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 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.
Bachrach
Gilad
G
Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1.
Colston
M Joseph
MJ
Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1.
Bercovier
Herve
H
Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, PO Box 12272, Israel2.
Bar-Nir
Dror
D
Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, PO Box 12272, Israel2.
Anderson
Colin
C
Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1.
Papavinasasundaram
K G
KG
Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics; D004261:DNA Replication; D004268:DNA-Binding Proteins; D018628:Gene Dosage; D008969:Molecular Sequence Data; D019907:Mycobacterium fortuitum / Q000235:genetics* / Q000254:growth & development; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D018741:Replication Origin; D012093:Replicon / Q000235:genetics*; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics
D004268:DNA-Binding Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases