Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India
Correspondence
Jaya S. Tyagi
jstyagi{at}aiims.ac.in
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ABSTRACT |
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These authors contributed equally to the work.
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INTRODUCTION |
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DevR mediates the genetic response of M. tuberculosis to oxygen limitation (Park et al., 2003) and is implicated in the bacterium's response to nitric oxide exposure (Voskuil et al., 2003
), two conditions encountered by bacteria in vivo and thought to be associated with latent tuberculosis (Nathan & Shiloh, 2000
; Wayne & Sohaskey, 2001
). Analysis of the transcriptional regulation of devR-devS is required to define the function of these genes in establishing and maintaining tuberculosis latency in human beings. To begin to study the regulation of this system, we characterized the transcription of the Rv3134c-devR-devS locus under aerobic conditions. We show here that there are two regions with promoter activity in this operon that map upstream of Rv3134c and devR, respectively, and transcription occurs from upstream of both genes to produce transcripts under basal conditions. Sequence analysis indicates the presence of sequences with similarity to consensus sites for sigma factors and DevR-binding sites in this region. DevR protein bound specifically to the promoter regions of this operon and regulated devR and Rv3134c promoter activity in Mycobacterium smegmatis and Escherichia coli. Our findings suggest the potential for complex regulation of the Rv3134c-devR-devS operon under inducing conditions.
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METHODS |
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Primer extension.
Primer extension was carried out using an appropriate gene-specific reverse primer and 10 µg RNA from exponential-phase cultures of M. tuberculosis H37Rv. Briefly, the 32P end-labelled primer was annealed to RNA at 60 or 65 °C for 45 min and cDNA was synthesized at 37 °C for 60 min using 50 units Stratascript Reverse Transcriptase as described by the manufacturer. cDNA was recovered by ethanol precipitation, resuspended in 4 µl of 40 µg RNase ml1 and incubated at room temperature for 3 min; the reaction was stopped by adding formamide gel loading buffer.
The sequencing reaction was performed with plasmid pDP2, which contained 1·45 kb of devR upstream region cloned into pDK16 (Bagchi et al., 2003), as template and oligonucleotide R3 or R9c primers and the fmolR DNA sequencing system (Promega) to map the transcription start points (Tsps) upstream of Rv3134c and devR. To map the Tsp upstream of devS, the sequencing ladder was obtained using plasmid pJT53.34 (Kinger & Tyagi, 1993
) and oligonucleotide Srev3. The sequences of the primers used for primer extension are listed in Table 2
and their positions are indicated in Fig. 1
(a). The sequencing reactions were set up according to the manufacturer's guidelines and were analysed on a 6 % denaturing polyacrylamide gel along with the primer extension products. Results are representative of at least triplicate experiments with two to four individual RNA preparations.
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DevR autoregulation was also assessed in E. coli DH5 using a two-plasmid system. For this, DNA fragments corresponding to the Rv3134c and devR upstream regions (the same as those used for M. smegmatis experiments) were cloned upstream of the promoterless lacZ gene in pDK16 (A. K. Tyagi & D. K. Kaushal, unpublished) to generate pSC5 and pSC6, respectively (Table 2
, Fig. 1a
). Plasmid pDK16 contains a multiple cloning site upstream of lacZ, a kanamycin-resistance cassette, the L5 att-int region for integration into the mycobacterial genome and a ColE1 origin for replication in E. coli. Plasmids pSC5 and pSC6 were transformed along with the DevR expression construct pDSR217P (see below) into E. coli and selected on LB agar plates containing kanamycin and ampicillin. The cultures were grown overnight with shaking at 37 °C, diluted 100-fold into fresh LB medium, grown to an OD595 of 0·60·7 and induced with 0·1 mM IPTG for 3 h.
-Galactosidase assay was performed as described by Miller (1972)
. The mean activity of three independent experiments is reported.
