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
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ABSTRACT |
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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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INTRODUCTION |
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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 mycobacterialEscherichia 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.
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METHODS |
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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
.
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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 (35 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.
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RESULTS AND DISCUSSION |
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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.
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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 HindIIIKpnI fragment (coordinates 11131) 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.
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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 32623771), 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.
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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 23, which is in agreement with the accepted copy number of 35 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.
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ACKNOWLEDGEMENTS |
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Received 17 May 1999;
revised 28 October 1999;
accepted 8 November 1999.