Analysis of the internal replication region of a mycobacterial linear plasmid
Mathieu Picardeau1,
Corinne Le Dantec1 and
Véronique Vincent1
Laboratoire de Référence des Mycobactéries, Institut Pasteur, 75724 Paris Cedex 15, France1
Author for correspondence: Mathieu Picardeau. Tel: +33 1 45 68 83 60. Fax: +33 1 40 61 31 18. e-mail: mpicard{at}pasteur.fr
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ABSTRACT
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Linear plasmids have previously been identified by the authors in mycobacteria, the telomeres of which have terminal inverted repeats and covalently attached proteins. In this study, the replication of these unusual molecules was investigated by studying a 25 kb linear plasmid from the slow-growing species Mycobacterium celatum called pCLP. An internal region of pCLP responsible for replication in Mycobacterium smegmatis was identified. The nucleotide sequence of the minimum replication region of pCLP, which was 2·8 kb long, contained a putative replication gene, rep, and a putative origin of replication consisting of an 18 bp direct repeat and an AT-rich region. A short section of the pCLP replication region was also found to have sequence identity with the replication regions of mycobacterial circular plasmids, suggesting that these linear and circular plasmids are related. It was found that pCLP replicated in Mycobacterium bovis BCG and was compatible in M. smegmatis with pAL5000- and pJAZ38-derived plasmids from Mycobacterium fortuitum, which belong to two different compatibility groups. Thus, this new Escherichia colimycobacteria shuttle vector may be used in both slow- and fast-growing mycobacteria and in co-transformation experiments with other mycobacterial vectors.
Keywords: mycobacteria, linear plasmid, shuttle vector, replication
Abbreviations: Ap, ampicillin; Hg, hygromycin; Km, kanamycin; Sm, streptomycin; Sp, spectinomycin
The GenBank accession number for the nucleotide sequence and putative ORFs of the replication origin region of pCLP determined in this work is AF144883.
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INTRODUCTION
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Chromosomal DNA and many plasmids are linear in the genus Streptomyces (Hinnebusch & Tilly, 1993
). Linear plasmids have recently been described in the other actinomycetes Planobispora, Rhodococcus and Mycobacterium (Crespi et al., 1992
; Dabrock et al., 1994
; Kalkus et al., 1993
; Kebeler et al., 1996; Kosono et al., 1997
; Picardeau & Vincent, 1997
; Polo et al., 1998
). All these linear replicons belong to a class of genetic elements called invertrons (Hinnebusch & Tilly, 1993
; Sakaguchi, 1990
) which have terminal inverted repeats with 5' ends covalently linked to a terminal protein. The invertron-like structure is also found in some bacteriophages, adenoviruses and mitochondrial linear plasmids (Hinnebusch & Tilly, 1993
; Sakaguchi, 1990
). The unusual structure of these linear elements raises several questions about their replication mechanism. Previous studies have demonstrated that the replication of linear plasmids and of linear chromosomes in Streptomyces spp. proceeds bidirectionally from a central origin towards the telomeres (Chang & Cohen, 1994
; Chang et al., 1996
; Fischer et al., 1998
; Musialowski et al., 1994
; Shiffman & Cohen, 1992
; Zakrzewska-Czerwinska & Schrempf, 1992
). This internally initiated replication leaves single-strand gaps at the 3' ends (Chang & Cohen, 1994
). The 3' strand overhang then folds back, due to the presence of multiple terminal palindromes, resulting in a DNA duplex, thereby providing a recognition site for the terminal protein, which serves as a primer to complete DNA synthesis at the telomeres (Qin & Cohen, 1998
).
Several circular plasmids have been detected in mycobacteria living in the environment or as opportunistic pathogens, but never in the Mycobacterium tuberculosis complex. The replication origins of circular plasmids from Mycobacterium avium, Mycobacterium fortuitum and Mycobacterium scrofulaceum have been described, 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. Some of the E. colimycobacteria shuttle vectors cannot replicate in the fast-growing Mycobacterium smegmatis strain mc2155 which is commonly used as a cloning host for studying mycobacterial genes (Snapper et al., 1990
). Moreover, pAL5000-derived plasmids are the only class that replicate in both slow- and fast-growing species.
