[Homologous recombination in Streptomyces lincolnensis B48]

To study frequency and mechanism of homologous recombination in Streptomyces, an E. coli plasmid which cannot replicate in Streptomyces was transformed into Streptomyces lincolnensis B48. After homologous recombination between delta lincomycin biosynthetic genes inactivated by thiostrepton resistant gene (tsr) carried on pYYE04al and homologous sequences on the chromosome, S. lincolnensis YY1 and S. lincolnensis, YY2 were obtained on SMA with low thiostrepton concentration. Hybridization of chromosomal DNA samples of S. lincolnensis YY1, S. lincolnensis YY2, standard S. lincolnensis and S. lincolnensis YYc digested with SmaI with the probe of tsr gene gave signal corresponding to a fragment of 1.5 kb in the former two; Nevertheless, hybridization of chromosomal DNA digested with Hind III and Sma I using the probe of delta lacZ' gene resulted in positive fragment of 4.4 kb only in S. lincolnensis YY2. Southern hybridizations indicate that S. lincolnensis YY1 is the result of homologous exchange while S. lincolnensis YY2 comes from bomologous recombination. To prove the existence of E. coli replicon and ampicillin resistant gene on the chromosome of S. lincolnensis YY2, its DNA digested with SphI was ligated and then transformed into E. coli JM83 competent cell. Two transformants named pSLE1 grew on the plate containing ampicillin. It's confirmed that pSLE1 is a part of pYYE04a1 from its digestion with different enzymes.