Development of an intergeneric conjugal transfer system for rimocidin-producing Streptomyces rimosus

To develop an intergeneric conjugation system for rimocidin-producing Streptomyces rimosus.

High efficiencies of conjugation [10(-2)-10(-3) transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40 degrees C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24-h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l(-1) MgCl(2) was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector.

Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium.

The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.