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

Gilad Bachracha,1, M. Joseph Colston1, Herve Bercovier2, Dror Bar-Nir2, Colin Anderson1 and K. G. Papavinasasundaram1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Although Mycobacterium leprae and Mycobacterium tuberculosis were among the first bacteria to be recognized as the aetiological agents of human disease, knowledge of mycobacteria at the molecular level has, until recently, lagged far behind that of other pathogenic bacteria. However, the re-emergence of tuberculosis as a major public health problem, even in highly developed countries, has stimulated the application of modern molecular genetic methods to understanding the molecular basis of pathogenicity of mycobacteria (reviewed by Pelicic et al., 1998 ), culminating in the sequencing of the entire M. tuberculosis genome (Cole et al., 1998 ).

The development of transformation techniques permitting the introduction and expression of genes in mycobacteria has been the cornerstone of mycobacterial molecular genetics (Hatfull, 1993 ). A variety of plasmids have been used, although the most extensively studied of these is pAL5000, isolated from Mycobacterium fortuitum (Labidi et al., 1984 , 1985 ). Other mycobacterial plasmids, such as pMSC262 from Mycobacterium scrofulaceum (Meissner & Falkinham, 1984 ; Qin et al., 1994 ), pLR7 from Mycobacterium avium (Crawford & Bates, 1984 ; Beggs et al., 1995 ) and pJAZ38 from M. fortuitum (Gavigan et al., 1997 ), have been isolated and used to develop additional mycobacterial–Escherichia coli shuttle vectors. Large linear plasmids have also been found in a few mycobacterial species, notably a 25 kb plasmid, pCLP, in Mycobacterium celatum (Picardeau & Vincent, 1997 , 1998 ). Some of these plasmids have a restricted mycobacterial host range, limiting their usefulness; for example pLR7 and pMSC262 are unable to replicate in the widely used fast-growing, non-pathogenic Mycobacterium smegmatis mc2155 (Beggs et al., 1995 ). Thus there is still a need to develop further genetic tools for gene manipulation in mycobacteria.

In this report, we describe the isolation and characterization of a stable, low-copy plasmid, pMF1, from M. fortuitum. This plasmid is able to replicate in M. smegmatis and M. tuberculosis and is compatible with derivatives of pAL5000, but not with those of pJAZ38, making it a useful vector to further advance genetic studies in mycobacteria.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
E. coli strain XL-1 Blue (Stratagene) was used as a host for all plasmid constructions. Mycobacterial strains used were as follows: M. fortuitum 110 (Labidi et al., 1984 ) was provided by H. David, Pasteur Institute, France; M. fortuitum 10394 is a plasmid-free strain and was provided by M. J. Garcia, Universidad Autonoma de Madrid, Spain; M. smegmatis mc2155 (Snapper et al., 1990 ) and M. tuberculosis H37Rv are standard strains maintained at the National Institute for Medical Research, London, UK.

Growth media for E. coli and mycobacteria, and mycobacterial transformation protocols have been described previously (Papavinasasundaram et al., 1998 ). For mycobacteria, whenever necessary, antibiotics were used at the following concentrations: hygromycin (Hyg), 50 µg ml-1; kanamycin (Kan), 25 µg ml-1 for M. smegmatis and M. tuberculosis and 75 µg ml-1 for M. fortuitum. Plasmids used in this study are listed in Table 1.


View this table:
[in this window]
[in a new window]
 
Table 1. Plasmids used in this work

 
Recombinant DNA techniques.
Plasmid DNA was prepared using Qiaprep spin miniprep or maxiprep kits (Qiagen). When extracting plasmids from mycobacteria, cells were incubated for 30 min at 65 °C after the addition of lysis buffer and then the kit protocol was followed without further modification. Published protocols were used for the isolation of mycobacterial DNA and for Southern hybridization (Papavinasasundaram et al., 1998 ).