We have recently characterized linear plasmids in the slow-growing species M. avium, Mycobacterium xenopi, Mycobacterium branderi and Mycobacterium celatum (Picardeau & Vincent, 1997
, 1998
), and in the fast-growing species M. fortuitum (C. Le Dantec, M. Picardeau & V. Vincent, unpublished results). The 25 kb plasmid pCLP from the opportunistic pathogen M. celatum has been cloned and its telomeres sequenced. The telomeres have structural features in common with other actinomycete linear plasmids (Picardeau & Vincent, 1998
). In this study, we cloned and characterized the internal region of pCLP, which is able to replicate in a circular form in M. smegmatis mc2155, and in the slow-growing species Mycobacterium bovis BCG. The nucleotide sequence of the replication region includes two ORFs encoding putative replication and partition proteins, and a short region with a sequence similar to that of the origin regions of mycobacterial circular plasmids. Our vector was compatible with plasmids derived from the circular plasmids pAL5000 and pJAZ38 from M. fortuitum.
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METHODS
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Bacterial strains, plasmids and culture conditions.
The M. celatum strain 4 used in this study is a clinical isolate kindly provided by W. R. Butler, which we have previously shown to contain two linear replicons, one of about 25 kb designated pCLP, and another of 320 kb (Picardeau & Vincent, 1998
). Cultures of mycobacteria were grown in 7H9 Middlebrook liquid medium at 37 °C with antibotics added to the media as required. The bacterial strains and plasmids used in this study are listed in Table 1
.
Cloning and electrotransformation.
The recombinant plasmids pMPV7 and pMPV3 used in this study correspond to the insertion of the two XbaI fragments of pCLP (the extremities of which are blunt-ended) between the XbaI and HincII restriction sites of pBluescript II KS(+/-) (Stratagene) (Picardeau & Vincent, 1998
). The 10 and 14·5 kb inserts of pMPV7 and pMPV3, respectively, correspond to the entire length of the linear plasmid pCLP (Picardeau & Vincent, 1998
). These recombinant plasmids were digested with BstEII, KpnI and XhoI to construct a restriction endonuclease map of pCLP (Fig. 1
). Seven restriction fragments from 1 to 6 kb in size were subcloned into a kanamycin (Km)-resistant pUC19 derivative called pPV8 (Table 1
). We also performed PCR assays using primers Poa (5'-ACC AAT GAG CAG TAA GCA GC-3'), Pob (5'-GCA GCA GCG ACA AAG ATG GG-3') and Poc (5'-CAT CGG GCT GCG GGA AAC CC-3'). The PCR mixture and the amplification reactions were performed as previously described (Picardeau & Vincent, 1998
). The products amplified using primers Poa/Poc and Pob/Poc (see Fig. 5
) were inserted into the pGEM-T Easy vector (Promega) and the resulting inserts were released by EcoRI digestion and inserted into pPV8.

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Fig. 1. Determination of the minimum region required for replication of pCLP in M. smegmatis. A restriction endonuclease map of pCLP is shown at the top of diagram. The approximate locations of the restriction sites are indicated. DNA fragments tested for autonomous replication are indicated by solid bars. Plasmids used for transformation are indicated in Table 1 . The ability (+) or inability (-) of the clone to replicate in M. smegmatis mc2155 is shown on the right.
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Fig. 5. Genetic organization of the replication region of pCLP. The putative genes parA and rep and their transcription orientation are indicated by open arrows. Arrowheads indicate the 18 bp repeat sequences. A+T indicates the AT-rich segment of the replication region. The line in bold type corresponds to the minimum replication region of pCLP (pCL5). The location of primers Poa, Pob and Poc are indicated. The amplified product Poa/Poc (pCRa) facilitated plasmid replication in M. smegmatis, but the amplified product Pob/Poc (pCRb) prevented plasmid replication.
