Unité de Génétique Mycobactérienne1, and Unité de Bactériologie Moléculaire et Médicale2, Institut Pasteur, F-75724 Paris Cedex 15, France
Instituto de Biotecnologia, CICV-INTA, Moron, Argentina3
Author for correspondence: Jean-Marc Reyrat. Tel: +33 1 45688828. Fax: +33 1 45688843. e-mail: jmreyrat{at}pasteur.fr
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ABSTRACT |
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Keywords: exported product, protein family, repeats, Mycobacterium
The GenBank accession numbers for the sequences reported in this paper are AF213152 (Mycobacterium smegmatis), AF213153 (Mycobacterium marinum), AF213154 (Mycobacterium ulcerans), AF213155 (Mycobacterium xenopi) and AF315789 (Mycobacterium avium). The accession numbers for the partial sequences of the erp gene of Mycobacterium tuberculosis clinical isolates are AF165856 (Benin), AF165857 (R.C.A.), AF165858 (Vietnam) and AF165859 (Tahiti).
a Present address: Dept of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
b Present address: University of Minnesota, Duluth School of Medicine, Duluth, MN, USA.
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INTRODUCTION |
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We show here that the erp gene is present in all mycobacterial species tested and is therefore not a pathogen-specific gene. The erp gene was found not only in pathogenic mycobacteria, but also in saprophytic mycobacteria such as Mycobacterium smegmatis, in environmental opportunistic pathogenic mycobacteria such as Mycobacterium avium, Mycobacterium marinum and Mycobacterium xenopi, and in extracellular-toxin-producing mycobacteria such as Mycobacterium ulcerans. This allowed the definition of a new protein family that seems specific to the genus Mycobacterium.
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METHODS |
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Cloning of the M. smegmatis and M. xenopi erp genes.
Chromosomal DNA was isolated from M. smegmatis and M. xenopi as described previously (Pelicic et al., 1996 ). Five micrograms of genomic DNA from either species was digested with SalI (M. smegmatis) or PstIXbaI (M. xenopi) and DNA fragments in the 2 kb or 4 kb size range (M. smegmatis or M. xenopi, respectively) were excised from the gel, purified and ligated to pBluescript II SK vector DNA linearized with the respective restriction enzymes and dephosphorylated, in the case of SalI. Colony replicas of the transformants were made on Hybond-N+ nylon membranes (Amersham Pharmacia) and screened by hybridization with a 32P-labelled PCR fragment probe generated by the amplification of genomic DNA with primers erp-8 and erp-9. Recombinant plasmid DNA from clones yielding strong hybridization signals were recovered by using Qiagen mini-columns. Screening of these partial genomic libraries allowed the identification of recombinant plasmids carrying the M. smegmatis 2 kb SalI insert (plasmid pML-2A1) or the M. xenopi PstIXbaI 4·5 kb insert (plasmid pEPX6).
DNA sequence of erp genes and flanking regions.
Sequences of double-stranded plasmid DNA or PCR-generated fragments were determined by the dideoxy chain-termination method using a 373-B DNA Analysis System (Applied Biosystems). Oligonucleotide sequences are listed in Table 1.
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Sequencing in TB isolates.
Five clinical isolates originating from France (hp030496 13), Benin (be290396 18), Vietnam (be290396 29), Tahiti (ta080296 11) and the Central African Republic (rca30696 16) with a distinct spoligotype profile were chosen (Goguet de la Salmonière, 1999 ), and the central region of the erp gene corresponding to amino acids 92235 was amplified by PCR using primers erpRf and erpRr and sequenced.
Construction of the M. smegmatis erp insertional mutant.
A M. smegmatis erp mutant was generated by insertion of a kanamycin (aph) resistance cassette into the erp coding region. DNA from plasmid pML-2A1, a suicide vector in mycobacteria, was partially digested with BamHI and ligated to a 1264 bp BamHI DNA fragment containing a kanamycin resistance cassette from plasmid pUC4K (Taylor & Rose, 1988 ). The ligation mixture was used to transform E. coli DH5
and colonies harbouring the desired constructs were selected on LB plates supplemented with kanamycin. Plasmid pML-2A1::aph contains the aph fragment inserted in the BamHI site at position 503 of the erp gene (relative to the start codon). One microgram of pML-2A1::aph DNA was introduced by electroporation into M. smegmatis mc2155, essentially as described by Pelicic et al. (1996)
. The electroporated bacteria were plated on 7H10 plates supplemented with kanamycin. Colonies were screened by PCR with oligonucleotide primers flanking the insertion site (primers erp-11 and erp-R) in order to identify double-recombination events.
Construction of complemented strains.
Constructs for complementation of the M. smegmatis erp::aph insertional mutant were made in the integrative vector pNIP40b (Berthet et al., 1998 ). Vector DNA was linearized with XbaI and dephosphorylated with 0·1 U shrimp alkaline phosphatase (USB) prior to ligation. For the M. smegmatis mc2155 or the M. leprae complementation constructs (pML-30 and pML-40, respectively), the following strategy was used: a 426 bp M. tuberculosis MT103 PCR fragment generated with primers erp-C1 and erp-C3, containing the erp regulatory region and signal peptide up to a unique EcoRI site (at position 129 in relation to the start codon), was ligated to either (1) an 805 bp PCR fragment generated with primers erp-C4 and erp-C6, containing the M. smegmatis erp coding sequence, starting at position 120 in relation to the start codon and ending at the stop codon, or (2) a 586 bp PCR fragment generated with primers erp-C5 and erp-C7, containing the M. leprae erp coding sequence, starting at position 132 in relation to the start codon and ending at the stop codon. The ligation mixtures were used to transform E. coli DH5
and transformants were selected on LB plates supplemented with hygromycin and kanamycin. Plasmid DNA was used to transform the M. smegmatis mc2155 erp::aph mutant strain by electroporation. Transformants were selected on 7H10 plates supplemented with hygromycin and kanamycin.