The minimal replication region of pMF1 was amplified by PCR in a reaction mix using the Expand High Fidelity PCR system (Boehringer Mannheim) containing 1·5 mM MgCl2, 200 µM dNTPs and 300 nM each of the primers PMF23 (5'-TTTTaagcttGGGAGCCGGATGGGTAGTTG-3') and PMF24 (5'-TTTaagcttCCTCCGTGGCGTAATCGTC-3') (lower case letters in both sequences indicate the HindIII restriction site; see below) and 10 ng template DNA (plasmid pBP1). This PCR reaction was carried out in thin-walled PCR tubes in a GeneAmp PCR system 9700 thermal cycler (PE Applied Biosystems) using the following temperature regime: 1 cycle of denaturation at 94 °C for 3 min; 10 cycles of 94 °C for 15 s, 63 °C for 30 s and 72 °C for 2 min; 30 cycles of 94 °C for 15 s, 63 °C for 30 s and 72 °C for 2 min with a cycle extension of 20 s; and a final elongation step at 72 °C for 7 min.

Nucleotide sequences were determined using an ABI PRISM 377 DNA sequencer with the dRhodamine dye terminator cycle sequencing kit (PE Applied Biosystems). For all other DNA manipulations, standard protocols were followed (Sambrook et al., 1989 ).

Nucleotide sequence and data analysis.
The nucleic acid sequences obtained were assembled into a contig and analysed using the biocomputing software Lasergene (DNASTAR) and the University of Wisconsin Genetics Computer Group (GCG) package. Homology searches were performed using the BLAST network service and non-redundant protein and nucleotide sequence databases (Altschul et al., 1990 ) at the National Centre for Biotechnology Information (NCBI), Bethesda, MD, USA.

Stability of pMF1 derivatives, plasmid copy number and compatibility with pAL5000.
These experiments were carried out as described by Gavigan et al. (1997) except that the diluted cultures were plated on 7H11 agar supplemented with appropriate concentrations of antibiotics as necessary. Relative copy number of the plasmid pBP4 in M. smegmatis mc2155 was determined as single-cell resistance to Kan (Gavigan et al., 1997 ). For copy number comparisons, M. smegmatis mc2155 strains carrying the single-copy integrating vector pMV306 (Stover et al., 1991 ) and the replicating vector pMV261 (3–5 cell copies) (Stover et al., 1991 ) were used. Compatibility of the pMF1 derivative pBP8 (similar to pBP4 but carrying a Hyg resistance gene) was tested with the pAL5000-derivative pMV261 and the pJAZ38-derivative pJAZ56 plasmids.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Isolation and subcloning of the M. fortuitum plasmid pMF1
We isolated a 9·2 kb cryptic plasmid pMF1 from M. fortuitum strain 110 and subcloned the entire 9·2 kb fragment obtained by partial HindIII digestion, and the 5 kb and 4·2 kb HindIII fragments to construct the plasmids pGB9, pGB5 and pGB4, respectively. Whereas pGB9 and pGB4 were able to replicate in M. smegmatis mc2155 and to produce Kanr colonies after electroporation, pGB5 could not, suggesting that the replication region of pMF1 was located within the 4·2 kb HindIII fragment. This fragment was subcloned into pBluescript II KS(+) to create pBP1. The aph gene of pMV261 (conferring Kan resistance) was cloned into pBP1 to construct pBP4, and this plasmid was transformed into electrocompetent cells of M. smegmatis, M. tuberculosis H37Rv and M. fortuitum with high efficiency [approx. 1x106 Kanr colonies (µg plasmid DNA)-1], confirming that all the elements necessary for plasmid replication were located within the 4·2 kb HindIII fragment.

