Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

C. Viana-Niero1, P. E. de Haas2, D. van Soolingen2 and S. C. Leão1

1 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo – Escola Paulista de Medicina (UNIFESP-EPM), Rua Botucatu, 862 3° andar, 04023-062, São Paulo, Brazil
2 Diagnostic Laboratory of Infectious Diseases and Perinatal Screening, National Institute of Public Health and the Environment (RIVM), PO Box 1, 3720 BA, The Netherlands

Correspondence
S. C. Leão
sylvia{at}ecb.epm.br


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.


The nucleotide sequences determined in this study have been deposited in GenBank under accession numbers AY462249AY462261.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Mycobacterium tuberculosis genome contains four highly related genes encoding phospholipase C enzymes (Cole et al., 1998). Three genes, plcA, plcB and plcC, are organized in tandem (plcABC locus). The fourth gene, plcD, is located in a different region. In the laboratory strain M. tuberculosis H37Rv, plcD is truncated by insertion of a copy of IS6110 and by deletion of a 7·9 kb fragment, designated RvD2 (Gordon et al., 1999; Brosch et al., 2002). An intact copy of plcD is present in M. tuberculosis CDC1551, Mycobacterium bovis and M. bovis bacille Calmette–Guérin (BCG).

M. tuberculosis plc genes present significant similarity with Pseudomonas aeruginosa genes plcH and plcN, which encode haemolytic and non-haemolytic phospholipase C enzymes, respectively (Leão et al., 1995; Ostroff et al., 1990). The {beta}-haemolysis exhibited by Escherichia coli extracts overexpressing recombinant PlcA suggests enzymic activity of M. tuberculosis Plc proteins (Leão et al., 1995). Moreover, sphingomyelinase and phospholipase C activities were detected in cell extracts from M. tuberculosis and in Mycobacterium smegmatis overexpressing recombinant PlcA or PlcB (Johansen et al., 1996). The product of the plcA gene was detected in cell extracts from M. tuberculosis grown in vitro and inside macrophages (Matsui et al., 2000).

Bacterial phospholipase C enzymes are recognized as important virulence factors of P. aeruginosa (Berka et al., 1981), Bacillus cereus (Gilmore et al., 1989), Clostridium perfringens (Logan et al., 1991) and Listeria monocytogenes (Vasquez-Boland et al., 1992), playing roles in intracellular survival, cytolysis and cell-to-cell spread (Titball, 1993). The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown. Recent contributions to this subject were the demonstration that triple plcABC and quadruple plcABCD M. tuberculosis mutants were attenuated in the late phase of mouse infection (Raynaud et al., 2002), and the observation that a plc{Delta}ABC{Delta}tlyA M. tuberculosis mutant, lacking plcABC and the haemolysin gene, is more virulent in mice than strain H37Rv, resulting in a higher bacterial load in organs and diminished survival (Smith et al., 2002). These results are not necessarily contradictory, since the strains and procedures used in both plc mutant studies were different. Nevertheless, both studies suggested participation of phospholipase C genes in the virulence of M. tuberculosis.

Genetic variations in plcABC and in plcD were observed within the M. tuberculosis complex. M. tuberculosis complex subspecies include M. tuberculosis, Mycobacterium africanum, M. bovis (along with the attenuated M. bovis BCG) and Mycobacterium microti. Recently proposed subspecies include Mycobacterium pinnipedii, the seal bacillus (Cousins et al., 2003), ‘Mycobacterium tuberculosis subsp. Canettii’ (‘M. canettii’) (Pfyffer et al., 1998) and M. bovis subsp. caprae (Aranaz et al., 1999; Niemann et al., 2002). Comparative genomics of the complete DNA sequence of M. tuberculosis H37Rv and M. bovis BCG disclosed a total of 16 M. bovis BCG-specific deletions, or regions of difference (RD) (Mahairas et al., 1996; Brosch et al., 1998; Behr et al., 1999; Gordon et al., 1999). Hybridization experiments on a DNA microarray (Behr et al., 1999) and using ordered bacterial artificial chromosome (BAC) libraries (Gordon et al., 1999) revealed that an 8·9 kb fragment containing plcABC genes, RD5 deletion (Gordon et al., 1999) or RD7 deletion (Behr et al., 1999) is absent from M. bovis BCG relative to M. tuberculosis H37Rv. This region is also absent from virulent M. bovis (Behr et al., 1999; Gordon et al., 1999).

Primers designed to amplify internal RD5 portions were used in PCRs to evaluate the occurrence of RD5 deletion in M. tuberculosis clinical isolates and in other members of the complex. Primers PT1/PT2 were used to amplify a 396 bp region of mtp40, a fragment situated at the 3' end of plcA. This product was amplified from the reference strains M. tuberculosis H37Rv (TMC102) and M. tuberculosis H37Ra (ATCC 25177), but not from M. bovis (TMC410), M. bovis BCG Pasteur (TMC1011), M. microti (NCTC 8710) or M. africanum (ATCC 25240) (Del Portillo et al., 1991). The mtp40 product was amplified from 54·6 % of 105 M. africanum isolates (Viana-Niero et al., 2001), and also from human, pig, llama and ferret M. microti isolates, but not from vole isolates or the hyrax strain (van Soolingen et al., 1998). This product was detected in mycobacteria isolates from seals (Liébana et al., 1996) and in ‘M. canettii (Brosch et al., 2002), but not in M. bovis subsp. caprae (Niemann et al., 2002). Parsons et al. (2002) studied 88 members of the M. tuberculosis complex, using a different pair of primers to amplify a 152 bp product of plcA. They concluded that RD5 is present in most, but not all, isolates of M. tuberculosis, M. africanum and M. microti, and consistently absent from M. bovis and M. bovis BCG isolates.