Construction of plasmids expressing DevR.
devR was amplified from M. tuberculosis DNA using devRBamf and devRBamr primers (Table 2, BamHI sites are underlined), digested with BamHI and cloned into the BamHI site of pGEX-4T1 (Amersham), generating pSCDevR. Plasmids pSC2 and pSC4, which expressed M. tuberculosis DevR at a physiological level in M. smegmatis, were constructed by digesting pSC1 and pSC3 at their unique NheI site and inserting the devR gene along with 327 bp of upstream sequences derived from pDSdevR (Malhotra et al., 2004
).
For E. coli lacZ reporter assays, DevR was overexpressed from pDSR217P, generated from pDSR217 (Saini et al., 2004) by replacing the pMB1 origin of replication in the latter with a p15A origin of replication derived from pGP1-2 (Tabor & Richardson, 1985
). In this way pDSR217P was compatible with pDK16 in E. coli cells carrying both plasmids.
DevR purification.
DevR was overexpressed as an N-terminally GST-tagged 49·2 kDa fusion protein from pSCDevR in E. coli BL23(DE3). Briefly, 600 ml of culture was grown at 37 °C to an OD595 of 1·0 and induced at 25 °C for 5 h with 0·1 mM IPTG. The induced cells were harvested by centrifugation, resuspended in phosphate-buffered saline (pH 7·4) containing protease inhibitor cocktail (Roche) and sonicated (duty cycle 70, three pulses of 2 min each, Branson Sonifier 450, USA). Briefly, DevR was purified by affinity chromatography using 2 ml GST.Bind.Resin (Novagen) previously equilibrated with wash/bind buffer (43 mM Na2HPO4, 14·7 mM KH2PO4, 1·37 M NaCl and 27 mM KCl pH 7·3). The lysate was mixed thoroughly with the resin and incubated at room temperature for 30 min. The flowthrough was collected and then the column was washed with 3035 bed volumes of the same buffer. Bound DevR was eluted from the column with GST elution buffer (10 mM reduced glutathione dissolved in 50 mM Tris/HCl pH 8·0). Protein-containing fractions were pooled, dialysed against buffer containing 50 mM Tris/HCl pH 8·0, 50 mM NaCl, 50 % (v/v) glycerol and 0·1 mM DTT and stored at 20 °C. The concentration of the dialysed protein was determined by the bicinchoninic acid method (Pierce) using BSA as standard. Purified GST-DevR (henceforth referred to as DevR) was used for the EMSA experiments (see below).
Electrophoretic mobility shift assays (EMSAs).
Purified recombinant DevR was used to assess protein binding to two promoter fragments derived from the Rv3134c-devR-devS operon. The radiolabelled promoter DNA fragments for these assays were generated by PCR from M. tuberculosis DNA using primers that were end-labelled with [-32P]ATP (specific activity
30005000 Ci mmol1,
110185 TBq mmol1; BRIT, India) using T4 polynucleotide kinase. PCR products were purified from the gel before use in EMSAs. The primers and probes that were used are listed in Table 2
. In standard reactions, DevR protein was incubated with binding buffer containing 25 mM Tris/HCl (pH 8·0), 0·5 mM EDTA, 20 mM KCl, 6 mM MgCl2, 5 % glycerol, 0·1 mM DTT and 1 µg poly (dI-dC) for 20 min on ice. Three nanograms of 32P-labelled DNA was added in a final reaction volume of 20 µl and incubated on ice for another 30 min. DNAprotein complexes were separated on a 5 % non-denaturing polyacrylamide gel by electrophoresis in 0·25x TBE at 150 V at 48 °C and the DNAprotein complexes were visualized by autoradiography.
M. smegmatis protein extract preparation and immunoblot analysis.
Lysates were prepared from exponential-phase and stationary-phase cultures of recombinant M. smegmatis carrying pFPV27 vector, pSC1 and pSC2 as described by Saini et al. (2004). Lysates containing 5 µg protein were electrophoresed on 12·5 % SDS-PAGE gels and subjected to immunoblotting using 1 : 4000 dilution of high-titre polyclonal anti-DevR sera as described by Saini et al. (2004)
.
Measurement of GFP fluorescence.
M. smegmatis cultures carrying the various recombinant gfp constructs (pSC series) and pFPV27 vector were grown in DTA containing kanamycin (starting OD595 0·01) under aerobic conditions at 37 °C until they attained stationary phase (6070 h). Samples were taken from mid-exponential and stationary-phase cultures and GFP fluorescence was assessed in a spectrofluorimeter using an excitation wavelength of 483 nm and an emission wavelength of 515 nm (Molecular Devices).
The inserts of all reporter constructs and the DevR expression plasmid were verified for accuracy by DNA sequencing.
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RESULTS |
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The Rv3134c-devR-devS operon is transcribed from several promoters
Two primer extension products were obtained using primer R9c and M. tuberculosis RNA from mid-exponential-phase cells grown under aerobic conditions. These included a prominent product, T1Rv3134c, at 240 and a minor Tsp, T2Rv3134c, at 286 (Fig. 2a, b). Since multiple promoters are common in M. tuberculosis, and devR upstream sequences supported DevR expression in devR mutant strains complemented with DevR (Malhotra et al., 2004
; Parish et al., 2003
), we searched for additional Tsps upstream of devR using primer R3. Two primer extension products, T2devR and T1devR, were obtained upstream of devR at 273 and 120/119 (Fig. 2c, d
). Product T1devR corresponded to a doublet. We also searched for Tsps upstream of devS using primer Srev3 and no signal was detected (data not shown). Annealing of primers to RNA at 65 °C gave similar primer extension products. These data show that the Rv3134c-devR-devS operon is transcribed from Tsps located upstream of both Rv3134c and devR and that the major transcript originated upstream of Rv3134c. The location of various Tsps in relation to the coding regions is indicated in Fig. 1(a)
. The four primer extension products are likely to represent individual Tsps driven by their independent promoters. It cannot however be ruled out that the proximal Tsps, T1devR and T1Rv3134c, are produced by processing from the distal Tsps, T2devR and T2Rv3134c, respectively. In the absence of specific data on transcript processing, we consider each of the Tsps to be independently generated.
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Six copies of the DevR consensus motif 5'-TTSGGGACTWWAGTCCCSAA-3' (Park et al., 2003) or 5'-WRGGGACNTTNGNCCCYN-3' (Florczyk et al., 2003
) were also identified in the region encompassing the devR upstream region, Rv3134c coding sequences and Rv3134c upstream region (see Fig. 1
) using MyPatternFinder, suggesting that DevR could bind at multiple sites in this region. Motifs 2 and 3 displayed >80 % identity with that described by Florczyk et al. (2003)
. To detect motifs similar to that proposed by Park et al. (2003)
, scores were calculated as described and motifs with scores >4 are indicated in Fig. 1(c)
. These putative binding sites varied in their location with respect to the Tsps or sigma factor consensus elements; they were arranged upstream as well as downstream of Tsps or in translated and untranslated regions (Fig. 1c
).
DevR autoregulation
For functional characterization of the Rv3134c-devR-devS regulatory region, we constructed plasmids pSC1 and pSC3 (Fig. 1a) containing transcriptional fusions between gfp and DNA sequences. Each plasmid carried two Tsps (and two promoters) and putative DevR-binding sequences. The plasmids were electroporated into M. smegmatis and the levels of gfp expression were compared over a 6070 h period that encompassed the exponential and stationary phases of growth. M. smegmatis has been used frequently to study the activity of M. tuberculosis promoters (Himpens et al., 2000
; Sala et al., 2003
; Verma et al., 1999
) because the transcriptional machinery is well conserved between the two organisms (Bashyam et al., 1996
). Further, the M. tuberculosis Rv3134c-devR promoter region was shown earlier to be active in M. smegmatis (Bagchi et al., 2003
). The data indicated that the Rv3134c promoters fragment (Rv3134cpro) carried the major promoters, which were notably stronger than the devR promoters (devRpro). Rv3134cpro remained active throughout and GFP fluorescence increased as the cells advanced into the stationary phase. The increase with time in GFP fluorescence from the Rv3134c promoters indicates that they are induced during stationary phase. The high activities of the Rv3134c promoters correlated with the strength of the Tsp signals detected upstream of Rv3134c by primer extension (Table 3
, Fig. 2
). The precise contribution of each Tsp to the total transcript pool awaits further assessment of the individual promoters.
