recA gene suggests that only mutants with residual activity remain viable.
Temperature-sensitive integration plasmids carrying internal fragments of the Streptomyces lividans TK24 recA gene were constructed and used to inactivate the chromosomal recA gene of S. lividans by gene disruption and gene replacement. Integration of these plasmids resulted in recA mutants expressing C-terminally truncated RecA proteins, as deduced from Southern hybridization experiments. Mutants FRECD2 in which the last 42 amino acids, comprising the variable part of bacterial RecA proteins, had been deleted retained the wild-type phenotype. The S. lividans recA mutant FRECD3 produced a RecA protein lacking 87 amino acids probably including the interfilament contact site. FRECD3 was more sensitive to UV and MMS than the wild-type. Its ability to undergo homologous recombination was impaired, but not completely abolished. Integration of the disruption plasmid pFRECD3 in S. coelicolor "Müller" caused the same mutant phenotype as S. lividans FRECD3. In spite of many attempts no S. lividans recA mutants with deletions of 165 C-terminal amino acids or more were isolated. Furthermore, the recA gene could not be replaced by a kanamycin resistance cassette. These experiments indicate a crucial role of the recA gene in ensuring viability of Streptomyces.
Muth
G
G
Universität Tübingen, Germany.
Frese
D
D
Kleber
A
A
Wohlleben
W
W
D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005804:Genes, Lethal; D016254:Mutagenesis, Insertional; D009154:Mutation; D010641:Phenotype; D011938:Rec A Recombinases / Q000235:genetics*; D017384:Sequence Deletion; D013045:Species Specificity; D013302:Streptomyces / Q000235:genetics*
D011938:Rec A Recombinases