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Plasmid constructs were introduced into E. coli DH5
by electroporation (Gene Pulser unit; Bio-Rad), selected on solid Luria-Bertani medium supplemented with 20 µg Km ml-1, 2 mM IPTG and 0·004% X-Gal, and plasmids were recovered using a Qiagen Midi kit. Electrocompetent cells of M. smegmatis and M. bovis BCG were prepared as previously described (Pelicic et al., 1996
). Briefly, M. smegmatis and M. bovis BCG cultures were grown to exponential phase; pellets were washed three times in 10% glycerol and resuspended in 10% glycerol. The competent cells were electroporated in the presence of 2 µg vector DNA by using a Gene Pulser (Bio-Rad) and then transferred to 5 ml 7H9 Middlebrook liquid medium, in which they were incubated for 4 h (M. smegmatis) or 24 h (M. bovis BCG) at 37 °C before plating.
Plasmids in M. smegmatis and M. bovis BCG transformants were detected by Southern analysis and electroporation of E. coli competent cells with total genomic DNA of M. smegmatis or M. bovis BCG transformants.
DNA extraction and Southern analysis.
Mycobacterial strains were grown in 5 ml Middlebrook 7H9 broth containing 1 mg D-cycloserine ml-1 and incubated overnight at 37 °C. Cells were heated for 20 min at 80 °C, centrifuged and resuspended in 250 µl 25% (w/v) sucrose/50 mM Tris pH 8/50 mM EDTA containing 500 µg lysozyme ml-1. Incubation was continued for an extra night. Aliquots (250 µl) of a solution consisting of 100 mM Tris, 1% (w/v) SDS and 400 µg proteinase K ml-1 were added and the mixtures incubated for 4 h at 55 °C. DNA was extracted with phenol/chloroform (50:50, v/v), precipitated with absolute ethanol and dissolved in TE (10 mM Tris, 1 mM EDTA, pH 8).
Two micrograms of genomic DNA was digested with SacI or PvuII, subjected to electrophoresis overnight in a 1% agarose gel and transferred onto nylon membranes (Hybond-N+; Amersham). Membranes were hybridized overnight at 65 °C in Rapid hybridization buffer (Amersham) with pCL4D radiolabelled with [
-32P]dCTP using a commercial kit (Megaprime; Amersham). The membranes were then washed as previously described (Picardeau & Vincent, 1997
).
Stability of pCL4D and compatibility studies.
M. smegmatis cells carrying one of the Km-resistant plasmids pCL4D or pB4 were grown in 5 ml Middlebrook 7H9 broth with no antibiotic selection pressure for 24 h at 37 °C. The culture was then diluted 1:100 and the bacteria grown in fresh antibiotic-free medium for a further 24 h; this procedure was repeated three times. After each dilution, the cells were plated on agar plates with and without Km and the proportion of resistant cells was taken as a measure of the number of cells carrying the plasmid. The same procedure was used for compatibility studies. To determine the number of plasmid-carrying cells, M. smegmatis mc2155 transformants were grown on Middlebrook 7H10 agar plates containing 20 µg Km ml-1 and 20 µg streptomycin (Sm) ml-1 (for co-transformation with pJAZ42 plus pCL4D) or Km and hygromycin (Hg) (50 µg ml-1) (for co-transformation with pB4 plus pBSh-D) for 4 d at 37 °C. Each experiment was repeated twice.
Plasmid copy number determination.
The relative copy number of pCL4D in M. smegmatis mc2155 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. 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 mc2155 chromosome (Ainsa et al., 1997
).
Sequencing experiments.
We sequenced double-stranded plasmid DNA by the dideoxy chain-termination method (Sanger et al., 1977
) using a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems), a model 9600 GenAmp PCR system (Perkin-Elmer) and a model 373 stretch DNA analysis system (Applied Biosystems). We used universal and reverse primers and a DNA walking strategy along the linear plasmid as an insert. Nucleotide sequences were analysed using the GCG software package (University of Wisconsin, Madison, WI, USA) and we searched for sequence similarities using the BLAST algorithm (Altschul et al., 1997
).