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DevR binds to DNA fragments containing DevR boxes in the Rv3134c-devR-devS operon
DevR was shown to bind to a conserved DNA motif located upstream of the acr gene of M. tuberculosis (Park et al., 2003). In view of the modulation of promoter activity by DevR in gfp reporter assays and the detection of putative DevR-binding motifs by computer analysis, we evaluated DevR binding to sequences of the Rv3134c-devR-devS operon. Two DNA fragments containing the Rv3134c and devR promoters were analysed. The Rv3134cpro fragment was generated by PCR using primers R7 and R9c and it contained T1Rv3134c and T2Rv3134c Tsps and four putative DevR-binding sites (Fig. 1a
). Purified DevR bound to radiolabelled Rv3134cpro promoter probe DNA and retarded its mobility. The extent of binding and DNA retardation increased with the protein concentration used, and nearly complete DNA binding was observed with 25 µM protein (Fig. 4
a). This suggests that additional DevR was bound, perhaps to the multiple DevR-binding sites present in the fragment, when increasing amounts were added in the reaction. The devRpro promoter probe, which encompassed the two devR upstream Tsps and one potential DevR-binding site, was also analysed in EMSAs. Compared to the Rv3134cpro probe, DevR retarded very low amounts of the devRpro fragment at equivalent protein concentrations (Fig. 5
a). Binding to both the promoter probes could be specifically competed with a 100-fold excess of the corresponding unlabelled DNA. In assays containing a 100-fold excess of poly(dI-dC), specific binding to both the promoter fragments was retained (Figs 4b and 5b
). Purified GST protein failed to bind DNA (not shown), indicating that DevR in the recombinant fusion protein bound to the promoter probes in a sequence-specific manner. Also, DevR failed to bind to other fragments from this operon that mapped inside the devR gene and lacked putative DevR-binding sites (not shown). The differential affinities to DevR of the two promoter fragments were confirmed through reciprocal competition experiments where DevR was incubated (a) with labelled Rv3134cpro in the presence of an excess of unlabelled devRpro, or (b) labelled devRpro in the presence of an excess of unlabelled Rv3134cpro. In reaction (a), binding to Rv3134cpro was competed by 20-fold excess of devRpro DNA while in the latter reaction,
5-fold excess of unlabelled Rv3134cpro DNA was sufficient to compete for binding with DevR (not shown).
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DISCUSSION |
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Many two-component systems are transcribed from more than one promoter: for example phoPQ in Salmonella typhimurium (Soncini et al., 1995), bvgAS of Bordetella pertussis (Scarlato et al., 1990
) and phoPR in Bacillus subtilis (Paul et al., 2004
). This strategy allows for differential usage and regulation of promoters under diverse environmental conditions to facilitate bacterial adaptation. The potential for regulation of the Rv3134c-devR-devS operon by more than one sigma factor was suggested since the Tsps were preceded by matches, albeit inexact, to the consensus elements for SigA and SigC. Intriguingly, an exact copy of the consensus motif proposed for the primary housekeeping sigma factor SigA (Manganelli et al., 2004
) was not found in the entire M. tuberculosis genome (D. Sharma and others, unpublished data). This suggests that there exists considerable flexibility in promoter recognition and a search for promoter sequences must necessarily accommodate mismatches in sequence or spacing of the bipartite elements.
