Institute of Biotechnology1, Institute of Pathobiology2, Institute of Virology3, CICVyA/INTA, 1712 Castelar, Argentina
Author for correspondence: Angel A. Cataldi. Tel: +54 11 4621 1447 or 0199. Fax: +54 11 4481 2975. e-mail: acataldi{at}correo.inta.gov.ar
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ABSTRACT |
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Keywords: repressor, tetR, reporter
Abbreviations: mce3; Rv1963; TSP, transcription start point; BAC, bacterial artificial chromosome
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INTRODUCTION |
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Unfortunately, many aspects of the infection and disease process of tuberculosis at the cellular and molecular level still remain unknown. According to Smith (1984) , to infect and cause disease pathogenic mycobacteria must be able to (i) colonize the hosts lung tissues, (ii) enter the hosts cells, (iii) multiply in the environment of the hosts tissues, (iv) resist or interfere with the hosts defence mechanisms and (v) cause damage to the hosts tissues. Despite the formidable advances in molecular biology in recent years, the identification of the virulence factors of pathogenic mycobacteria (i.e. M. tuberculosis, Mycobacterium bovis and Mycobacterium leprae) has been delayed, when compared to the identification of the virulence factors of other infectious bacteria. Factors contributing to this situation include the lack of a phenotype clearly associated with virulence in pathogenic mycobacteria and the scarcity of genetic tools for transforming and mutating M. tuberculosis. Steps to solving the latter problem have been taken, as improved genetic tools have allowed the identification of virulence-related genes in M. tuberculosis (Camacho et al., 1999
; Cox et al., 1999
). The most-employed virulence-associated attribute of M. tuberculosis is its persistence in the infected organism or in cultured cells (Bange et al., 1999
).
We have previously identified a 12·7 kb region in the genome of M. tuberculosis that is absent from the genome of M. bovis (Fisanotti et al., 1997 ; Zumarraga et al., 1999
). This 12·7 kb fragment was present in all of the M. tuberculosis strains tested and was absent from all of the M. bovis, Mycobacterium microti and Mycobacterium africanum strains tested. The region is located near the 3' end of the RD2 element described by Mahairas et al. (1996)
, a 14 kb genomic locus present in M. bovis but absent from some strains of M. bovis BCG, suggesting that this region suffers from genetic instability. Sequence analysis of ORFs within the 12·7 kb fragment of the M. tuberculosis genome demonstrated that it mostly encodes exported proteins. One of the ORFs is highly homologous to the invasin-like protein described by Riley and colleagues (Arruda et al., 1993
; Chitale et al., 2001
). This region was also described by Gordon et al. (1999)
as RD7. The presence of genes encoding an invasin-like protein and many membrane or secreted proteins within the 12·7 kb region suggests that this region may play an essential role in the hostpathogen interaction of M. tuberculosis. The ORFs within the region are organized as a putative operon, which is similar in its sequence and organization to three other M. tuberculosis regions (operons mce1, mce2 and mce4) described by Cole et al. (1998)
. The lack of similarity of the genes encoded by this putative operon to genes from other bacterial species suggests that they may play a specific role in the physiology or virulence of M. tuberculosis. Flesselles et al. (1999)
reported that a BCG strain mutated in mce2 exhibits a reduced ability to invade the non-phagocytic epithelial cell line HeLa, which supports the idea of a role for mce operons in virulence. Harboe et al. (1999)
demonstrated production of the mce1-encoded proteins using cell extracts from M. tuberculosis and M. bovis BCG. Mce1 proteins are recognized by the antibodies of TB patients (Ahmad et al., 1999
), indicating in vivo expression of the mce1 operon. At the genetic level, the four mce operons appear to have evolved from a common ancestor. The four operons may play different roles in the infection process, may be expressed at different times along growth phases or the infection process, or they may simply serve to protect essential virulence genes against mutations. In the present study, the regulation of the expression of the mce3 operon was investigated, to gain an insight into the role of this operon in M. tuberculosis. Our results show the presence of a repressor that controls mce3 transcription.