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RESULTS
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Identification of the internal region of the linear plasmid required for replication
We first constructed an endonuclease restriction map of pCLP by single and double digestion with the restriction enzymes BstEII, KpnI and XhoI (Fig. 1
). A set of recombinant plasmids was constructed by inserting restriction fragments covering the complete sequence of pCLP into a pUC19 derivative containing the Km gene from Tn903 as a selective marker. These recombinant plasmids were used to transform M. smegmatis, to test their ability to replicate in mycobacteria. Transformation was also performed with pB4, which carries a known mycobacterial origin of replication from the M. fortuitum circular plasmid pAL5000 (Ranes et al., 1990
), as a control. The plasmid containing fragment D, designated pCL4D, replicated in M. smegmatis, but plasmids containing fragments A, B, C and E did not (Fig. 1
). The efficiency of transformation of M. smegmatis with pCL4D was 2 x 104 bacteria (µg DNA)-1. We then subcloned fragment D, using XhoI, to determine the minimum replication region. Deletion of the first kilobase of fragment D did not prevent plasmid replication (Fig. 1
). Therefore, the region necessary for autonomous replication in M. smegmatis was located in a 4 kb fragment extending from the XhoI restriction site to the KpnI site (Fig. 1
). The banding patterns obtained by probing Southern blots of M. smegmatis transformant colonies with pCL4D demonstrated that pCL4D was neither integrated into the M. smegmatis chromosome nor in a linear form, but was instead present as an autonomously replicating circular plasmid (Fig. 2
).

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Fig. 2. Southern blot analysis of digested DNA of M. smegmatis mc2155 transformed with pCL4D. DNA in all lanes was digested with SacI and probed with pCL4D. Lanes: 16, individual colonies of M. smegmatis transformants; T-, untransformed M. smegmatis mc2155; T+, original plasmid pCL4D. The arrow indicates the expected size for the linear pCL4D construct.
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We tested whether the replication region of pCLP could replicate in slow-growing species by transforming M. bovis BCG with pCL4D, which resulted in around 102 transformants (µg DNA)-1. Again, Southern blots of digested DNA from M. bovis BCG transformant colonies demonstrated that pCL4D was not integrated into the chromosome (Fig. 3a
). Southern blots of non-digested DNA from M. bovis BCG transformant colonies revealed patterns that could correspond to the relaxed and supercoiled DNA (which migrates more rapidly than the relaxed counterpart) of pCL4D (Fig. 3b
). DNA was also extracted from transformants and used to transform E. coli, which resulted in E. coli transformant colonies from which pCL4D-derived plasmids could be isolated (Fig. 3c
), further indicating that pCL4D was present in M. bovis BCG as an autonomously replicating circular plasmid. However, plasmids from almost all the M. bovis BCG transformants displayed structural modifications, which included insertion and deletions (Fig. 3
). For example, the recombinant plasmid extracted from a M. bovis BCG clone and used to transform E. coli resulted in a deletion of almost 3 kb (Fig. 3c
).

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Fig. 3. Analysis of plasmids recovered from M. bovis BCG transformed with pCL4D. (a) Southern blot analysis of PvuII-digested DNA of M. bovis BCG transformed with pCL4D. DNA in all lanes was digested with PvuII and probed with pCL4D. Lanes: 1, M. bovis BCG transformant; 2, untransformed M. bovis BCG; 3, original plasmid pCL4D (expected sizes are indicated on the right). The weak band indicated by a small arrow is better visualized after a longer exposure. (b) Southern blot of non-digested DNA of M. bovis BCG transformants probed with pCL4D. Lanes 15, individual colonies of M. bovis BCG transformants. The arrows indicate the relaxed (top of the gel) and supercoiled (bottom of the gel) DNA of the pCL4D circular molecule. (c) Restriction of plasmid extracted from E. coli transformed with total DNA from one M. bovis BCG transformant. Lanes: 1, original plasmid pCL4D; 2, recombinant plasmid recovered from E. coli. DNA was digested with SacI or SalI. Lane M, molecular mass marker; sizes of the fragments are indicated on the left.