To find out whether the pMF1-derived plasmids integrate into the chromosome, we carried out Southern hybridization analysis of genomic DNA isolated from pBP6 transformants of M. fortuitum, M. smegmatis and M. tuberculosis. Plasmid pBP6 is derived from plasmids pBP4 and pKP38 and contains the first 500 bp of the mycobacterial recA sequence (see Table 1). The 0·5 kb recA fragment was used as a probe. The Southern analysis of PstI-digested DNA (Fig. 1) and EcoRI-digested DNA (data not shown) confirmed that the plasmid had not randomly integrated into the chromosome in all the three species studied. In addition, reisolation of the plasmid from the transformants clearly indicated that the pBP4 and pBP6 plasmids replicated extrachromosomally in mycobacteria.



View larger version (71K):
[in this window]
[in a new window]
 
Fig. 1. Southern analysis of PstI-digested genomic DNA isolated from pBP6 transformants of M. smegmatis mc2155 (lane 1), M. fortuitum 10394 (lane 3) and M. tuberculosis H37Rv (lane 5). DNA from the untransformed parent strains (lanes 2, 4 and 6, respectively) was included for comparison. The 0·5 kb PstI fragment, corresponding to the N-terminal region of the M. smegmatis recA gene, was used as a probe. Fragment sizes are indicated on the left in kb.

 
Subcloning and sequencing the replication region of pMF1
The nucleotide sequence of the 4·2 kb HindIII DNA fragment in the pBP1 plasmid was determined on both strands from additional subclones and by primer walking. Computer analysis of the sequence revealed three important elements associated with plasmid replication and stability (Fig. 2): (i) ORF1, encoding a potential replication protein; (ii) ORF2, with homology to the resolvase gene (res), known to play a role in plasmid stability; and (iii) a stretch of conserved DNA present upstream of ORF1 thought to contain the pMF1 site of replication.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 2. Genetic organization of the 4·2 kb HindIII fragment carrying the elements for replication of the pMF1 plasmid. The ability of the plasmids carrying either the entire fragment or its derivatives to confer replication in M. smegmatis mc2155 is indicated on the right. The hatched box indicates the proposed location of the plasmid origin of replication. The ORFs rep and res, encoding proteins with homology to Rep proteins and resolvase, respectively, are indicated. Only relevant restriction sites are shown.

 
ORF1 was located between nucleotides 1787 and 2875 and encoded a protein of 362 amino acids with a predicted molecular mass of 39·3 kDa. A BLAST search of the NCBI database with the translated sequence revealed homology with the Rep proteins of the mycobacterial plasmids pCLP, pJAZ38 and PLR7, but not with the RepA and RepB proteins of pAL5000 (Labidi et al., 1985 ; Stolt & Stoker, 1996a ). The pMF1 Rep protein displayed highest homology with the putative Rep protein of the M. celatum plasmid pCLP (Picardeau et al., 2000 ; GenBank accession no. AF144883), showing 49% identity through most of the sequence. In contrast, it showed only about 18–20% identity with the Rep proteins of pLR7, pJAZ38 and pMSC262 plasmids. The protein sequence of the pMSC262 Rep is based on the corrected ORF (complement of 812–1654; EMBL accession no. D14416) (Beggs et al., 1995 ). Alignment of pMF1, pCLP, pJAZ38, pLR7 and pMSC262 Rep proteins using the DNASTAR CLUSTAL analysis software showed that all these Rep proteins had conserved residues, mainly in their N-terminal region (Fig. 3a). The homology of pMF1 Rep with these Rep proteins suggested that ORF1 of pMF1 encodes a Rep protein in mycobacteria. The N-terminal region of the pLR7 Rep is reported to contain helix–turn–helix motifs, which are characteristic features of DNA-binding regulatory proteins (Beggs et al., 1995 ). Among the Rep proteins of mycobacterial plasmids, only the pAL5000 RepB protein (119 residues) has been purified and its role in plasmid replication characterized (Stolt & Stoker, 1996b ). This protein binds to a high-affinity site upstream of the repAB genes to regulate its own expression, and to a low-affinity site further upstream which is suggested to contain its ori site. Purification of the pMF1 Rep protein will help to clarify whether it also has dual regulatory functions in controlling plasmid replication.