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METHODS |
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DNA manipulations.
Standard methods were used for restriction-endonuclease digestion of plasmids, for DNA ligations and for other manipulations. Isolation of plasmid DNA was performed using the Wizard Minipreps SV Kit, according to the manufacturers instructions (Promega). DNA from M. tuberculosis was prepared according to van Soolingen et al. (1991) .
Construction of lacZ reporter fusions.
Details for the plasmids, vectors and primers used in this study can be found in Table 1. To create fusions with the E. coli lacZ gene, the regions containing the Rv1963Rv1964 intergenic fragment including or not including the Rv1963 gene were amplified by PCR and cloned into the promoter-probe vector pJEM15 (Timm et al., 1994
). The resulting plasmids were called p1963-P3 (plus Rv1963) and pP3 (minus Rv1963). All amplified fragments were cloned into either pGEM-T (Promega) or pPCRII-TOPO (Invitrogen), before being transferred to pJEM15. Fragments (100 and 206 bp in length) from upstream of the Rv1964 gene start codon were also fused to a promoterless lacZ gene, resulting in plasmids pP3-100 and pP3-200, respectively (Table 1
).
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Different deletions of the Rv1963Rv1964 intergenic region were obtained as follows. The pPCRII-TOPO intermediate constructs p1963-P3 and pP3 were separately digested with NheI and SalI; the digest products were then ligated. The inserts from the resulting plasmids were transferred to BamHI-linearized pJEM15 (see Table 1), resulting in p1963-P3
Nhe and pP3
Nhe (for NheI deletions), and p1963-P3
Sal and pP3
Sal (for SalI deletions). An XmaIAgeI deletion was obtained by digestion of p1963-P3 with XmaI and AgeI. The digest products were ligated and introduced into linearized pJEM15, resulting in pP3
Xma/Age (Table 1
).
Cloning and expression of Rv1963.
Rv1963 was amplified from M. tuberculosis BAC DNA and cloned into pRSET-A (Table 1). The resulting plasmid, pRSET1963, was introduced into E. coli BL21(
DE3). E. coli BL21(pRSET1963) was then grown in LB broth at 28 °C. Expression of the Rv1963 gene was induced by the addition of 0·1 mM IPTG to the growth medium at the mid-exponential phase of growth. Soluble cell extracts from the culture were prepared by Fast Prep FP120 (Qbiogene) bead-beater disruption (40 s at 6·0 m s-1, using Lysing Matrix B). Proteins separated by SDS-PAGE (Cataldi et al., 1994
) were assayed by Western blotting using anti-histidine as the primary antibody (1:3000 dilution; Amersham Pharmacia) and alkaline-phosphatase-conjugated anti-mouse-IgG as the secondary antibody (1:2000 dilution; Sigma).
PCR amplification.
PCR amplifications were performed using Taq DNA polymerase (Promega) under standard conditions in a total volume of 50 µl. dNTPs were used at a concentration of 0·2 mM each; 20 pmol of each primer was used. The protocol used for amplification was as follows; denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1 min, 1 min annealing at a temperature dependent on the primer pair used (Table 1) and elongation at 72 °C for 1 min, with a final elongation at 72 °C for 10 min. A total of 2 ng of genomic M. tuberculosis DNA or BAC DNA was used as template.
RNA preparation.
Total RNA from M. tuberculosis H37Rv was isolated using the FastRNA Kit-Blue (Qbiogene). Briefly, 50 ml of a culture was harvested during the exponential phase of growth by centrifugation at 3000 r.p.m. for 10 min. The cell pellet was resuspended in 100 µl diethyl pyrocarbonate (DEPC)-treated water and transferred to a 2 ml screw-cap microcentrifuge tube containing 0·1 mm diameter zirconium beads and FastRNA reagents (Qbiogene). Cells were disrupted by using a Fastprep FP120 bead-beater for 20 s at a speed of 6·0 m s-1. Total RNA was then extracted from the cells following the manufacturers instructions. After agarose-gel electrophoresis of the total RNA and staining of the gels with ethidium bromide, the different bands of rRNA were clearly visible, indicating that the RNA preparations were of a high integrity.