IS6110 is a repetitive element frequently used in the epidemiology of tuberculosis. Transposition of this element generates genome plasticity of members of the tubercle bacilli (Fang et al., 1999; Warren et al., 2000). Genetic variations caused by IS6110 insertions at different points in plcABC and plcD were observed in M. tuberculosis clinical isolates (Vera-Cabrera et al., 1997, 2001; Warren et al., 2000), indicating that these domains represent preferential regions for integration (Warren et al., 2000). Considering the potential role that the plc genes may have in the virulence of the tubercle bacillus, the study of genetic variations affecting the four genes in isolates cultured from patients may provide insights into the significance of phospholipase C enzymes for the pathogenicity of the bacillus. Genetic polymorphisms affecting both plcABC and plcD loci in 25 representative M. tuberculosis clinical isolates, three M. africanum and four M. canettii’ strains are described here.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial isolates.
Twenty-five M. tuberculosis isolates, belonging to a collection of 790 strains from the RIVM (Bilthoven, The Netherlands) isolated from patients with active tuberculosis in 1998, were included in this study. The mtp40-associated RFLP was determined in all 790 M. tuberculosis isolates and 50 distinct banding patterns were distinguished. At least two isolates from each mtp40-RFLP type were subjected to PCR analysis using mtp40-specific primers. Isolates from 14 out of the 50 mtp40-RFLP types were negative in this PCR, and comprised 54/790 of the strains (6·8 %). Ten mtp40-PCR-negative and 13 mtp40-PCR-positive isolates were selected from the five most prevalent mtp40-RFLP types in our collection. In addition, two mtp40-RFLP type strains with mtp40 PCR fragments larger than the normal size (approx. 2 kb) were also included. The most prevalent mtp40-RFLP types represented 85 % of all 790 strains investigated. M. tuberculosis H37Rv and M. bovis BCG Pasteur were used as controls (Table 1).


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Table 1. Isolates included in the study and results of PCR with primers PT1/PT2 (mtp40), AD/AR (plcA), BD/BR (plcB), CD/CR (plcC) and DD/DR (plcD)

+, Positive; –, negative; +*, ~2·0 kb product; +**, ~3·0 kb product. IS6110 flanking sequences in the plcABC and plcD loci determined by sequence analysis are shown, with direct repeats in bold. ND, Not determined. X, Not applicable to these strains. Positions of insertions in the plcABC locus were obtained from M. tuberculosis H37Rv MTCY98 (GenBank accession no. Z83860); positions of insertions in the plcD locus were obtained from M. tuberculosis CDC1551 (GenBank accession no. AE007040).

 
Three M. africanum and four ‘M. canettii’ isolates from the National Reference Laboratory for Mycobacteria at Institut Pasteur (Paris, France) culture collection were kindly provided by Dr Véronique Vincent. These strains were characterized extensively using biochemical tests, oxyR-PCR, pncA-PCR, and molecular typing methods, such as IS6110-RFLP typing and spoligotyping (Viana-Niero et al., 2001) (Table 1).

Cultures were grown on Löwenstein–Jensen (LJ) solid medium or in Middlebrook 7H9 (Difco) liquid medium supplemented with albumin, glucose and catalase (Difco) and kept frozen at –70 °C in aliquots of 7H9-ADC with 15 % (v/v) glycerol. DNA was prepared as described by van Embden et al. (1993).

PCR and sequencing.
For primer design, sequences for plcABC from M. tuberculosis H37Rv and plcD from M. tuberculosis CDC1551 were obtained from GenBank (http://www.ncbi.nlm.nih.gov/). Primers were designed using OLIGO (version 5.0; National Biosciences) and are depicted in Table 2. Each of the primer pairs AD/AR, BD/BR, CD/CR and DD/DR amplified an individual plc gene and amplification specificity was evaluated by restriction analysis of the amplified fragments (data not shown). Internal primers AD2/AR2, BD2/BR2, CD2/CR2 and DD3/DR3, PT1/PT2 primers specific for the mtp40 sequence, and IS1/IS2 primers from IS6110 and PPE1 from Rv2356c (H37Rv) were used to address specific sequencing issues (Table 2). PCR amplicons were sequenced in an automated ABI Prism 377 sequencer (Perkin-Elmer) using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Sequences were compared using BLAST (http://www.ncbi.nlm.nih.gov/blast).


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Table 2. Sequences and localization of primers used in this work

 
Southern blot analysis of plc polymorphisms.
Southern blotting was performed as described by van Embden et al. (1993). Briefly, 2 µg DNA was digested using PvuII and subjected to electrophoresis in 0·8 % (w/v) agarose in 0·5xTBE pH 8·0, at 2 V cm–1. A mixture of PvuII-digested supercoiled DNA ladder (Invitrogen) and HaeIII-digested {phi}X174 DNA (Invitrogen) was used as internal DNA size marker. DNA was blotted onto nylon membranes (Hybond-N-plus; Amersham Biosciences) and probed, in different experiments, with probes complementary to each plc gene. Probes were prepared by PCR using primers AD/AR, BD/BR, CD/CR or DD/DR. Blots were also probed with the internal size marker. Probes were covalently labelled with peroxidase by glutaraldehyde and detected using the ECL Direct System (Amersham) according to the manufacturer's instructions. Membranes were exposed to an X-ray film (X-OMAT; Kodak). Fingerprints were analysed using GELCOMPAR II version 2.5 software (Applied Maths). Fingerprint autoradiograms were superimposed on internal marker autoradiograms for normalization.

