Cloning and expression of the origin of replication of mycobacteriophage D29 in Mycobacterium smegmatis

Libraries of mycobacteriophage D29 genes were obtained by transforming Escherichia coli with constructs derived from pUC19. The genomic libraries were used to transform Mycobacterium smegmatis MC²155, and one of the vectors designated pRM64 was found to stably replicate in the mycobacterial recipients. The pRM64 vector contained a 2.65-kb fragment that was used as a probe and was then located on the physical map of D29. Vectors containing this fragment replicated stably in M. smegmatis for at least 144 generations in medium without the selective agent used (kanamycin); vectors not containing the fragment did not replicate in the recipient M. smegmatis. This is the first report showing that the cloning of the OriR of a mycobacteriophage allowed the stable replication of shuttle vectors for mycobacterial genetics.