Identification and characterization of a central replication origin of the mega-plasmid pSCATT of Streptomyces cattleya.

Abstract

Streptomyces linear plasmids often contain an internal replication origin. In this study, a new replication origin was identified and confirmed in the 1.8-Mb plasmid pSCATT of Streptomyces cattleya DSM46488. The real-time qPCR results indicated that the copy number of pSCATT was one copy per chromosome. The identified replication origin oriC1-II was found to locate in the central region of pSCATT and was 2 kb in size. This replication origin consists of a protein-coding gene SCATT_p08010 with an unknown function and the upstream non-coding sequence. Deletion or disruption analysis of SCATT_p08010 or the upstream non-coding sequence revealed that both SCATT_p08010 and the non-coding sequence were essential for replication. However, the identified replication origin was shown to endow the plasmid with the ability to replicate in a circular model but not in a linear model in S. lividans. Interestingly, the knockout of the replication origin did not result in the curing of pSCATT, indicating that there might be other replication origins present in the mega-plasmid. The experimental validation of the central replication origin oriC1-II might be helpful for the investigation of the replication mechanism of the mega-plasmid and the genome evolution of Streptomyces.