A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon

A 9.2 kb cryptic Mycobacterium fortuitum plasmid, pMF1, was isolated from s train 110 and its restriction map constructed. A 4.2 kb HindIII fragment of pMF1 was found to support replication in mycobacteria and this fragment wa s cloned and sequenced to characterize the replication elements of the plas mid. Computer analysis identified a putative Rep protein (362 amino acids) with high homology to the putative Rep protein of the Mycobacterium celatum plasmid pCLP and limited homology, mostly in the N-terminal region, to the Rep proteins of Mycobacterium avium pLR7, M. fortuitum pJAZ38 and Mycobact erium scrofulaceum pMSC262. A region containing a putative ori site was loc ated upstream of the rep gene; this region displayed high homology at the n ucleotide level with the predicted ori of pCLP and pJAZ38. A plasmid carryi ng the 4.2 kb HindIII fragment and a kanamycin resistance marker, designate d pBP4. was maintained as a single-copy plasmid in Mycobacterium smegmatis and was stably inherited in the absence of antibiotic selection. Plasmid pB P4 was incompatible with the pJAZ38 replicon but was compatible with the wi dely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the pla smid was able to replicate in the pathogen Mycobacterium tuberculosis, maki ng it a useful tool for gene expression studies. To provide a choice of res triction sites and easy manipulation, a 2.1 kb fragment containing the mini mal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.