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

Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK¹
Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, PO Box 12272, Israel²

Author for correspondence: K. G. Papavinasasundaram. Tel: +44 20 8959 3666. Fax: +44 20 8913 8528. e-mail: kpapavi{at}nimr.mrc.ac.uk

A 9·2 kb cryptic Mycobacterium fortuitum plasmid, pMF1, was isolated from strain 110 and its restriction map constructed. A 4·2 kb HindIII fragment of pMF1 was found to support replication in mycobacteria and this fragment was cloned and sequenced to characterize the replication elements of the plasmid. 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 Mycobacterium scrofulaceum pMSC262. A region containing a putative ori site was located upstream of the rep gene; this region displayed high homology at the nucleotide level with the predicted ori of pCLP and pJAZ38. A plasmid carrying the 4·2 kb HindIII fragment and a kanamycin resistance marker, designated pBP4, was maintained as a single-copy plasmid in Mycobacterium smegmatis and was stably inherited in the absence of antibiotic selection. Plasmid pBP4 was incompatible with the pJAZ38 replicon but was compatible with the widely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the plasmid was able to replicate in the pathogen Mycobacterium tuberculosis, making it a useful tool for gene expression studies. To provide a choice of restriction sites and easy manipulation, a 2·1 kb fragment containing the minimal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.

Keywords: mycobacterial plasmid pMF1, incompatibility, copy number, stability, resolvase

Abbreviations: Hyg, hygromycin; Kan, kanamycin; SCR, single cell resistance

The EMBL accession number for the sequence determined in this work is AJ238973.

^a Present Address: Department of Oral Biology, The Hebrew University-Hadassah Faculty of Dental Medicine, Jerusalem, PO Box 12272, Israel.

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