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Escherichia coli -Streptomyces rimosus conjugation system

Production of oxytetracycline (OTC) by S. rimosus R6 is genetically unstable (Gravius et al., Appl. Environ. Microbiol., 59, 2220, 1993), making it difficult to manipulate the otc gene cluster within its chromosomal location (N. PeriÊ et al., this meeting). For that reason, a strategy to manipulate the cluster by "chromosomal building" (O'Connor et al., Science, 244, 1307, 1989) is being evaluated, by in vivo recombination of otc genes in E. coli and their subsequent translocation to an ectopic site on the S. rimosus R6 chromosome. The standard protocol for E. coli - Streptomyces spp. conjugation (Mazodier et al. J. Bacteriol., 171, 3583, 1989) gave poor transfer to S. rimosus, but compartively efficient transfer (one exconjugant among 104 - 103 c.f.u.) was obtained using an E. coli mutant triply defective in DNA methylation as a donor. Transfer functions were provided on a RK2- derived plasmid. In such a system, a derivative of pSET152, [an integrating plasmid based on C31 (Bierman et al. Gene, 116, 43, 1992)] was successfully delivered to the chromosome of numerous S. rimosus strains using freshly germinated spores as well as mycelial fragments. Preliminary restriction maps of the chromosomal C31 attB site, and of the attB site of integrating vector pSAM2 will be presented.

Escherichia coli; Streptomyces rimosus; conjugation

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