1 Department of Medical Microbiology, Barts and the London, Queen Mary's School of Medicine and Dentistry, Turner Street, London E1 2AD, UK
2 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
3 GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
4 Department of Pathology and Infectious Diseases, Royal Veterinary College, Royal College Street, London NW1 0TU, UK
Correspondence
Tanya Parish
t.parish{at}qmul.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mycobacterium tuberculosis is a sophisticated pathogen that can persist in the human host for many years. The bacteria are exposed to many different conditions during the infection and disease process and must have well-tuned mechanisms to respond to any given environment. For example, the bacteria can be found growing both intracellularly (in macrophages) or extracellularly (in the granuloma). Since the function of 2CRs is to adapt to different external conditions, they are likely to play an important role in the ability of M. tuberculosis to sense and respond to different host environments.
The current TB vaccine (Mycobacterium bovis BCG), a live attenuated strain, is not ideal due to its variable efficacy. Several approaches are being taken to try to improve the vaccine, of which one is to rationally attenuate M. tuberculosis. A genetic knock-out in one of the 2CRs may be a valuable component of such a vaccine strain, as many relevant genes may be affected at the same time. In addition, identifying genes that are regulated by these systems may lead to the identification of new virulence factors. This will increase our knowledge of pathogenesis and may allow for the development of novel interventions.
M. tuberculosis has 11 2CRs, as identified from the genome sequence (Cole et al., 1998), and several of these have been implicated in virulence. For example, PhoP and Prr mutants are attenuated (Ewann et al., 2002
; Perez et al., 2001
), whereas DevR, TcrXY, TrcS and KdpDE mutants are hypervirulent (Parish et al., 2003
), showing that these systems do play an important role during infection. The senX3regX3 system, originally identified by degenerate PCR, was the first reported example of an M. tuberculosis 2CR (Wren et al., 1992
). The two genes are separated by a small intergenic region which contains three repeats of a MIRU (mycobacterial interspersed repeat unit), although they are very likely to be co-transcribed. Phosphorylation of the regulator by the sensor has been demonstrated and there is some evidence that the system is auto-regulated with RegX3 binding to its own promoter (Himpens et al., 2000
). However, the genes that this system controls have not been identified. We have investigated the role of this system in virulence and used a mutant strain lacking a functional senX3regX3 system in order to characterize the regulon.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction and confirmation of the senX3 deletion strain.
The senX3regX3 deletion was constructed using previously published methods (Parish & Stoker, 2000). Plasmids used in this study are described in Table 1
. A deletion delivery construct (pSOUP25) was made using the pNIL and pGOAL series vectors (maps available on request) (Parish & Stoker, 2000
). The deletion constructed encompassed the 3' end of the senX3 gene, the intergenic region and the 5' end of the regX3 gene (Fig. 1
). Mutants were constructed using a two-step strategy as described previously. Briefly, 15 µg of vector DNA was pre-treated with UV to stimulate homologous recombination and used to electroporate M. tuberculosis. Single cross-over strains were selected on agar containing hygromycin, kanamycin and X-Gal. An individual colony was streaked out onto agar (without antibiotics) to allow the second cross-over to occur. Cells were resuspended in media and serial dilutions were plated onto X-Gal and sucrose. Sucrose-resistant, white colonies were tested for kanamycin sensitivity and analysed by PCR and Southern hybridization. PCR primers regP1 (5'-GGTAATTGTTTGAGATCCCAC-3') and regP3 (5'-GTCCGCTAGCCCTCGAGTTTG-3') were used to PCR-amplify the whole operon to distinguish strains carrying the wild-type (2·3 kb) from the deletion allele (1·4 kb) (Fig. 2
). The PCR product from the deletion strain was sequence-verified. Genomic DNA was prepared according to the method of Belisle & Sonnenberg (1998)
. Southern hybridization was carried out using the AlkPhos Direct kit (Amersham) according to the manufacturer's instructions in order to confirm the expected genotype (Fig. 2
).
|
|
|
Infection assays.
Viable stocks of wild-type M. tuberculosis and mutants were grown in 10 ml of liquid medium until an OD600 value of between 0·5 and 1·0 was reached. Bacteria were washed once in Dulbecco's PBS (Sigma) and resuspended in 5 ml sterile PBS. Aliquots were used fresh for the THP1 infections or stored at -70 °C until use. At the time of inoculation, serial dilutions were plated to determine the input c.f.u. value.
Macrophage infection assays
The THP1 macrophage-like human cell line and bone-marrow-derived macrophages (BMDMs) were used for in vitro assays. Macrophage viability over the assay time was typically greater than 95 %. Antibiotics were not added to the cells, since M. tuberculosis does not replicate in the medium in this assay.
THP1 infection.
THP1 cells were maintained in culture, treated with phorbol 12-myristate 13-acetate to induce differentiation, washed and then infected as described by Lukey (2001). Extracellular bacteria were removed by washing several times. Determination of the initial inoculum was assessed by plating serial dilutions, and the number of intracellular bacteria was monitored over 7 days.
BMDMs.