The requirement for DevR during induction of Rv3134c-devR-devS indicates autoregulation of the operon during hypoxia (Park et al., 2003). In the present study, it was noted that the Rv3134c and devR promoters exhibited striking differences in their activities and their modulation by DevR under aerobic conditions. DevR activated the weak devR promoters and repressed the stronger Rv3134c promoters. The repressing effect of DevR on Rv3134c promoters was observed in both M. smegmatis and E. coli. Interestingly, 35 and 10 motifs having 4/6 identity with the E. coli
70 consensus sequences exactly overlapped the putative M. tuberculosis SigA consensus element predicted upstream of Tsp T2Rv3134c (Fig. 1b
). The differential regulation of devR and Rv3134c promoters could be attributed to differences in their affinity for DevR. The presence of four potential protein-binding sites in the Rv3134cpro promoter fragment could explain the stronger binding of DevR with the Rv3134cpro in comparison to the devRpro fragment, which contained only one putative binding element. When the two promoters were assessed separately, the Rv3134c promoters were repressed while the devR promoters were activated in the presence of DevR. The overall effect of DevR in the context of the intact operon awaits evaluation in M. tuberculosis. Further experiments in M. tuberculosis under inducing conditions (hypoxia or nitric oxide exposure, for example) will help delineate the role of DevS sensor kinase and DevR itself in regulating the activity of this operon.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bashyam, M. D., Kaushal, D., Dasgupta, S. K. & Tyagi, A. K. (1996). A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements. J Bacteriol 178, 48474853.
Calamita, H., Ko, C., Tyagi, S., Yoshimatsu, T., Morrison, N. E. & Bishai, W. R. (2005). The Mycobacterium tuberculosis SigD sigma factor controls the expression of ribosome-associated gene products in stationary phase and is required for full virulence. Cell Microbiol 7, 233244.[Medline]
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[CrossRef][Medline]
Das Gupta, S. K., Bashyam, M. D. & Tyagi, A. K. (1993). Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector. J Bacteriol 175, 51865192.[Abstract]
Dasgupta, N. & Tyagi, J. S. (1998). Identification of a restriction fragment length polymorphism associated with a deletion that maps in a transcriptionally active open-reading frame, orfX, in Mycobacterium tuberculosis Erdman. Tuber Lung Dis 79, 7581.[CrossRef][Medline]
Dasgupta, N., Kapur, V., Singh, K. K., Das, T. K., Sachdeva, S., Jyothisri, K. & Tyagi, J. S. (2000). Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis. Tuber Lung Dis 80, 141159.[CrossRef][Medline]
Ewann, F., Locht, C. & Supply, P. (2004). Intracellular autoregulation of the Mycobacterium tuberculosis PrrA response regulator. Microbiology 150, 241246.[CrossRef][Medline]
Florczyk, M. A., McCue, L. A., Purkayastha, A., Currenti, E., Wolin, M. J. & McDonough, K. A. (2003). A family of acr-coregulated Mycobacterium tuberculosis genes shares a common DNA motif and requires Rv3133c (dosR or devR) for expression. Infect Immun 71, 53325343.
Haydel, S. E., Benjamin, W. H., Jr, Dunlap, N. E. & Clark-Curtiss, J. E. (2002). Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator. J Bacteriol 184, 21922203.
He, H. & Zahrt, T. C. (2005). Identification and characterization of a regulatory sequence recognized by Mycobacterium tuberculosis persistence regulator MprA. J Bacteriol 187, 202212.
Himpens, S., Locht, C. & Supply, P. (2000). Molecular characterization of the mycobacterial SenX3-RegX3 two-component system: evidence for autoregulation. Microbiology 146, 30913098.[Medline]
Hu, Y. & Coates, A. R. (2001). Increased levels of sigJ mRNA in late stationary phase cultures of Mycobacterium tuberculosis detected by DNA array hybridisation. FEMS Microbiol Lett 202, 5965.[CrossRef][Medline]
Kinger, A. K. & Tyagi, J. S. (1993). Identification and cloning of genes differentially expressed in the virulent strain of Mycobacterium tuberculosis. Gene 131, 113117.[CrossRef][Medline]
Lewin, B. (1997). Transcription. In Genes VI, pp. 287334. New York: Oxford University Press.