Primer extension.
This was performed using the primer Low861 (Table 1). Ten picomoles of the non-phosphorylated primer were labelled by using T4 polynucleotide kinase (Promega) in the presence of [
-32P]ATP. The specific activity of the primer was 8000 c.p.m. pmol-1. M. tuberculosis H37Rv RNA (6 µg) and the labelled primer (0·1 pmol, 33000 c.p.m. pmol-1) were mixed in 7 µl of 50 mM Tris/HCl (pH 8·3) containing 0·1 M KCl. The reaction was then incubated at 94 °C for 1 min, at 56 °C for 10 min and then on ice for 15 min. The mixture was adjusted to a final volume of 12 µl by the addition of 1 µl of a mixture containing the dNTPs (2·5 mM each), 0·5 µl of RNAsin (Promega), 2·2 µl of 5xreverse transcriptase buffer [0·25 M Tris/HCl (pH 8·3), 0·2 M KCl, 36 mM magnesium acetate, 0·01 M DTT], 0·8 µl of DEPC-treated water and 0·5 µl avian myeloblastosis virus reverse transcriptase (Promega). Reverse transcription was performed at 42 °C for 45 min; the reaction was stopped by the addition of 5 µl of stop buffer to the reaction mixture.
The reverse-transcription products were separated by PAGE (6% acrylamide gel containing 8 M urea), and were run alongside the sequencing products obtained with the Low861 primer. The gels were fixed by immersion in a mixture containing 5% (v/v) methanol and 5% (v/v) acetic acid. They were then exposed to X-ray film (Kodak X-Omat RS) for 24 h at -70 °C.
Computer analyses.
Amino-acid-sequence alignments were generated by searching public databases using BLASTP (http://www.ncbi.nlm.nih.gov/BLAST). M. smegmatis BLAST searches (http://www.tigr.org/tdb/mdb/mdbinprogress.html) were used to identify an Rv1963 orthologue in this bacterium. Domain analysis was performed by using PFAM (http://www.cgr.ki.se/Pfam). Prokaryotic promoter sequences were searched by using the prokaryotic option of the Neural Network Promoter Prediction algorithm of the Berkeley Drosophila Genome Project (http://www.fruitfly.org/seq_tools/promoter.html).
Measurements of ß-galactosidase activity.
For in vitro determinations of ß-galactosidase activity, 1 ml of a recombinant M. smegmatis culture or 1 ml of a recombinant M. tuberculosis culture was pelleted, resuspended in 1 ml buffer Z [0·1 M sodium phosphate (pH 7·0), 10 mM KCl, 1 mM MgSO4 and 50 mM mercaptoethanol], disrupted using a Fast Prep FP120 bead-beater (40 s at 6·0 m s-1, using Lysing Matrix B) and centrifuged at 13000 r.p.m. to obtain the soluble cell extract. ß-Galactosidase measurements were performed on the soluble cell extract, as described by Miller (1972) ; results are expressed in Miller units [A420x1000/reaction time (min)xA550].