Preparation of protein samples for SDS-PAGE and immunoblotting.
Protein samples were prepared from bacteria grown in 25 ml Sauton medium containing 0·05% Tween 80, under agitation. Briefly, the culture supernatants were obtained after centrifugation at 4000 g for 10 min, and filtered through a 0·2 µm Millex-GV (Millipore) filter to remove any remaining cells, and the proteins were precipitated with 17% (v/v) TCA and analysed by SDS-PAGE and Western blotting.
SDS-PAGE and immunoblotting.
Proteins were resolved by SDS-PAGE using 12% polyacrylamide gels (Laemmli, 1970 ) and transferred to Hybond-C membranes (Amersham Pharmacia). A rabbit Erp antiserum (Berthet et al., 1998
) was used as first antibody at a 1:5000 dilution, and bound antibodies were revealed using a horseradish-peroxidase-coupled donkey anti-rabbit antibody (Amersham Pharmacia) at 1:10000 dilution. Detection was performed with the ECL system (Amersham Pharmacia).
Computer methods.
The amino acid sequence alignments used to produce the identity between orthologues were generated by ALIGN from the Wisconsin Package, version 9.1-Unix with a PAM250 matrix, whereas multiple alignments were generated using PILEUP (Pearson & Lipman, 1988 ). Domain analysis was performed using ProDom (Corpet et al., 2000
). Boxshade (http://www.ch.embnet.org/software/box_html) was used to highlight similarity between proteins. Public databases were searched using either BLASTP or PSI-BLAST algorithms (Altschul et al., 1997
).
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RESULTS AND DISCUSSION |
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Differences between species in the sequence of the central region of the erp gene led us to look for sequence variation between characterized isolates of M. tuberculosis. Five clinical isolates from France, Benin, Vietnam, Tahiti and the Central African Republic with different spoligotype profiles were studied (Goguet de la Salmonière, 1999 ). The central region of the erp gene was amplified by PCR and sequenced. No differences in DNA sequence were observed, despite the different spoligotype profiles and geographical origins, demonstrating that the repeated region was not subject to allelic variation.
During the completion of this work, the genomic sequences of M. smegmatis (http://www.tigr.org/tdb/mdb/mdbinprogress.html) and Mycobacterium paratuberculosis (http://www.cbc.umn.edu/cgibin/blasts/AGAC.restrict/blastn.cgi) were released, showing the erp gene to be present in both species, thereby confirming and extending our data. It is thus very likely that erp is common to all members of the genus Mycobacterium.
Conservation of the erp genomic environment
The completed genome sequence of M. tuberculosis shows that erp lies between glf (Rv3809) and csp (Rv3811), two genes probably involved in cell-wall elaboration (Cole et al., 1998 ; Weston et al., 1998
). Using in silico methods, when the sequences were available (M. tuberculosis, M. bovis, M. leprae or M. avium), or through PCR amplification with glf- and csp-specific primers for M. smegmatis, M. xenopi and M. ulcerans, we showed that the genetic context of the erp locus is conserved in all mycobacteria tested (Fig. 2
). However, in M. leprae, numerous point mutations in the csp gene probably lead to the absence of an active product, suggesting that csp may not be required for a strictly intracellular lifestyle. Nevertheless, the extensive genomic conservation observed suggests a strong selective pressure that has maintained this locus unchanged from saprophytic to pathogenic mycobacteria.
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Phenotypic characterization of the M. smegmatis erp::aph mutant
To demonstrate that the 36 kDa protein of M. smegmatis reacting with the anti-Erp serum was indeed due to the Erp homologue, an erp::aph derivative with a kanamycin cassette inserted within the erp gene was constructed by allelic exchange. This erp mutant was then transformed with pIPX70, an integrative vector expressing TB erp under its own promoter, which had already been used to complement the erp mutant of M. tuberculosis (Berthet et al., 1998 ). Fifty micrograms of total protein contained in the culture supernatants of M. smegmatis mc2155, the M. smegmatis erp::aph mutant and the isogenic complemented strain was probed with the anti-Erp antiserum. No cross-reacting band was detected in the M. smegmatis erp mutant strain (Fig. 3
), demonstrating that this cross-reaction is indeed due to the product of the erp gene of M. smegmatis.
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The requirement for Erp in M. tuberculosis for a successful murine infection is clear (Berthet et al., 1998 ). However, this ubiquity in the mycobacterial genus shows that it is not a pathogen-specific gene. It is, however, possible to speculate that the Erp protein, which has an extracellular localization, may have an important role in cell-wall structure. Several studies have shown that genes encoding cell-wall-related products are involved in the virulence of the tubercle bacillus (Camacho et al., 1999
; Cox et al., 1999
; Armitige et al., 2000
; Glickman et al., 2000
). One future challenge is the identification of the molecular function of the Erp protein and its role in M. tuberculosis virulence.
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ACKNOWLEDGEMENTS |
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Received 22 February 2001;
revised 30 March 2001;
accepted 5 April 2001.