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Characteristics of the E. colimycobacteria shuttle vector pCL4D
The single-cell resistance to Km (Gavigan et al., 1997
) of M. smegmatis mc2155 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. This plasmid was therefore about as stable as pB4, the stability of which was also assessed in the absence of selection pressure (Fig. 4a
). We tested whether our pCL4D construct was compatible with a pJAZ38-derived replicon and a pAL5000-derived replicon in M. smegmatis. We co-transformed M. smegmatis with pJAZ42 (a pJAZ38-derived plasmid expressing Sm resistance; Gavigan et al., 1997
) and pCL4D. M. smegmatis transformants [5x102 transformants (µg DNA)-1] expressing both Km and Sm resistance were obtained. Every day, the M. smegmatis transformants were diluted in antibiotic-free medium, cultured for a few generations and then tested for antibiotic resistance. Our results indicated that these two plasmids coexisted in M. smegmatis and were stable for at least 40 generations (Fig. 4b
). In a similar experiment, we co-transformed M. smegmatis with a pCLP-derived plasmid and pB4 (containing the complete sequence of pAL5000 and expressing Km resistance; Ranes et al., 1990
). Fragment D of pCL4D was inserted into a pUC derivative vector containing a Hg resistance cassette (Table 1
). We obtained 7x102 M. smegmatis transformants (µg DNA)-1 expressing both Km and Hg resistance, even after 4 d growth (data not shown).
Sequencing of the replication region of pCLP and identification of a putative origin of replication
We determined the complete nucleotide sequence of the 4 kb fragment G of pCLP, which conferred autonomous replication in M. smegmatis (Fig. 1
). The genetic organization of the origin of replication region of pCLP is shown schematically in Fig. 5
. The nucleotide sequence contains two possible ORFs encoding proteins of 214 and 350 amino acids (Fig. 5
). The deduced amino acid sequence of the first ORF was up to 40% identical to homologues of the ParA partition protein of a wide variety of bacteria, including Pseudomonas spp. and Agrobacterium tumefaciens. The second putative ORF encodes a putative protein the carboxy-terminal region of which is about 25% identical to the Rep protein of a circular plasmid from a Rhodococcus sp. and to the circular plasmid pLR7 from M. avium. The region of sequence identity between the putative Rep proteins encoded by pLR7 and pCLP includes the previously identified helixturnhelix motif (Beggs et al., 1995
). Analysis of the replication region of pCLP also identified a 67 bp region more than 80% identical to the nucleotide sequences of the replication regions of the mycobacterial circular plasmids pLR7, pJAZ38 and pMSC262; it includes a 20 bp sequence that is absolutely identical (except for 1 bp in pJAZ38) (Fig. 5
). In all of these plasmids, the conserved region was located about 50 bp upstream from the start codon of the putative rep genes. No sequence similarity was detected between the replication region of pCLP and pAL5000 from M. fortuitum, which has been completely sequenced (Rauzier et al., 1988
). We found no consensus sequences typical of the origin of replication of bacterial circular plasmids (Del Solar et al., 1998
). However, the most striking feature of the replication region was the presence of an 18 bp sequence, 5'-TCC GAA ACC CGC TTA GCG-3', as a direct repeat (Fig. 5
). The overall GC content of the replication region was 66%, similar to that of mycobacterial chromosomal DNA, but a 40 bp region with a GC content of 37% was found in the vicinity of the 18 bp direct repeats, which may facilitate strand separation at the initiation site of replication. Sequence analysis of the pCLP replication region led to the identification of a SalI restriction site located upstream from the candidate origin of replication (Fig. 5
). This restriction site was used to construct a plasmid, pCL5, containing only the candidate origin of replication and the putative rep gene (Table 1
, Fig. 5
). This construct replicated in M. smegmatis and M. bovis BCG, suggesting that the region containing the putative parA gene is not necessary for replication. We defined the location of the origin of replication of pCLP by constructing plasmids with (plasmid pCRa) and without (plasmid pCRb) the candidate origin of replication (Table 1
, Fig. 5
). M. smegmatis transformants were obtained with pCRa, which contains the minimum replication region of pCL5 (Fig. 5
). However, no Km-resistant transformants were obtained with pCRb, which contains the region conserved in other mycobacterial replicons and the putative rep gene (Fig. 5
). Therefore, the deleted fragment, which contains a non-coding sequence including the 18 bp direct repeat and the AT-rich region, may be the origin of replication of pCLP.