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 3. Homology of pMF1 (AJ238973; 362 residues) with the mycobacterial plasmids pCLP (AF144883, 350 residues), pJAZ38 (U84216, 368 residues), pLR7 (U18777, 360 residues) and pMSC262 (D14416, 281 residues; ORF as corrected by Beggs et al., 1995 ) (the EMBL accession numbers of each plasmid sequence and the predicted length of the corresponding Rep protein are given in parentheses). (a) N-terminal region of the deduced replication protein sequences. (b) Conserved DNA region of the plasmids near the putative ori site, present upstream of the rep gene. Residues/nucleotides that are identical to the pMF1 sequence are shown on a black background. The alignment was generated using the CLUSTAL method of the program Megalign (DNASTAR).

 
Even though the pMF1 Rep displayed only 18% identity with the pJAZ38 Rep protein, comparison of the DNA sequence present upstream of the rep genes indicated that a 235 bp segment of pMF1 (coordinates 1349–1583) was 70% identical to the corresponding region of pJAZ38 (coordinates 388–623) and 67% identical to that of pCLP (coordinates 2162–2383). A 72 bp segment of pMF1 (coordinates 1534–1605) was 62% identical to that of pMSC262 and 57% identical to that of pLR7. Alignment of sequences of the five plasmids in this 72 bp segment revealed high sequence conservation (Fig. 3b), indicating that all these plasmids have similar ori sites and therefore are likely to belong to one incompatibility group.

Homology between putative ori regions of various plasmids suggested that the pMF1 ori is likely to be located between coordinates 1349 and 1605. However, a deletion of the HindIII–KpnI fragment (coordinates 1–1131) of pBP4 eliminated the ability of the generated plasmid (pBP5) to replicate in M. smegmatis (Fig. 2). The plasmid pBP5 contained the conserved nucleotide region upstream of the rep gene, the rep gene and the downstream regions. This suggested that the ori site or the elements associated with the ori required for plasmid replication, are also located upstream of the KpnI site. To determine the boundaries of the replication elements, the DNA fragment located between nucleotides 904 and 3000 of pBP1 (Fig. 2) was amplified by PCR and cloned into pBP9 to construct pBP10 (Fig. 4). This plasmid replicated in mycobacteria indicating that the minimal origin of replication and the Rep protein are located between nucleotides 904 and 3000 in the pMF1 sequence.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. pBP10 mycobacterial vector. Ap, ampicillin resistance; Kan, kanamycin resistance; oriE, E. coli origin of replication; oriM, mycobacterial origin of replication. Useful restriction sites are also indicated.

 
Database searches also revealed that ORF2 (coordinates 3262–3771) displayed high homology to res genes; a 293 bp DNA sequence (coordinates 3351–3644) was found to be 64% identical to the gene encoding the invertase/recombinase-like protein in the Thiobacillus ferrooxidans plasmid pTF5 (coordinates 8944–9237 in the ORF 8819–9391; EMBL accession no. U73041) (Dominy et al., 1997 ). It is thought that the Res proteins help in multimer resolution during plasmid replication, thus ensuring segregation of the plasmids to daughter cells (Dodd & Bennett, 1987 ). BLAST analysis of the database with the pMF1 Res protein sequence revealed a possible frameshift in the N terminus of the protein (between amino acids 18 and 27). The frameshift mutation was not introduced during the cloning of pBP4 since the parent plasmid, pGB9, had the same frameshift and identical sequences in the remainder of the gene. If this gene is expressed, then it is likely to be missing the first conserved N-terminal region unless the expression occurs by translational frameshifting. The importance of the pMF1 res gene with regard to plasmid stability was investigated further (see below).

