The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

Abstract

The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and approximate copy number of 20-50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S.lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.