BMDMs from BALB/c mice were isolated and infected in the absence of antibiotics as described previously (Smith et al., 2001). Macrophage monolayers were pre-stimulated with IFN
(Gibco) at a concentration of 200 units ml-1 for 4 h prior to infection. Cells were infected for 4 h and washed six times in warm tissue culture medium to remove extracellular bacteria. The infection dose was assayed independently by plating the inoculum. The number of viable mycobacteria was assessed by lysis of the macrophage monolayer with 1 ml sterile distilled water containing 0·1 % Triton X-100 per well, followed by plating serial dilutions.
Infection of mice and tissue analysis.
Mice were infected with 1x106 viable mycobacteria in 200 µl pyrogen-free saline via a lateral tail vein. Where appropriate, infected mice were killed by cervical dislocation in accordance with humane end point protocols under the Animals Scientific Procedures Act, 1986 (UK). Median survival times were calculated for each group and statistical analysis was performed using KaplanMeier plots and Log Rank tests of survival. For tissue analysis, lungs, livers and spleens were collected aseptically and passed through a 100 micron pore-size sieve (Falcon) in 7H9 medium containing 0·05 % (w/v) Tween 80. Serial 10-fold dilutions were plated and c.f.u. were counted after 4 weeks. Statistical analysis was performed using Student's t-test.
Microarray analysis.
Wild-type and mutant M. tuberculosis were grown in 100 ml media in roller bottles to late-exponential phase (7 days). Cells were harvested by centrifugation and RNA was prepared according to the method of Movahedzadeh et al. (2001). Fluorescently labelled cDNA was prepared from total RNA by direct incorporation of fluorescent nucleotide analogues during a first-strand reverse transcription (RT) reaction as described previously (Betts et al., 2002
). Wild-type RNA was labelled with Cy3-dCTP and mutant RNA was labelled with Cy5-dCTP, and they were compared directly by competitive hybridization. DNA microarrays used consisted of 3649 PCR-amplified ORF-specific DNA fragments, representing 93 % of the predicted 3924 M. tuberculosis H37Rv ORFs, and hybridizations were performed as described previously (Betts et al., 2002
). Slides were scanned using a ScanArray 3000 instrument (GSI Lumonics) and the resulting images were analysed using GENEPIX PRO 3.0 software (Axon Instruments). RNA was isolated from three separate cultures and duplicate hybridizations were carried out for each, making a total of six hybridizations. Data from GENEPIX were analysed in GENESPRING (Silicon Genetics). Data points were excluded from the analysis if the spots were flagged as absent or marginal by GENEPIX. For each slide, the normalization was as follows: each gene's measured intensity (mutant) was divided by the control channel value (wild-type) in each sample to give the ratio of expression; when the control channel value was below 10·0 the datum point was considered bad; the 50th percentile of all measurements was used as a positive control for each sample; each measurement for each gene was divided by this synthetic positive control (assuming that this was at least 0·01); normalized values below 0 were set to 0. Genes were defined as being differentially regulated where there was a greater than twofold change in at least four of the hybridizations and where P<0·05 by Student's t-test. The t-test was conducted on the six experiments as a group. Data from published array experiments were imported into GENESPRING. Cluster analysis was then performed to group the genes depending upon their expression pattern using GENESPRING.
Promoter activity assays.
The promoter region of the senX3regX3 operon was amplified using primers regP1 and regP2 (5'-CAGCGCCGAGAACACAGTCAC-3') and cloned into pGEM-EasyT vector (Promega). The promoter region was subcloned as a blunt-ended fragment in the forward orientation into the ScaI site of the promoter-probe shuttle vector pSM128 to make plasmid pIKL-R1. The plasmid was electroporated into wild-type and Tame15 strains, and transformants were selected on streptomycin. Transformants were grown in 10 ml liquid medium to late-exponential phase before assaying for -galactosidase activity as described previously (Parish et al., 2001
). Three independent transformants were each assayed in duplicate.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Growth characteristics
We first looked at the growth characteristics of the mutant in axenic culture (Fig. 3). The mutant behaved erratically, in that on some occasions growth resembled the wild-type strain (Fig. 3a
) whilst on other occasions there seemed to be a clear defect (Fig. 3b
). Small differences in the inoculum with respect to growth phase, number of bacteria or other factors may be responsible for the inconsistent pattern of growth. Since M. tuberculosis is such a slow-growing organism, very small differences in the initial inoculum could be magnified during prolonged growth. The failure of the mutant to grow to the same optical density value as the wild-type was noted on numerous occasions but was not perfectly reproducible. Thus, we concluded that the mutant had a subtle growth defect which we could not quantify precisely.
|
|
|
|
The reason for the attenuation of Tame15 is not yet known. It is possible that the attenuation may result from a general decrease in the growth rate; however, this is true of many strains, e.g. auxotrophic strains are attenuated because their growth is severely restricted by amino acid availability (Smith et al., 2001). Nevertheless, Tame15 is an attenuated strain, whatever the root cause. The difference seen in the SCID mouse model was not due to small variations in inoculum size, since the experiment was conducted twice with similar results (inocula sizes 1x106 vs 2x106 and 4x106 vs 2x106, respectively).