Cloning and sequencing of a plcC-specific PstI restriction fragment.
DNA from isolate 97-1177 was digested using PstI at 37 °C for 2 h, and subjected to electrophoresis in 0·8 % (w/v) agarose, along with the internal size marker. DNA was blotted onto a nylon membrane and hybridized with probes from plcC, IS6110 and the internal size marker, as described previously. The size of the fragment hybridizing with both the plcC and the IS6110 probe was calculated using GELCOMPAR II. Agarose gel slices around the selected band of PstI restriction fragment were excised from a second gel, purified using the Gel Extraction Kit (Qiagen) and ligated into PstI-digested dephosphorylated (CIAP; Invitrogen) pBluescript SK+ (Stratagene), using T4 DNA ligase (Invitrogen) at 16 °C, overnight. Ligated DNA was transformed into electrocompetent E. coli DH5{alpha} cells, which were plated onto LB-agar plates containing 100 µg ampicillin ml–1, 0·25 mM IPTG and 1 mM X-Gal. Colony lifts were carried out on Whatman 541 filters and filters were hybridized with the plcC probe, labelled with [{alpha}-32P]dCTP with Ready-To-Go DNA Labelling Beads (Amersham), according to the protocol described by Maas (1983). Positive colonies visualized by exposure to autoradiographic film were confirmed by PCR with primers CD2/CR2 and by PstI digestion of the plasmid DNA. The recombinant plasmid, isolated using the Qiaprep Spin miniprep kit (Qiagen), was sequenced using M13 primers, as described previously.

RNA purification and RT-PCR.
Two-week-old cultures in Middlebrook 7H9 ADC medium were used for RNA isolation using FastRNA kit-BLUE (QBiogene) and a FastPrep Instrument (QBiogene), according to the manufacturer's instructions. DNA was removed by DNase I (Invitrogen). RT-PCR was performed with 1 µg RNA using the Access RT-PCR System (Promega) and internal primers specific for each plc gene. Amplicons were sequenced. Control of RT-PCR was performed using primers from atpB, a gene encoding a key component of the proton channel, which is transcribed during axenic growth and after infection of human macrophages (Graham & Clark-Curtiss, 1999).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PCR revealed polymorphisms in the four plc genes
PCR assays were set up using primer pairs AD/AR, BD/BR, CD/CR and DD/DR, for amplification of a 1599 bp fragment from plcA, a 1611 bp fragment from plcB, a 1610 bp fragment from plcC and a 1478 bp fragment from plcD, respectively (Fig. 1). As expected, the plcD amplicon was not detected in M. tuberculosis H37Rv and the plcA, plcB and plcC amplicons were not detected in M. bovis BCG Pasteur.



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Fig. 1. Schematic representation of the plcABC and plcD loci. PvuII restriction sites (arrows) were obtained from M. tuberculosis H37Rv MTCY98 (GenBank accession no. Z83860) and M. tuberculosis CDC1551 (GenBank accession no. AE007040). A, B, C, D, fragments generated by amplification using primers AD/AR, BD/BR, CD/CR and DD/DR, respectively. These fragments were used as probes in Southern blot hybridizations. 1–8, Fragments detected by hybridization of Southern blots: 1, 574 bp; 2, 2156 bp; 3, 580 bp; 4, 751 bp; 5, 4657 bp (according to M. tuberculosis H37Rv genome data) or 2983 bp (according to empirical data from this work); 6, >=4 kb (not determined); 7, 538 bp; 8, 3900 bp.

 
Results of PCR are summarized in Table 1. Three of the 25 M. tuberculosis, two of the M. africanum and the four M. canettii’ isolates produced amplicons of all four plc genes. Products from plcA, plcB and plcC, but not from plcD, were amplified from six M. tuberculosis isolates and one M. africanum isolate. The only product amplified from five isolates of M. tuberculosis was plcD. Four M. tuberculosis isolates did not produce any plc amplicon.

Instead of the normal mtp40 product of 396 bp, two isolates, 97-858 and 97-2433, produced amplicons of approximately 2 kb in size with primers PT1/PT2. plcC products larger than expected, of approximately 3 kb, were generated from isolates 97-742, 97-818 and 97-1505. Isolates 97-432 and 97-1389 produced plcD fragments of approximately 3 kb in size. In all cases, re-amplification of these amplicons using primers IS1/IS2 generated products of 123 bp, confirming the insertion of IS6110 elements in the mtp40 region of plcA, and in plcC and plcD, respectively.

To characterize the polymorphisms detected by PCR in the plcABC and plcD genomic regions of these selected isolates, plc-based fingerprints were obtained by the use of restriction endonuclease PvuII and specific DNA probes, homologous to the sequences of each of the four plc genes.

Southern hybridization and sequence analysis confirmed insertions and deletions
All isolates were subjected to Southern hybridization analysis. Polymorphisms in plc genes detected by PCR and hybridization were analysed by sequencing of selected amplicons.

Fig. 1 shows the physical map and PvuII restriction sites of plcABC obtained from analysis of M. tuberculosis H37Rv cosmid MTCY98 (GenBank accession no. Z83860) and plcD from M. tuberculosis CDC1551 cosmid MT1799 (GenBank accession no. AE007040). Fragments A, B, C and D were generated by PCR using primers AD/AR, BD/BR, CD/CR and DD/DR, respectively, and were used as probes in Southern blot hybridization (Fig. 1). Information about PvuII fragment sizes was used to identify specific bands hybridizing with each probe. Empirical results showed that the size of the band located at the 5' region of plcA, calculated using GELCOMPAR II, was 2983 bp instead of 4657 bp, the expected size according to M. tuberculosis H37Rv genome data (Fig. 2).