Malhotra, V., Sharma, D., Ramanathan, V. D. & 8 other authors (2004). Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis. FEMS Microbiol Lett 231, 237245.[CrossRef][Medline]
Manganelli, R., Dubnau, E., Tyagi, S., Kramer, F. R. & Smith, I. (1999). Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis. Mol Microbiol 31, 715724.[CrossRef][Medline]
Manganelli, R., Provvedi, R., Rodrigue, S., Beaucher, J., Gaudreau, L. & Smith, I. (2004). factors and global gene regulation in Mycobacterium tuberculosis. J Bacteriol 186, 895902.
Miller, J. H. (1972). Experiments in Molecular Genetics, pp. 352355. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Nathan, C. & Shiloh, M. U. (2000). Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A 97, 88418848.
Nystrom, T. & Neidhardt, F. C. (1992). Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Escherichia coli. Mol Microbiol 6, 31873198.[Medline]
Nystrom, T. & Neidhardt, F. C. (1994). Expression and role of the universal stress protein, UspA, of Escherichia coli during growth arrest. Mol Microbiol 11, 537544.[Medline]
Parish, T., Smith, D. A., Kendall, S., Casali, N., Bancroft, G. J. & Stoker, N. G. (2003). Deletion of two-component regulatory systems increases the virulence of Mycobacterium tuberculosis. Infect Immun 71, 11341140.
Park, H. D., Guinn, K. M., Harrell, M. I., Liao, R., Voskuil, M. I., Tompa, M., Schoolnik, G. K. & Sherman, D. R. (2003). Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 48, 833843.[CrossRef][Medline]
Paul, S., Birkey, S., Liu, W. & Hulett, F. M. (2004). Autoinduction of Bacillus subtilis phoPR operon transcription results from enhanced transcription from EA- and E
E-responsive promoters by phosphorylated PhoP. J Bacteriol 186, 42624275.
Raman, S., Hazra, R., Dascher, C. C. & Husson, R. N. (2004). Transcription regulation by the Mycobacterium tuberculosis alternative sigma factor SigD and its role in virulence. J Bacteriol 186, 66056616.
Roberts, D. M., Liao, R. P., Wisedchaisri, G., Hol, W. G. & Sherman, D. R. (2004). Two sensor kinases contribute to the hypoxic response of Mycobacterium tuberculosis. J Biol Chem 279, 2308223087.
Saini, D. K., Malhotra, V., Dey, D., Pant, N., Das, T. K. & Tyagi, J. S. (2004). DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR. Microbiology 150, 865875.[CrossRef][Medline]
Sala, C., Forti, F., Di Florio, E., Canneva, F., Milano, A., Riccardi, G. & Ghisotti, D. (2003). Mycobacterium tuberculosis FurA autoregulates its own expression. J Bacteriol 185, 53575362.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scarlato, V., Prugnola, A., Arico, B. & Rappuoli, R. (1990). Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis. Proc Natl Acad Sci U S A 87, 67536757.
Soncini, F. C., Vescovi, E. G. & Groisman, E. A. (1995). Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon. J Bacteriol 177, 43644371.
Tabor, S. & Richardson, C. C. (1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A 82, 10741078.
Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L. & Falkow, S. (1996). Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. Gene 173, 4752.[CrossRef][Medline]
Verma, A., Sampla, A. K. & Tyagi, J. S. (1999). Mycobacterium tuberculosis rrn promoters: differential usage and growth rate-dependent control. J Bacteriol 181, 43264333.
Voskuil, M. I., Schnappinger, D., Visconti, K. C., Harrell, M. I., Dolganov, G. M., Sherman, D. R. & Schoolnik, G. K. (2003). Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198, 705713.
Wayne, L. G. & Sohaskey, C. D. (2001). Nonreplicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 55, 139163.[CrossRef][Medline]
Received 8 July 2005;
revised 24 August 2005;
accepted 21 September 2005.
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