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DISCUSSION
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We have previously demonstrated that mycobacterial linear plasmids have an invertron-like structure similar to that of other replicons in actinomycetes (Picardeau & Vincent, 1998
). In this study, we identified the origin of replication of pCLP, which is located in a region about one third of the way along the sequence. The replication region of this linear plasmid had several features in common with those of circular plasmids. Firstly, pCL4D, which contains the internal origin of replication of pCLP, was stably maintained in mycobacteria as a circular plasmid. Previous studies of linear chromosomes and linear plasmids in Streptomyces spp., which also have a terminal protein covalently bound to each 5' end, have shown that these linear DNA structures initiate replication from a centrally located origin and replicate after circularization (Chang & Cohen, 1994
; Fischer et al., 1998
; Musialowski et al., 1994
; Zakrzewska-Czerwinska & Schrempf, 1992
). In contrast, the linear replicons with an invertron-like structure found in organisms other than actinomycetes, such as the Bacillus subtilis phage
29, initiate the replication of full-length DNA strands at the telomeres by a protein-primed strand-displacement mechanism (Salas, 1991
). The similar terminal structure of the linear plasmids in actinomycetes suggests that, like the linear replicons of Streptomyces spp., linear plasmids in mycobacteria may have another replication mechanism, in addition to the internal replication origin, for filling in the ends of lagging-strand DNA (Qin & Cohen, 1998
). The termini of pCLP contain several palindromes (Picardeau & Vincent, 1998
) that may be involved in such a mechanism. The structural features of the replication region of pCLP are similar to those of circular plasmids. Indeed, homologues of bacterial circular DNA partition and replication proteins were found in the replication region of pCLP. We identified a possible ParA homologue, which appears not to be essential for the replication of pCLP. The ParA and ParB proteins encoded by the P1 plasmid are involved in the partitioning of stable low-copy-number plasmids. In the P1 partition cycle, ParB binds to an AT-rich sequence, parS, to form a complex that interacts with ParA, stimulating the ATPase activity of the Par A (Davis et al., 1992
). All three elements, parA/parB/parS, are absolutely essential for plasmid stability. The low copy number of pCL4D in M. smegmatis, and the loss of pCL4D by 15 % of M. smegmatis transformant cells after 40 generations, may be due to the presence of an incomplete partitioning system, consisting of only the ParA homologue. ParA homologues have also been found in the candidate origins of replication of Borrelia burgdorferi linear and circular plasmids (M. Picardeau, J. R. Lobry & B. Hinnebusch, unpublished results). The other putative ORF in the replication region of pCLP encodes a putative protein that is a homologue of the Rep protein of the circular plasmids of other actinomycetes. Subcloning experiments with pCL4D suggest that this ORF is necessary for replication. Rep-like proteins have in common a helixturnhelix motif, typical of DNA-binding proteins (Brennan & Matthews, 1989
), and may therefore be involved in the initiation of replication. A region was identified that was almost completely conserved, upstream from the putative rep genes of the linear plasmid pCLP and mycobacterial circular plasmids pLR7 from M. avium, pJAZ38 from M. fortuitum and pMSC262 from M. scrofulaceum. As pCL4D and pJAZ38, both containing this conserved region, are compatible in M. smegmatis, this sequence is presumably not involved in the incompatibility process. In bacterial circular plasmids, the origins of replication usually contain directly repeated sequences that act as binding sites for the Rep protein and AT-rich regions, facilitating strand separation (Del Solar et al., 1998
). The region conserved in pCLP, pLR7, pJAZ38 and pMSC262 contains no repeats or AT-rich regions. Its location, upstream from the putative rep gene, suggests that this region may have control properties. A candidate origin of replication was found between the putative parA and rep genes of pCLP; it contains direct repeats, which may act as a Rep-binding site, and in its vicinity there are AT-rich segments. The replication regions of the linear plasmids pSCL and pSLA2 of Streptomyces spp. also contain long juxtaposed direct repeats (Chang et al., 1996
). Cloning experiments in which the candidate origin of replication of pCLP was not used, but the conserved region upstream from the putative rep gene was, abolished plasmid replication, further suggesting that the region containing the 18 bp direct repeat and the AT-rich segments is the origin of replication and is required with the Rep protein for plasmid replication.