Stability of pMF1 derivatives in M. smegmatis
The pMF1-derivative pGB9 remained stable in M. smegmatis for 4 d (40 generations) in the absence of antibiotic selection (Fig. 5). This is similar to the reported stability of other mycobacterial plasmids (Gavigan et al., 1997 ). The stability of pBP4 and pBP10 was two to three orders of magnitude lower then pGB9 (Fig. 5), indicating that pBP4 and pBP10 may be lacking regions which confer higher plasmid stability. The pGB9 plasmid carried the Tn5 aph gene whereas pBP4 and pBP10 plasmids carried the aph of Tn903. However, it is reported that both aph genes impose a similar burden on the mycobacterial cell and do not alter the stability of the plasmid expressing them (Stolt & Stoker, 1996a ). It is important to note that although pBP4 included ORF2 encoding the putative res gene (coordinates 3262–3771), it was not found to be more stable in M. smegmatis than pBP10, which lacked this region. This suggests that either the res gene is not expressed or that even if expressed, with translational frameshifting, it alone is not sufficient to confer increased plasmid stability. It will be interesting to see whether correcting the ORF encoding the putative pMF1 Res improves the stability of the pBP4 plasmid in mycobacteria.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Stability of pMF1-derivatives pGB9 ({blacksquare}), pBP4 ({blacktriangleup}) and pBP10 ({bullet}) in M. smegmatis mc2155. Plasmid-carrying strains were grown in Dubos broth in the absence of antibiotic selection and dilutions were plated on 7H11 agar with and without Kan. Plasmid loss over 4 d (40 generations) is expressed as a percentage of plasmid-carrying cells (c.f.u. on 7H11 plus Kan as a percentage of c.f.u. on antibiotic-free medium). The experiment was performed in triplicate with consistent results; data from a typical experiment are shown.

 
Very little is known about stability mechanisms of mycobacterial plasmids. It is interesting to note that the sequenced region of the pCLP plasmid contains parA encoding a putative partitioning protein present about 1·5 kb upstream of the rep gene (EMBL accession no. AF144883). It is suggested that the ATPase activity of ParA is required for an essential energetic step in the orderly segregation of the E. coli P1 plasmid copies to daughter cells (Davis et al., 1996 ; Bouet & Funnell, 1999 ). We did not find any parA homologue within the 4·2 kb HindIII fragment of pMF1. It is possible that gene(s) controlling plasmid stabilization functions are located in the additional 5 kb HindIII fragment present in pGB9 which confer increased plasmid stability.

The pMF1-derivative pBP8 is incompatible with M. fortuitum pJAZ38-derivative pJAZ56 but compatible with the pAL5000-derivative pMV261
Competent cells prepared from M. smegmatis carrying pBP8 (identical to pBP4 but expressing Hyg resistance instead of Kan resistance) were electroporated with pMV261 or with pJAZ56 and plated on medium containing either Kan or Hyg or both. Whereas pBP8-containing cells transformed with pMV261 grew readily on all three plates, pBP8 transformants electroporated with pJAZ56 grew only on plates containing either Hyg or Kan but not on plates containing both antibiotics. This demonstrated that the electroporation of pJAZ56 was successful but that pBP8 and pJAZ56 could not coexist in the same cell. Even though the pMF1 and pJAZ38 Rep proteins showed only 18% identity, the plasmids were incompatible presumably due to the additional homology at their replication origins (Fig. 3b). This conservation of nucleotides in the ori region of plasmids pMF1, pCLP, pJAZ38, pLR7 and pMSC262, and the inability of pMF1 and pJAZ38 to coexist suggested that these plasmids can be grouped into one incompatibility unit.

