Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production

Abstract

Cosmid pOJ436, containing large inserts of Saccharopolyspora spinosa ( Ss) DNA, was transferred by conjugation from Escherichia coli to Ss and integrated into the chromosome, apparently by homologous recombination, at high frequencies (10 −5 to 10 −4 per recipient). Transfer was mediated by the plasmid RP4 (RK2) transfer functions in E. coli, and the RK2 oriT function located on pOJ436 [Bierman et al., Gene 116 (1992) 43–49]. pOJ436 lacking Ss DNA, or containing a small insert (approx. 2 kb) of Ss DNA, conjugated from E. coli and integrated at either of two bacteriophage øC31 attB sites at low frequency (approx. 10 −7 per recipient). Exconjugants containing homologous inserts or inserts at the øC31 attB sites were stable in the absence of antibiotic selection, and most produced control levels of tetracyclic macrolide A83543 factors. Some exconjugants contained similar kinds of large deletions and were defective in macrolide production.