For determination of the ß-galactosidase activity in an infected macrophage cell line, the murine macrophage-like cell line J774 was cultivated in RPMI medium 1640 with 25 mM HEPES buffer and L-glutamine supplemented with 10% fetal calf serum in 5% CO2 at 37 °C in T25 flat-bottomed cell-culture flasks. The cell line was incubated for 1824 h until a density of 5x106 cells per flask was reached. Recombinant M. tuberculosis H37Rv strains were grown in Middlebrook 7H10 medium containing 20 µg kanamycin ml-1. The cultures were then harvested, resuspended in RPMI medium, vortexed, sonicated for 1 min in an ultrasonic cleaner and allowed to settle. The upper part of each of the bacterial suspensions (supposedly free of clumps of bacteria) was used to infect the J774 cell line. The OD650 value for the upper portion of the suspension was taken, and it was assumed that an OD650 value of 0·1 was equal to 107 c.f.u. ml-1 (data not shown). This suspension was then used to replace the medium that the J774 cells were in, and infection of the J774 cells was performed at an m.o.i. of 100. Infected cells were incubated for 3 h, then washed five times with fresh RPMI medium. At 0, 1, 4 and 6 days post-infection, the J774 cells were scraped and lysed with 1 ml of 1% Triton X-100 for 15 min. The resulting suspension was centrifuged to obtain the mycobacterial pellet, and ß-galactosidase activity was determined for the mycobacterial cells, as described above. Activity was related to the number of bacteria as determined by plating onto Middlebrook 7H11 agar supplemented with kanamycin and OADC. As a control, the ß-galactosidase activity of the bacterial strains that were used for macrophage infection but grown in vitro was determined.
Gel-shift assay.
A 100 bp and a 206 bp fragment from upstream of the start codon of Rv1964 (from mce3; see Construction of lacZ reporter fusions) and a 139 bp fragment from upstream of Rv0586 of the mce2 promoter region (obtained using primers P2up and P2low; Table 1) were used as probes in this assay. Fragments were labelled with [
-32P]ATP by using the polynucleotide kinase enzyme. Non-incorporated nucleotides were eliminated from the mixture containing the labelled probes by using Wizard PCR Prep Columns (Promega). Labelled probes were incubated with the soluble cell extract from recombinant E. coli BL21 overexpressing Rv1963 or from E. coli BL21(pRSET) (negative control) in binding buffer [4% glycerol (v/v), 1 mM MgCl2, 0·5 mM EDTA, 0·5 mM DTT, 50 mM NaCl, 10 mM Tris/HCl (pH 7·5), 0·05 mg salmon sperm DNA ml-1] for 20 min at room temperature. The samples were separated by PAGE [4% acrylamide gel containing 1xTBE (0·05 M Tris base, 0·05 M boric acid, 1 mM EDTA-Na2.2H2O), 0·1% bisacrylamide and 2·5% glycerol] for 3 h at 100 V, after a 30 min pre-run in a 0·5xTBE buffer tank. The gels were then dried and exposed to X-ray film.
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RESULTS |
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DISCUSSION |
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Even though the expression of Mce proteins has been demonstrated both in vitro (Harboe et al., 1999 ) and in vivo (Ahmad et al., 1999
), nothing is known about the regulation of production of Mce proteins. The observation of a regulatory-type gene in the vicinity of the mce3 operon prompted us to determine whether this putative regulator controls mce expression. The 0·9 kbp intergenic region between Rv1963 and Rv1964 should be the location of the mce3 promoter and the target of regulatory proteins. Progressive deletions and cloning of this region helped us to map the promoter region in a fragment extending from the Rv1964 start codon to 206 bp upstream of it. A neural-network search for putative prokaryotic promoters located a probable promoter extending from -170 to -230 upstream of the ATG start of the first mce3 ORF. As deduced from gel-shift assays, it was also observed that Mce3R binds to this region.