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Fig. 2. Southern blot hybridization with probes A (plca), B (plcb), C (plcc) and D (plcd) analysed using GELCOMPAR II version 2.5. (a) Hybridization patterns; (b) specific bands hybridizing with each plc probe. Non-marked fragments are the result of cross-hybridization with fragments from other plc genes. Lanes: 1, M. tuberculosis H37Rv; 2, 97-1344, 97-286, 97-1894, 97-432, 97-365, 97-535, 97-438, 97-969, 97-269, 97-1674, 140030063, 140030065, 140030068, 140010059, 140010105, 140010107, 140010110; 3, 97-858; 4, 97-2433; 5, 97-742; 6, 97-818, 97-1505; 7, 97-803, 97-1177, 97-1200, 97-1289; 8, 97-488; 9, 97-1389; 10, 97-279, 97-1438, 97-66, 97-464; 11, M. bovis BCG Pasteur; 12, 97-438, 97-535, 97-969, 140030065; 13, 97-365; 14, M. tuberculosis H37Rv; 15, 97-279, 97-2438, 97-488, 97-858, 97-1894; 16, M. bovis BCG Pasteur; 17, 97-2433; 18, 97-286, 97-66, 140030063, 140030068, 140010059, 140010105, 140010107, 140010110; 19, 97-1389; 20, 97-432; 21, 97-803, 97-818, 97-1505, 97-1289, 97-742, 97-1177, 97-1200, 97-1674; 22, 97-269.

 
Banding patterns generated by hybridization with the four probes confirmed PCR results showing integrity of all four plc genes in three M. tuberculosis isolates, two M. africanum isolates and all four ‘M. canettii’ isolates. The presence of an intact plcABC locus and the lack of one or more of the plcD-hybridizing bands confirmed PCR results in six M. tuberculosis isolates and one M. africanum isolate (Fig. 2).

In isolates 97-858 and 97-2433, Southern hybridization using probe A was consistent with the presence of a copy of IS6110 in the mtp40 region of plcA (Fig. 2). Sequence analysis of the amplicon from isolate 97-858 revealed the site of insertion at position 22 319, which is related to the sequence of cosmid MCTY98 (GenBank accession no. Z83860), and a direct repeat of three nucleotides TGT : TGT at the ends of the insertion element (Table 1 and Fig. 3a). The exact insertion site in isolate 97-2433 was not determined.



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Fig. 3. Schematic representation of polymorphisms in the plcABC locus. The upper map represents positions 16 800 to 31 225 of MTCY98 (GenBank accession no. Z83860) from M. tuberculosis H37Rv and annotated genes. Black arrows represent plc genes and hatched arrows represent unrelated genes. White boxes indicate IS6110 elements and closed arrows within white boxes represent direction of transcription of the transposase gene. Dotted lines represent deletion regions. Small open arrows (not to scale) placed above the line represent primers used for amplification and sequencing. (a) Isolate 97-858; (b) isolate 97-742; (c) isolates 97-818 and 97-1505; (d) isolates 97-803, 97-1289, 97-1177 and 97-1200; (e) isolate 97-1389; (f) isolate 97-488.

 
In isolates 97-818 and 97-1505, which produced a plcC amplicon of 3·0 kb, hybridization using probe C revealed substitution of the 2156 bp PvuII band by two bands of approximately 1·7 kb in size (Fig. 2). Sequence analysis of the 3·0 kb plcC amplicons from isolates 97-818 and 97-1505 revealed insertion of a copy of IS6110 at position 19 849 of plcC (related to GenBank sequence MCTY98) and duplication of four nucleotides at the site of insertion, with one nucleotide change, GCAG : GCAA (Table 1 and Fig. 3c). A similar polymorphism was observed in isolate 97-742, in which the 2156 bp PvuII band was substituted by two bands of approximately 1·5 kb and 1·9 kb in size (Fig. 2). Insertion of a copy of IS6110 was confirmed by sequence analysis at position 19 668 of plcC, with a TGA : TGA direct repeat (Table 1 and Fig. 3b).

Six isolates, 97-1289, 97-1177, 97-1200, 97-803, 97-488 and 97-1389, lacked plcA- and plcB-hybridizing bands. Southern hybridization with probe C disclosed shifts in the 2156 bp band. Amplicons of approximately 1 kb in size were produced using primers IS2/CR2. Sequence analysis of these amplicons revealed insertion of IS6110 elements in plcC at position 19 849 in four isolates, position 19 500 in one isolate and position 19 916 in one isolate (Table 1 and Fig. 3d, e, f).

In four isolates, 97-66, 97-279, 97-464 and 97-1438, Southern hybridizations with probes A, B and C revealed a single plcC-hybridizing band of approximately 1·3 kb in size. Generation of a 450 bp product using primers CD2/CR2 and lack of hybridization of the plcC probe with the 574 bp restriction fragment confirmed that these isolates retain only the central part of plcC.

In isolates 97-432 and 97-1389, amplification using primers DD/DR resulted in a fragment of 3·0 kb in size. Hybridization using probe D was suggestive of the presence of an intervening sequence in plcD (Fig. 2). Sequence analysis of the amplicon from isolate 97-432 revealed the insertion of a copy of IS6110, with the right imperfect repeat inserted at position 2611 and the left repeat at position 2698 (relative to the sequence of cosmid MT1799; GenBank accession no. AE007040), and deletion of 87 bp from plcD. No direct repeats were detected at the ends of the IS element (Table 1 and Fig. 4a).



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Fig. 4. Schematic representation of polymorphisms in plcD, between positions 1 and 14 648 from M. tuberculosis CDC1551 (GenBank accession no. AE007040) and corresponding maps of M. tuberculosis H37Ra and H37Rv. (a) Isolate 97-432; (b) isolates 97-269, 97-803, 97-818, 97-1505, 97-1289, 97-742, 97-1177, 97-1200 and 97-1674. See legend to Fig. 3 for explanation of symbols.

