Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum

Abstract

In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc 2 155, making it a useful tool for mycobacterial genetics.The genus Mycobacterium comprises a wide range of species, from the pathogenic slow growers, such as the causative agents of tuberculosis and leprosy, to fast-growing mycobacteria found in the environment, such as Mycobacterium fortuitum. Traditionally, mycobacterial genetics has not been developed because of the pathogenicity and slow growth rate of Mycobacterium tuberculosis. The global increase in the incidence of tuberculosis, however, has been accompanied by an increase in molecular studies of mycobacteria, and although genetic systems are steadily being developed, there is still a need for more tools in this area.Several plasmids have been detected in mycobacteria from both environmental organisms and opportunistic pathogens (4,5,17). They are especially common in Mycobacterium avium complex isolates recovered from infected humans with pulmonary infections or AIDS and in Mycobacterium scrofulaceum and the M. fortuitum complex of both clinical and environmental isolates (3,10). Of all of the plasmids isolated, the most extensively studied is the M. fortuitum plasmid pAL5000 (14,21,25). This plasmid has been used as the basis for several other mycobacterial vectors (6,8,9). The other two mycobacterial plasmids whose replication regions have been defined and sequenced are the M. scrofulaceum plasmid pMSC262 (20) and the M. avium plasmid pLR7 (2). These two replicons were found to show homology in the replication region. Related plasmids have been detected throughout the mycobacteria by hybridization studies with pLR7 as a probe (5).Several insertion sequences have been isolated from mycobacteria (18), but only one composite transposon, Tn610, has been isolated. This transposon was isolated from M. fortuitum and contains a sulfonamide-resistant determinant and two copies of insertion sequence IS6100 which show homologies with the IS6 family elements. This is the only mycobacterial mobile element whose mechanism of transposition is a replicative process leading to the formation of cointegrates by duplication of one IS element in a direct orientation. Tn611 is a kanamycin-resistant derivative of Tn610 (16). Hybridization of transposition events shows ...

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“…Replication seems to be initiated by the predicted product of repA, which shares 68.3% amino acid identity with RepA from the cryptic Mycobacterium fortuitum plasmid, pJAZ38 (10). Two different direct repeat regions were identified 500-1,000 bp upstream of repA, suggesting possible replication origins (ori).…”

Analysis of the internal replication region of a mycobacterial linear plasmid The GenBank accession number for the nucleotide sequence and putative ORFs of the replication origin region of pCLP determined in this work is AF144883.

“…The minimum concentration of Km necessary to inhibit cell growth was taken as the single-cell resistance. In this experiment, M. smegmatis strain EP10 (Ainsa et al, 1997) was used as a control because it contains only one copy of the Km resistance gene on its chromosome. M. smegmatis strain EP10 arose from the insertion of the gene from Tn903 confering resistance to Km into the M. smegmatis mc#155 chromosome (Ainsa et al, 1997).…”

“…Plasmid copy number determination. The relative copy number of pCL4D in M. smegmatis mc#155 was determined by single-cell resistance to Km as previously described (Gavigan et al, 1997 ;Stolt & Stoker, 1996). The minimum concentration of Km necessary to inhibit cell growth was taken as the single-cell resistance.…”

“…The single-cell resistance to Km (Gavigan et al, 1997) of M. smegmatis mc#155 transformants carrying pCL4D gave a relative copy number for pCL4D of 1 (data not shown). After 4 d growth in an antibiotic-free medium, which corresponds to more than 40 generations, 85 % of M. smegmatis transformant cells contained pCL4D.…”

“…The replication origins of circular plasmids from Mycobacterium avium, Mycobacterium fortuitum and Mycobacterium scrofulaceum have been described, 0002-3674 # 2000 SGM and this has facilitated the construction of shuttle vectors capable of replication in both mycobacteria and Escherichia coli (Beggs et al, 1995 ;Gavigan et al, 1997 ;Qin et al, 1994 ;Ranes et al, 1990). However, the genetic system for studying mycobacteria is still limited.…”