Using all of the published putative mycobacterial sequences, Mulder et al. (1997) calculated the mycobacterial promoter to consist of a -35 consensus sequence, TTGACG/A, and a -10 consensus sequence, TATA/GA/CT. Putative -10 (TATATG) and -35 (TAGCAA) sequences were identified in the predicted promoter region of the mce3 operon. The proposed -10 hexamer is identical to the corresponding sequence in E. coli and conserves the four first positions of the Mulder et al. (1997)
consensus sequence. The putative -35 sequence conserves three positions of the Mulder et al. (1997)
consensus sequence and is identical to other individual mycobacterial promoters. However, the proposed -10 sequence is far (32 bp) from the TSP identified by primer-extension experiments. Possible explanations for this large separation could be (i) an erroneous TSP was mapped because the 5' end of the transcript was processed or degraded, or (ii) non-conserved -10 and -35 sequences are present in the mce3 promoter region. As in many actinomycete promoters, mycobacterial promoters not carrying the canonical -35 and -10 sequences have been reported. Counting upstream from the proposed start site of the mce3 promoter region there is another, less consensual, sequence, TATTTA, at a distance of -12 bp.
In general, the TetR family of bacterial regulators are repressors; many of these proteins regulate genes that encode proteins involved in membrane processes such as osmoregulation (Rkenes et al., 1996 ), permeability (Ma et al., 1996
; Lucas et al., 1997
; Namwat et al., 2001
) and resistance to antibiotics or quaternary ammonium salts (Rouch et al., 1990
; Grkovic et al., 1998
), among other functions. The prototype TetR repressor is TetR from the Tn10 transposon of E. coli (Orth et al., 2000
). There are approximately 40 tetR-family regulatory genes in M. tuberculosis (http://www.sanger.ac.uk/Projects/M_tuberculosis). To the best of our knowledge, the Rv1963-encoded protein examined in this study (as Mce3R) is the first TetR-like regulatory protein whose function has been described in M. tuberculosis. As with other TetR regulators, a helixturnhelix motif and a tetR-family signature sequence reside in the first third of the Rv1963 gene. Most TetR-like regulators have molecular masses ranging from 21 to 25 kDa; however, Mce3R (Rv1963) has a molecular mass >25 kDa. We observed that other TetR-like proteins with molecular masses >25 kDa contain two consensus signatures, as does Mce3R (Rv1963). The last third of Mce3R (Rv1963) shows no homology to other proteins. The two TetR-like regulators most homologous to Mce3R (Rv1963), Rv2506 and Rv3557c, are in the vicinity of lipid-metabolism-related genes of the M. tuberculosis genome.
We could not find the inducer(s) of the mce3 operon; different conditions of stress (ethanol, SDS or low carbon) did not abolish repression of expression (data not shown). No removal of repression was observed when the J774 macrophage-like cell line was infected with recombinant M. tuberculosis. The possibility exists that removal of repression could not be observed due to the fact that in recombinant mycobacteria carrying the reporter gene the repressor is harboured in cis in multicopy plasmids. However, an increase in mce3 expression by M. tuberculosis H37Rv(pP3) and M. tuberculosis H37Rv(p1963-P3) was observed in the later stages of J774 infection, suggesting that additional factors may affect mce3 expression.
At present, it is not known whether the Rv1963-encoded protein regulates genes other than mce3, but gel-shift experiments indicated that Mce3R binds to the mce2 promoter region, raising the possibility that mce2 may also be negatively regulated by the Rv1963-encoded protein. However, the binding of the recombinant protein Mce3R to the mce2 promoter seems to be weaker than binding of this protein to the mce3 promoter, suggesting that fine-tuning of regulation of the mce operons may occur. Interestingly, a moderately homologous region of about 100 bp was found in the promoter-containing regions of mce2 and mce3 (data not shown).
The present study reveals interesting features of the control of expression of the mce operons of M. tuberculosis. It also poses a number of questions that need to be answered, such as (i) does the Rv1963-encoded regulatory protein have the same effect on mce promoters other than mce3, and (ii) which extracellular signals are required for mce expression? At present, it is not known whether the mce1 and mce4 promoter regions are also the target of the Rv1963-encoded trans-acting regulatory factor, but the high homology between the mce operons supports the idea of a broader regulatory mechanism.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 April 2002;
revised 9 May 2002;
accepted 26 June 2002.