 
The hybridization pattern generated by the probe complementary to plcD in nine isolates was indistinguishable (Fig. 2). The expected bands of 538 bp and 3·9 kb were not detected, suggesting deletion of the 5' region. Sequencing of these amplicons produced by amplification using primers IS2/DR2 confirmed the presence of an IS6110 element at position 2611, relative to the sequence of M. tuberculosis CDC1551 cosmid MT1799 (GenBank accession no. AE007040) (Table 1 and Fig. 4b). The size of the deleted fragment was not determined.

Polymorphisms in plc genes were characterized in a group of genetically related isolates
An interesting polymorphism in the plcABC locus was detected in a group of six genetically related isolates, 97-818, 97-1505, 97-803, 97-1289, 97-1177 and 97-1200. Analysis of the RFLP patterns generated upon hybridization with a probe complementary to the IS6110 element revealed similarities greater than 70 % in the IS6110-RFLP profiles (data not shown). All six isolates carried a copy of IS6110 in plcC at position 19 849. The orientation of the IS element was the same in all isolates. Four isolates, 97-803, 97-1289, 97-1177 and 97-1200, were mtp40-PCR-negative and presented deletions of plcA, plcB and part of plcC. The remaining two isolates retained the genes plcA and plcB. Polymorphism in the plcD genomic region was shared by all six isolates (Fig. 4b).

To understand the mechanism involved in the deletion of plc genes in this group of isolates, the sequences flanking the ends of the IS insertion were determined in isolate 97-1177. DNA was digested using PstI and hybridized in Southern blots, using probes complementary to plcC and IS6110 (data not shown). A 4·0 kb PstI DNA fragment, which hybridized with both probes, was cloned and sequenced. The right imperfect repeat of IS6110 was inserted in plcC at position 19 849, and the left repeat was inserted at position 28 509 from MTCY98 (Table 1 and Fig. 3b). Sequence analysis confirmed the deletion of a segment of 8·66 kb in size, containing the 5' region of plcC and the complete genes plcB, plcA, PPE38 and PPE39, and its substitution by a copy of the IS6110 element. No direct repeats were detected at the ends of the IS element, suggesting that deletion occurred by homologous recombination between two copies of IS6110. Identical polymorphism was observed in the other three isolates and was corroborated by sequence analysis of a product of 2·0 kb, generated by amplification with primers PPE1 and CR2.

Isolates 97-818 and 97-1505, which carry the IS element in plcC without subsequent gene deletion, presented a second copy of IS6110 at position 28 509. Sequence analysis of amplicons obtained by PCR using primers PPE and IS1 confirmed that the second IS element, located between genes PPE39 and PPE40, has the same orientation of the IS6110 copy inserted in plcC (Fig. 3a). These results, in conjunction, represent strong evidence that the homologous recombination between these two IS6110 elements resulted in deletion of the chromosomal domain located between them.

Analysis of transcription of plc genes
We also studied possible disturbances caused by insertions of IS6110 elements in the transcription of plc genes in selected isolates. Disruption of plcA or plcC by an IS6110 element did not impair transcription of adjacent plc genes in isolates 97-858 and 97-818, respectively (Fig. 5). Transcription of plcD was confirmed by RT-PCR in isolates with an intact plcD gene, such as M. bovis BCG Pasteur and isolate 97-279 (Fig. 5). Products of plc genes were not detected by RT-PCR in isolates 97-1289, 97-1177, 97-1200, 97-803 and 97-1389 (not shown).



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Fig. 5. RT-PCR analysis of plc genes in isolates 97-818, 97-858, 97-279 and M. bovis BCG Pasteur. R, RT-PCR products corresponding to genes atpB, plcA, plcB, plcC and plcD were amplified with primers atpb1/atpb2, AD2/AR2, BD2/BR2, CD2/CR2 and DD3/DR3, respectively. P, control PCR was performed with non-retrotranscribed RNA to confirm absence of DNA contamination. +, PCR was performed directly on DNA from M. bovis BCG Pasteur, as positive control.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The mtp40 sequence, located in the plcA opposite strand, was described before identification and sequencing of plc genes (Parra et al., 1991). It has been used in diagnostic PCR for identification of M. tuberculosis (Del Portillo et al., 1991) and, more recently, to evaluate the occurrence of RD5 deletion in clinical M. tuberculosis isolates or in isolates from other members of the M. tuberculosis complex (Gordon et al., 1999; Parsons et al., 2002). There have been conflicting results over the distribution of this sequence in members of the complex, and several researchers have described the existence of mtp40-negative M. tuberculosis isolates (Liébana et al., 1996; Weil et al., 1996; Vera-Cabrera et al., 1997). These mtp40-PCR negative results may be a consequence of RD5/RD7 deletion, other types of deletions, insertions of IS6110 elements or mutations in the primer region, and are indicative of the existence of polymorphisms in this genomic region.

To examine polymorphisms in plc genes in members of the M. tuberculosis complex that cause disease in humans, we analysed 25 selected M. tuberculosis clinical isolates and well-characterized isolates from M. africanum and ‘M. canettii’. This analysis revealed the presence of insertions inside the coding regions of plcA, plcC and plcD in M. tuberculosis isolates. No polymorphisms in the plcABC region were detected in M. africanum and ‘M. canettii’, but the small number of isolates analysed so far does not allow a definite conclusion. The finding of partial plcD deletion in one M. africanum isolate suggests that this region is also prone to polymorphisms in this subspecies.

The original report on IS6110 distribution in the genome of M. tuberculosis H37Rv indicated that insertions occurred preferentially in non-coding regions (Philipp et al., 1996). A recent analysis of data collated from various sources demonstrated that 57 of the 95 analysed insertions (60 %) occurred within coding regions (Sampson et al., 2001). The impact of these insertions on the phenotype is unknown, as most of the disrupted genes are multicopy genes present in the genome. Disruption of individual members in a family of genes may have limited impact on phenotype, depending on the activity of the protein encoded.

