USE OF RYSL FOR DOMINANCE SELECTION AND GENE REPLACEMENT IN STREPTOMYCES-ROSEOSPORUS

We developed a gene replacement system using the rpsL gene of Streptom yces roseosporus and demonstrated its utility by constructing a deleti on in the S. roseosporus glnA gene, A 1,3-kb BamHI fragment that hybri dized to the Mycobacterium smegmatis rpsL gene was subcloned from an S . roseosporus cosmid library and sequenced, Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm-5) to the Sm-r S . roseosporus TH149, The temperature-sensitive plasmid pRHB543 contain ing rpsL and the S. roseosporus gluA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S, roseosporus TH149, and recombinants containing single and double crossovers were obtained aft er a temperature increase, Southern hybridization analysis revealed th at single crossovers occurred in the glnA or rpsL genes and that doubl e crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.