However, unlike the case with pJAZ38, M. smegmatis carrying pBP8 plasmids was successfully transformed with pMV261 (Kanr) with high efficiency [~1x106 colonies (µg DNA) -1] to confer both Hyg and Kan resistance. Single colonies from these double antibiotic plates were subcultured into Dubos broth, grown in the absence of antibiotic selection and then plated to determine the proportion of Kanr, Hygr and Kanr+Hygr colonies. Results from three independent experiments revealed that more than 97% Hygr bacteria carried both plasmids for at least 30 generations, indicating that the pMF1 origin of replication present in pBP8 was compatible and stably maintained with the pAL5000 origin of pMV261. It has been shown that plasmids pMSC262 and pJAZ38 are also compatible with the pAL5000 replicon (Qin et al., 1994 ; Gavigan et al., 1997 ). This suggests that pAL5000 belongs to a second and different mycobacterial plasmid incompatibility group. Whilst the plasmids of the first incompatibility group (pMF1, pCLP, pJAZ38, pMSC262 and pLR7) code for only one Rep protein, plasmid pAL5000 encodes two replication proteins (RepA and RepB) (Stolt & Stoker, 1996a , b , 1997 ). The pMF1 Rep protein does not show any significant homology with the pAL5000 Rep proteins and there is no homology between their origins of replication. Not surprisingly, these two replicons were compatible.

Copy number of the pMF1 derivatives
Relative plasmid copy number for pBP4 in M. smegmatis was determined using a method based on single cell resistance (SCR) to increasing concentrations of Kan (Gavigan et al., 1997 ). As shown in Fig. 6, setting the SCR of pMV306 (an integrating single-copy plasmid; Stover et al., 1991 ) to 1, a copy number of 1·33 was obtained for pBP4. The relative copy number determined for pMV261 was 2–3, which is in agreement with the accepted copy number of 3–5 reported for pAL5000 (Stover et al., 1991 ). All these plasmids carry the same aph gene of Tn903 and therefore the resistance to increasing concentrations of Kan reflects the copy number rather than differences due to aph gene expression. The copy number obtained for pBP4 is similar to that reported for the plasmid pJAZ38 (Gavigan et al., 1997 ). Since plasmid copy number may affect the quantity of the cloned gene products and the stability of the cloned genes, use of single-copy vectors will be more appropriate in complementation studies. To this end, plasmid pBP10 with its smaller oriM will serve as a useful vector for genetic studies in M. smegmatis and M. tuberculosis.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6. SCR of M. smegmatis transformants expressed as percentage of cells surviving under increasing concentrations of Kan. The SCR of the strain carrying the pBP4 plasmid ({bullet}) was compared to that carrying the single-copy integrating plasmid pMV306 ({blacksquare}) and the replicating vector pMV261 (3–5 copies; {blacktriangleup}). The experiment was performed in triplicate with consistent results; data from a typical experiment are shown.

 

   ACKNOWLEDGEMENTS
 
We thank Pat Brooks and Farahnaz Movahezadeh for their help in the Containment III facility. We also thank the referees for bringing to our notice the sequence data of pCLP that was deposited in the database after submission of this manuscript, and Dr Carlos Martin, Universidad de Zaragoza, for supplying plasmid pJAZ56. G.B. was supported by a fellowship from The Heiser Foundation.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool.J Mol Biol 215, 403-410.[Medline]

Beggs, M. L., Crawford, J. T. & Eisenach, K. D. (1995). Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7.J Bacteriol 177, 4836-4840.[Abstract]

Bouet, J. Y. & Funnell, B. E. (1999). P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities.EMBO J 18, 1415-1424.[Abstract/Free Full Text]

Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[Medline]

Crawford, J. T. & Bates, J. H. (1984). Restriction endonuclease mapping and cloning of Mycobacterium intracellulare plasmid pLR7.Gene 27, 331-333.[Medline]

Davis, M. A., Radnedge, L., Martin, K. A., Hayes, F., Youngren, B. & Austin, S. J. (1996). The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells.Mol Microbiol 21, 1029-1036.[Medline]

Dodd, H. M. & Bennett, P. M. (1987). The R46 site-specific recombination system is a homologue of the Tn3 and {gamma}{delta} (Tn1000) cointegrate resolution system.J Gen Microbiol 133, 2031-2039.[Medline]