Insertions of IS6110 elements were detected in plcA and plcC. RT-PCR analysis provided evidence that disruption of either of these genes does not have a significant polar effect on transcription from neighbouring plc genes. Raynaud et al. (2002) obtained similar results using mutants that harboured transposon insertions in plcA, plcB or plcC.

In this work, discrete IS6110 insertions in plcC at positions 19 849 and 19 668 were detected. Insertion events at positions 19 589, 19 645 and 19 848, located in plcC, were observed in a previous study including 11 isolates (Vera-Cabrera et al., 2001). These findings indicate that this domain is a preferential integration region, defined as a chromosomal domain of <500 bp, where several points of IS6110 insertion have been identified in different clinical isolates (Warren et al., 2000).

A preferential insertion site was also detected in plcD. In 10 isolates, insertion of a copy of IS6110 was detected at position 2611, relative to M. tuberculosis CDC1551 cosmid MT1799 (GenBank accession no. AE007040). Insertions at position 2611 were reported by other authors in clones ISL540.9 and ISL0480.11 (GenBank accession nos AF126473 and AF077945, respectively) (Sampson et al., 1999), confirming that this site represents a hot spot for integration. Ho et al. (2000) studied 22 non-related clinical isolates and found 18 discrete IS6110 insertion sites in plcD and adjacent genes. Lari et al. (2001) detected an IS6110 insertion in plcD in one isolate and deletions of the entire RvD2 region in 15 out of 45 clinical M. tuberculosis isolates.

Detailed analysis of six genetically related isolates disclosed a genetic mechanism of deletion of a fragment located between two IS6110 elements. Deletion was accompanied by loss of the characteristic three- or four-nucleotide direct repeat flanking IS6110 elements. The observed deletion is most likely explained by the homologous recombination mechanism described by Fang et al. (1999), and this explanation agrees with findings of other authors (Ho et al., 2000).

Fang et al. (1999) reported deletions in a preferential locus for IS6110 insertion, named ipl, in clinical M. tuberculosis isolates, and attributed these deletions to homologous recombination between two IS6110 elements. This hypothesis was based on the assumption that a recent related ancestor had two IS6110 insertions, with the same orientation, in this locus. Homologous recombination of these two IS6110 sequences would have led to deletion of the DNA segment between them. To our knowledge, in this study, for the first time this hypothesis was substantiated by the finding of clinical isolates that are genetically related as determined by IS6110 typing and that could represent evolutionary intermediates.

According to Sampson et al. (2003), preferential integration of IS6110 elements apparently triggers IS6110-mediated deletion events, as observed in the DR region and in a 20 kb hypervariable region (Ho et al., 2000). Admitting that plcC and PPE genes are preferential insertion regions (Sampson et al., 1999; Warren et al., 2000), it could be speculated that recombination between two insertion sequences, one in a plc gene and the other in the vicinity of a neighbouring PPE gene, may have resulted in additional IS6110-mediated deletion events in plc genes, identified in this work.

Proximity and relative orientation of IS6110 elements are expected to influence recombination events within preferential integration regions, but other as-yet-unknown factors, such as IS6110 transposition activity, may also play a role.

Analysis of polymorphisms in the plcD–cutinase region of H37 strains provides some insights. The genomes of M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. tuberculosis CDC1551 contain a copy of IS6110 in front of the cutinase gene, in the same position and with the same orientation (Fig. 4). In H37Ra, a second copy of IS6110 is present in plcD. In M. tuberculosis H37Rv, a 7·9 kb fragment comprising the segment between position 2856 in plcD and the cutinase gene was deleted, leaving one copy of IS6110. The absence of flanking direct repeat elements strongly suggests that homologous recombination between two copies of IS6110 caused the deletion event (fragment RvD2). Therefore, the presence of two insertion elements, one in plcD and the other in the cutinase gene, triggered deletion in H37Rv but not in H37Ra, suggesting that IS6110 activity is in some way more pronounced in Rv than in Ra. Differences in IS6110 transposition activity between these two strains were confirmed by analysis of IS6110-RFLP patterns from 18 ATCC H37 variants (Bifani et al., 2000). Nine distinct but similar patterns were detected in 15 H37Rv isolates and a single pattern was detected in three H37Ra isolates.

In five isolates, insertions and/or deletions occurred simultaneously in all four plc genes. Even with extensive deletions in plcABC genes, a truncated fragment of the plcC gene was still detected by Southern blot hybridization. Transcription of this truncated plcC gene was not detected by RT-PCR. Since this is the first description of gene interruptions affecting expression of all members in the same gene family, implications on the fitness of these organisms are unknown at this time. Nevertheless, the fact that these strains were recovered from patients with active tuberculosis demonstrates that they retain the ability to cause disease.

Despite the mobile quality of the IS6110 element, its instability is not sufficiently high to support short-term variations in the plc genes. It is also very unlikely that the variability in the plc genes could be influenced by culture procedures. First of all, cultures are not frequently re-cultured after primary isolation. Strains with the same genotypes often have the same plc gene composition. In fact, the half-life of IS6110 RFLP is between 3 and 5 years (De Boer et al., 1999). This includes transpositions in all genomic regions. Alterations of the plc genes due to transposition of IS6110 would be much less frequent.

Insertions and deletions were identified in 84 % of the M. tuberculosis isolates studied and in one M. africanum isolate, with effects ranging from disruption of individual open reading frames to loss of complete genes. Insertions in nearby locations indicate that these genomic regions may be particularly susceptible to insertion events. These findings suggest that insertions of IS6110 in plc loci result in a biological advantage for the bacteria, which is maintained during subsequent strain diversification. Deletions may be subjected to positive selection when the bacteria no longer require the removed genes. The question whether interruptions of these genes by insertion or deletion may have important effects on the biological properties of clinical isolates still deserves an answer. Differences in the virulence and phospholipase C activity of these isolates are currently under evaluation.