The replication region of the mycobacterial linear plasmid is more similar to that of mycobacterial circular plasmids than to that of other linear replicons of Streptomyces spp. This suggests that the linear plasmids in actinomycetes may not have a common ancestor, but that they may have arisen from several independent events, such as genetic exchanges with bacteriophages or fungi (Hinnebusch & Tilly, 1993
), resulting in genetic entities with similar terminal structures.
Several of our results suggest that pCL4D may be useful as a shuttle vector for genetic studies in mycobacteria. (i) We obtained a high efficiency of transformation with pCL4D in M. smegmatis and M. bovis BCG. However, DNA rearrangements were observed in plasmids extracted from M. bovis BCG transformants and never in M. smegmatis. Although the restriction patterns of these plasmids showed that they underwent rearrangements, pCL4D-derived plasmids are still able to replicate in mycobacteria. This phenomenon of plasmid structural instability has previously been observed for pAL5000-derived plasmids expressing heterologous DNA in M. bovis BCG (Haeseleer, 1994
). Restriction analysis and sequencing of the rearranged plasmids will allow determination of the mechanisms of these structural modifications. This may help in constructing pCL4D-derived plasmids with more stable structures in M. bovis BCG. (ii) We found that this pCLP-derived plasmid can co-exist in M. smegmatis with pJAZ38- and pAL5000-derived plasmids, which have been shown to be compatible (Gavigan et al., 1997
). This finding suggests that the three plasmids belong to three different compatibility groups. (iii) Previous studies have shown that pMSC262 from M. scrofulaceum and pLR7 from M. avium cannot transform the hypertransformable mutant strain, M. smegmatis mc2155 (Beggs et al., 1995
; Qin et al., 1994
). Therefore, our plasmid, which replicated in both slow- and fast-growing species, may be useful for the introduction of genes into various species of mycobacteria. Thus, for example, pCL4D may be used in experiments requiring more than one plasmid together with plasmids derived from pAL5000, the most studied mycobacterial plasmid which has been extensively used for the construction of vectors.
In conclusion, as previously suggested, linear plasmids in actinomycetes may be evolutionary intermediates between circular plasmids and linear phage replicons (Shiffman & Cohen, 1992
). Future studies of linear plasmids in actinomycetes should focus on the characterization of their terminal proteins and investigation of their function. These terminal proteins have not been characterized in bacteria and their function is unknown; they may be part of the replication machinery and/or they may protect the extremities from exonuclease activity. Intergeneric transfer of linear plasmids has never been demonstrated in mycobacteria, but may be possible between mycobacteria, Streptomyces spp. and Rhodococcus spp., in which linear plasmids have also been found (Picardeau & Vincent, 1998
). Indeed, in Streptomyces spp., almost all the extensively studied linear replicons have been found to be conjugative plasmids. Further studies are required to define the role of these linear replicons in the spread of genes in the natural habitats of actinomycetes.
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ACKNOWLEDGEMENTS
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We thank C. Guilhot for plasmid pB4, C. Martin for plasmid pJAZ42 and strain EP10, and V. Pelicic for plasmid pPV8. We would also like to thank J. Rauzier for help with sequencing. M.P. is a recipient of a fellowship from Fondation Roux (Institut Pasteur). This work is part of the doctoral thesis in Microbiology of C.L.D.
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Received 9 August 1999;
revised 1 November 1999;
accepted 3 November 1999.