Three functional replication origins of the linear and artificially circularized plasmid SCP1 of Streptomyces coelicolor.

Previous reports showed that the large linear plasmid SCP1 of Streptomyces coelicolor A3(2) contains a 5.4 kb centrally located replication locus. We report here that SCP1 actually contains three internal replication loci. Subcloning of the 5.4 kb sequence identified a 3.2 kb minimal locus (rep1A/repB/iteron) that determined propagation in Streptomyces lividans. The two newly identified replication genes, rep2A and rep3A, resembled the rep gene of Streptomyces circular plasmid pZL12. Transcription start points of the three replication genes were determined. The three replication loci could independently determine propagation in linear mode in S. lividans. Individual and sequential deletions of the rep1A and rep3A genes were successful. The SCP1-derived linear plasmids with deletions of the rep1A and/or rep3A genes still propagated in similar copy numbers, were inherited largely stable and were transferred efficiently by conjugation in S. coelicolor. Interestingly, SCP1 can be artificially circularized to yield a 280 kb circular plasmid, circular SCP-1 (C-SCP1), which contains the three replication loci. Strikingly, the copy numbers, inheritance and transfer of C-SCP1 resembled that of the linear SCP1 plasmids. Transcripts of the rep1A, rep2A and rep3A genes in linear or artificially circularized SCP1 were detected at all the time points of strain growth.