   ACKNOWLEDGEMENTS
 
This work was supported by Centro Argentino Brasileiro de Biotecnologia (CABBIO) grant 480382/01-8 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and European Commission RDG (INCO-DEV Programme), project No. ICA4-CT-2001-10087. C. V.-N. is the recipient of a fellowship from FAPESP (00/02525-3). D. S. and S. C. L. are members of RELACTB (Tuberculosis Network for Latin America and The Caribbean).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aranaz, A., Liebana, E., Gomez-Mampaso, E. & 8 other authors (1999). Mycobacterium tuberculosis subsp. caprae subsp. nov. a taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain. Int J Syst Bacteriol 49, 1263–1273.[Abstract]

Behr, M. A., Wilson, M. A., Gill, W. P., Salamon, H., Schoolnik, G. K., Rane, S. & Small, P. M. (1999). Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520–1523.[Abstract/Free Full Text]

Berka, R. M., Gray, G. L. & Vasil, M. L. (1981). Studies of phospholipase C (heat labile hemolysin) in Pseudomonas aeruginosa. Infect Immun 34, 1071–1074.[Medline]

Bifani, P., Moghazeh, S., Shopsin, B., Driscoll, J., Ravikovitch, A. & Kreiswirth, B. N. (2000). Molecular characterization of Mycobacterium tuberculosis H37Rv/Ra variants: distinguishing the mycobacterial laboratory strain. J Clin Microbiol 38, 3200–3204.[Abstract/Free Full Text]

Brosch, R., Gordon, S. V., Billault, A., Garnier, T., Eiglmeier, K., Soravito, C., Barrel, B. G. & Cole, S. T. (1998). Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect Immun 66, 2221–2229.[Abstract/Free Full Text]

Brosch, R., Philipp, W. J., Stavropoulos, E., Colston, M. J., Cole, S. T. & Gordon, S. V. (1999). Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain. Infect Immun 67, 5768–5774.[Abstract/Free Full Text]

Brosch, R., Gordon, S. V., Marmiesse, M. & 12 other authors (2002). A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A 99, 3684–3689.[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.[CrossRef][Medline]

Cousins, D., Bastida, R., Cataldi, A. & 9 other authors (2003). Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov. Int J Syst Evol Microbiol 53, 1305–1314.[Abstract/Free Full Text]

De Boer, A. S., Borgdorff, M. W., de Haas, P. E. W., Nagelkerke, N. J. D., van Embden, J. D. A. & van Soolingen, D. (1999). Analysis of rate of change of IS6110 RFLP patterns of Mycobacterium tuberculosis based on serial isolates. J Infect Dis 180, 1238–1244.[CrossRef][Medline]

Del Portillo, P., Murillo, L. A. & Patarroyo, M. E. (1991). Amplification of a species-specific DNA fragment Mycobacterium tuberculosis and its possible use in diagnosis. J Clin Microbiol 29, 2163–2168.[Medline]

Eisenach, K. D., Cave, M. D., Bates, J. H. & Crawford, J. T. (1990). Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis 161, 977–981.[Medline]

Fang, Z., Doig, C., Kenna, D. T., Smittipat, N., Palittapongarnpim, P., Watt, B. & Forbes, K. J. (1999). IS6110-mediated deletions of wild-type chromosomes of Mycobacterium tuberculosis. J Bacteriol 181, 1014–1020.[Abstract/Free Full Text]

Gilmore, M. S., Cruz-Rodz, A. L., Leimeister-Wätcher, M., Kreft, J. & Goebel, W. (1989). A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage. J Bacteriol 171, 744–753.[Medline]

Gordon, S. V., Brosch, R., Billault, A., Garnier, T., Eiglmeier, K. & Cole, S. T. (1999). Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol Microbiol 32, 643–655.[CrossRef][Medline]

Graham, J. E. & Clark-Curtiss, J. E. (1999). Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci U S A 96, 11554–11559.[Abstract/Free Full Text]

Ho, T. B., Robertson, B. D., Taylor, G. M., Shaw, R. J. & Young, D. B. (2000). Comparison of Mycobacterium tuberculosis genomes reveals frequent deletions in a 20 kb variable region in clinical isolates. Yeast 17, 272–282.[CrossRef][Medline]

Johansen, K. A., Gell, R. E. & Vasil, M. L. (1996). Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria. Infect Immun 64, 3259–3266.[Abstract]

Lari, N., Rindi, L. & Garzelli, C. (2001). Identification of one insertion site of IS6110 in Mycobacterium tuberculosis H37Ra and analysis of the RvD2 deletion in M. tuberculosis clinical isolates. J Med Microbiol 50, 805–811.[Abstract/Free Full Text]

Leão, S. C., Rocha, C. L., Murillo, L. A., Parra, C. A. & Patarroyo, M. E. (1995). A species-specific nucleotide sequence of Mycobacterium tuberculosis encodes a protein that exhibits hemolytic activity when expressed in Escherichia coli. Infect Immun 63, 4301–4306.[Abstract]

Liébana, E., Aranaz, A., Francis, B. & Cousins, D. (1996). Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex. J Clin Microbiol 34, 933–938.[Abstract]

Logan, A. J., Williamson, E. D., Titball, R. W., Percival, D. A., Shuttleworth, A. D., Conlan, J. W. & Kelly, D. C. (1991). Epitope mapping of the alpha-toxin of Clostridium perfringens. Infect Immun 59, 4338–4342.[Medline]

