Gene transfer and transposition mutagenesis in Streptomyces roseosporus: Mapping of insertions that influence daptomycin or pigment production

Abstract

Streptomyces roseosporus, the producer of the cyclic lipopeptide antibiotic daptomycin, was shown to be a suitable host for molecular genetic manipulation. S. roseosporus does not appear to express significant restriction barriers based upon bacteriophage plaque formation studies. Plasmid DNA can be introduced into S. roseosporus by bacteriophage-FP43-mediated transduction and by conjugation from Escherichia coli. The streptomycete transposons Tn5096 and Tn5099, derived from IS493, transpose in S. roseosporus, and Tn5099-induced transposition mutants altered in the production of daptomycin, red pigment or black pigment were identified, and mapped to DraI and AsnI fragments. Three auxotrophic mutations (argB1, ade-1 and metB1) were identified among 100 individual Tn5096 insertions. Alignment and physical mapping of several Tn5099 insertions in DraI-E and AsnI-B fragments was facilitated by the presence of DraI and AsnI cleavage sites in Tn5099.