Dominy, C. N., Deane, S. M. & Rawlings, D. E. (1997). A geographically widespread plasmid from Thiobacillus ferrooxidans has genes for ferredoxin-, FNR-, prismane- and NADH-oxidoreductase-like proteins which are also located on the chromosome.Microbiology 143, 3123-3136.[Abstract]

Gavigan, J. A., Ainsa, J. A., Perez, E., Otal, I. & Martin, C. (1997). Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum.J Bacteriol 179, 4115-4122.[Abstract]

Hatfull, G. F. (1993). Genetic transformation of mycobacteria.Trends Microbiol 1, 310-314.[Medline]

Labidi, A., Dauguet, C., Goh, K. S. & David, H. L. (1984). Plasmid profiles of Mycobacterium fortuitum complex isolates.Curr Microbiol 11, 235-240.

Labidi, A., David, H. L. & Roulland-Dussoix, D. (1985). Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000.Ann Inst Pasteur Microbiol 136, 209-215.

Lydiate, D. J., Ashby, A. M., Henderson, D. J., Kieser, H. M. & Hopwood, D. A. (1989). Physical and genetic characterization of chromosomal copies of the Streptomyces coelicolor mini-circle.J Gen Microbiol 135, 941-955.

Meissner, P. S. & Falkinham, J. O.III (1984). Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum.J Bacteriol 157, 669-672.[Medline]

Papavinasasundaram, K. G., Movahedzadeh, F., Keer, J. T., Stoker, N. G., Colston, M. J. & Davis, E. O. (1997). Mycobacterial recA is cotranscribed with a potential regulatory gene called recX.Mol Microbiol 24, 141-153.[Medline]

Papavinasasundaram, K. G., Colston, M. J. & Davis, E. O. (1998). Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function.Mol Microbiol 30, 525-534.[Medline]

Pelicic, V., Reyrat, J.-M. & Gicquel, B. (1998). Genetic advances for studying Mycobacterium tuberculosis pathogenicity.Mol Microbiol 28, 413-420.[Medline]

Picardeau, M. & Vincent, V. (1997). Characterization of large linear plasmids in mycobacteria.J Bacteriol 179, 2753-2756.[Abstract]

Picardeau, M. & Vincent, V. (1998). Mycobacterial linear plasmids have an invertron-like structure related to other linear replicons in actinomycetes.Microbiology 144, 1981-1988.[Abstract]

Picardeau, M., Le Dantec, C. & Vincent, V. (2000). Analysis of the internal replication region of a mycobacterial linear plasmid.Microbiology 146, 305-313.[Abstract/Free Full Text]

Qin, M., Taniguchi, H. & Mizuguchi, Y. (1994). Analysis of the replication region of a mycobacterial plasmid, pMSC262.J Bacteriol 176, 419-425.[Abstract]

Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Snapper, S. B., Lugosi, L., Jekkel, A., Melton, R. E., Kieser, T., Bloom, B. R. & Jacobs, W. R.Jr (1988). Lysogeny and transformation in mycobacteria: stable expression of foreign genes.Proc Natl Acad Sci USA 85, 6987-6991.[Abstract]

Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R.Jr (1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.Mol Microbiol 4, 1911-1919.[Medline]

Stolt, P. & Stoker, N. G. (1996a). Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000.Microbiology 142, 2795-2802.[Abstract]

Stolt, P. & Stoker, N. G. (1996b). Protein–DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000.J Bacteriol 178, 6693-6700.[Abstract]

Stolt, P. & Stoker, N. G. (1997). Mutational analysis of the regulatory region of the Mycobacterium plasmid pAL5000.Nucleic Acids Res 25, 3840-3846.[Abstract/Free Full Text]

Stover, C. K., de la Cruz, V. F., Fuerst, T. R. & 11 other authors (1991). New use of BCG for recombinant vaccines. Nature 351, 456–460.[Medline]

Received 17 May 1999; revised 28 October 1999; accepted 8 November 1999.