Maas, R. (1983). An improved colony hybridization method with significantly increased sensitivity for detection of single genes. Plasmid 10, 296–298.[Medline]

Mahairas, G. G., Sabo, P. J., Hickey, M. J., Singh, D. C. & Stover, C. K. (1996). Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 178, 1274–1282.[Abstract]

Matsui, T., Carneiro, C. R. W. & Leão, S. C. (2000). Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity. Braz J Med Biol Res 33, 1275–1282.[Medline]

Niemann, S., Richter, E. & Rusch-Gerdes, S. (2002). Biochemical and genetic evidence for the transfer of Mycobacterium tuberculosis subsp. caprae Aranaz et al., 1999 to the species Mycobacterium bovis Karlson and Lessel 1970 (approved lists 1980) as Mycobacterium bovis subsp. caprae comb. nov. Int J Syst Evol Microbiol 52, 433–436.[Abstract/Free Full Text]

Ostroff, R. M., Vasil, A. I. & Vasil, M. C. (1990). Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa. J Bacteriol 172, 5915–5923.[Medline]

Parra, C. A., Lodoño, L. P., Del Portillo, P. & Patarroyo, M. E. (1991). Isolation, characterization, and molecular cloning of a specific Mycobacterium tuberculosis antigen gene. Infect Immun 59, 3411–3417.[Medline]

Parsons, L. M., Brosch, R., Cole, S. T. & 6 other authors (2002). Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis. J Clin Microbiol 40, 2339–2345.[Abstract/Free Full Text]

Pfyffer, G. E., Auckenthaler, R., van Embden, J. D. & van Soolingen, D. (1998). Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa. Emerg Infect Dis 4, 631–634.[Medline]

Philipp, W. J., Poulet, S., Eiglmeier, K. & 7 other authors (1996). An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae. Proc Natl Acad Sci U S A 93, 3132–3137.[Abstract/Free Full Text]

Raynaud, C., Guilhot, C., Rauzier, J., Bordat, Y., Pelicic, V., Manganelli, R., Smith, I., Gicquel, B. & Jackson, M. (2002). Phospholipases C are involved in the virulence of Mycobacterium tuberculosis. Mol Microbiol 45, 203–217.[CrossRef][Medline]

Sampson, S. L., Warren, R. M., Richardson, M., van der Spuy, G. D. & van Helden, P. D. (1999). Disruption of coding regions by IS6110 insertion in Mycobacterium tuberculosis. Tuber Lung Dis 79, 349–359.[CrossRef][Medline]

Sampson, S., Warren, R., Richardson, M., van der Spuy, G. D. & van Helden, P. D. (2001). IS6110 insertions in Mycobacterium tuberculosis: predominantly into coding regions. J Clin Microbiol 39, 3423–3424.[Free Full Text]

Sampson, S., Warren, R., Richardson, M., Victor, T. C., Jordaan, A. M., van der Spuy, G. D. & van Helden, P. D. (2003). IS6110-mediated deletion polymorphism in the direct repeat region of clinical isolates of Mycobacterium tuberculosis. J Bacteriol 185, 2856–2866.[Abstract/Free Full Text]

Smith, D. A., Parish, T., Smith, S. M., Dockrell, H. M., Stoker, N. G. & Bancroft, G. J. (2002). Deletion of mycobacterial phospholipases C and haemolysin alters virulence and inhibits T cell recognition of Mycobacterium tuberculosis H37Rv. In Fifth International Conference on the Pathogenesis of Mycobacterial Infections, Stockholm, Sweden, Abstract book, p. 11.

Titball, R. W. (1993). Bacterial phospholipases C. Microbiol Rev 57, 347–366.[Medline]

van Embden, J. D., Cave, M. D., Crawford, J. T. & 8 other authors (1993). Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 31, 406–409.[Abstract]

van Soolingen, D., van der Zanden, A. G., de Haas, P. E. & 7 other authors (1998). Diagnosis of Mycobacterium microti infections among humans by using novel genetic markers. J Clin Microbiol 36, 1840–1845.[Abstract/Free Full Text]

Vasquez-Boland, J.-A., Kocks, C., Dramsi, S., Ohayon, H., Geoffroy, C., Mengaud, J. & Cossart, P. (1992). Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect Immun 60, 219–230.[Abstract]

Vera-Cabrera, L., Hoard, S. T., Laszlo, A. & Johnson, W. M. (1997). Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis. J Clin Microbiol 35, 1190–1195.[Abstract]

Vera-Cabrera, L., Hernandez-Vera, M. A., Welsh, O., Johnson, W. M. & Castro-Garza, J. (2001). Phospholipase region of Mycobacterium tuberculosis is a preferential locus for IS6110 transposition. J Clin Microbiol 39, 3499–3504.[Abstract/Free Full Text]

Viana-Niero, C., Gutierrez, C., Sola, C., Filliol, I., Boulahbal, F., Vincent, V. & Rastogi, N. (2001). Genetic diversity of Mycobacterium africanum clinical isolates based on IS6110-restriction fragment length polymorphism analysis, spoligotyping, and variable number of tandem DNA repeats. J Clin Microbiol 39, 57–65.[Abstract/Free Full Text]

Warren, R. M., Sampson, S. L., Richardson, M., van der Spuy, G. D., Lombard, C. J., Victor, T. C. & van Helden, P. D. (2000). Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity. Mol Microbiol 37, 1405–1416.[CrossRef][Medline]

Weil, A., Plikaytis, B. B., Butler, W. R., Woodley, C. L. & Shinnick, T. M. (1996). The mtp40 gene is not present in all strains of Mycobacterium tuberculosis. J Clin Microbiol 34, 2309–2311.[Abstract]

Received 19 September 2003; revised 3 November 2003; accepted 4 November 2003.



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