Molecular cloning of a DNA segment essential for replication from Streptomyces hygroscopicus strain 10-22 and localization of its minimal functional region.

The 13 kb DNA segment responsible for replication has been isolated from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22 using a pBR322-derived Streptomyces replication-probe vector pIJ2703. The restriction map of the hybrid plasmid, pHZ54, has been constructed. Following a series of deletion and subcloning experiments, the minimal functional region for replication was located within a 2.86 kb DNA fragment. Subsequently, this region was further shortened into a 2 kb region by exonuclease III deletion. A few small bifunctional plasmids able to replicate in E. coli as well as in Streptomyces, were obtained during the above process. Some plasmids could be used as shuttle vectors.