Promoter activity
To look at promoter activity for the senX3regX3 system in the wild-type and mutant strain we cloned the upstream promoter region into the integrating vector pSM128 (Dussurget et al., 1999) (Fig. 7
). Using the
-galactosidase reporter gene, we were able to assess promoter activity under growth in aerobic cultures. Promoter activity was low in both strains, but unexpectedly the promoter activity was 2·8-fold increased in the mutant strain (wild-type, 2±0·4 units; Tame15, 5·6±0·7 units; P<0·00001).
|
Analysis of global gene expression
Two-component systems generally function as global regulators of gene expression in response to environmental conditions. Each system controls a set of genes, termed the regulon, in response to a particular signal. To identify potential members of the senX3regX3 regulon, we compared global gene expression in the mutant with that of the wild-type strain. The reporter assays confirmed that the system is expressed under aerobically grown conditions, so we would expect to see changes in expression of genes controlled by this 2CR under these conditions. Therefore, RNA was prepared from both strains grown in roller bottles (aerobic) and competitively hybridized to a whole-genome microarray. Genes showing a significant difference in expression in the mutant were identified and are given in Table 2. As expected from the deletion constructed, regX3 transcripts were significantly reduced; however, the senX3 transcript was unchanged (expression ratio not significantly different from 1). Thus, the change in senX3 promoter activity observed in the reporter gene was not reflected in the array data. This may be because of the low absolute activity of the promoter, so that the twofold increase was below the limit of detection of the microarray. We confirmed the absence of regX3 mRNA by quantitative RT-PCR and found that there was 100-fold less regX3 mRNA in the mutant strain (data not shown).
|
Differential gene expression
At this stage, it is not possible to determine which genes are directly controlled at the transcriptional level by RegX3 and which genes may be indirectly controlled, for example, via other regulators. However, we can say that all of the genes whose expression changes must rely on the senX3regX3 system in some way for normal expression. If RegX3 directly controls the transcription of these genes, then it must be a negative regulator of up-regulated genes, i.e. it represses the expression of such genes. In contrast, for those genes that are down-regulated in the mutant, RegX3 would act as a positive regulator (inducer). More genes were expressed at a lower level in the mutant than were de-repressed, indicating that a larger number of genes rely on the senX3regX3 system for normal expression. The possibility of regulatory cascades is raised by the array data since there are four potential transcriptional regulators whose expression changes, two going up in the mutant (Rv1990c and Rv2669) and two going down (Rv2488c and Rv2308). In addition, two other up-regulated genes show some similarity to anti-anti-sigma factors (Rv2638, Rv0516c). In yeast, several different types of networks of regulatory proteins have been identified (Lee et al., 2002) and it is likely that similar networks exist in bacteria.
Down-regulated genes
Most genes that were significantly down-regulated decreased by a factor of two- to threefold. More genes appear to be repressed than induced in the mutant and these fall into several categories. Many of the genes are involved in basic macromolecule biosynthesis, particularly DNA and RNA synthesis. We have previously seen that deletion of a 2CR can have an indirect effect on gene expression. With TrcS the cells appeared to be stressed and several genes were differentially expressed in response to the stress rather than as a direct result of the regulator deletion (Wernisch et al., 2003). The data for this strain look as if a similar stress response is occurring.
Several ribosomal proteins are down-regulated as are genes involved in DNA replication, repair and recombination (dnaB, ruvC, dnaQ, Rv3644c) as well as insertion elements. This would seem to indicate that the mutant would have a slower growth rate than the wild-type since it is less capable of synthesizing new DNA, RNA and protein. This is consistent with the growth phenotype observed earlier.
A number of genes involved in fatty acid degradation are repressed (fadE6, accD2, fadE14, fadE23) as are several probable oxidoreductases and dehydrogenases (Rv0183, Rv1714, Rv1812c). There are also a number of genes involved in cell-wall biosynthesis, including lipid biosynthesis (acpM, desA3, fbpC2) and the cell envelope (membrane and exported proteins; lprE, Rv0867c, Rv1433, Rv1457c). The alkyl-hydroperoxidases ahpC and ahpD are both down-regulated, whereas superoxide dismutase (sodA) is up-regulated, suggesting that there is a change in the type of oxidative stress the cells are facing internally, rather than a general increase in stress. Several members of the PE/PPE family are down-regulated as are a large number of conserved hypothetical and unknown proteins.
Up-regulated genes
Most genes that were significantly up-regulated increased by a factor of two- to threefold. However, the expression of one operon (Rv0096 to Rv0101) was highly elevated. The level of induction in the mutant was five- to 17-fold over that in the wild-type (Fig. 8). Since the Rv0096 operon was so highly induced, it seems likely that this operon is controlled directly by the RegX3 regulator rather than via an indirect effect, due to, for example, slowed growth rate or increased intracellular stress levels. The operon looks to consist of several genes comprising at least Rv0096 to Rv0100 and possibly Rv0101 and Rv0102 as well (Fig. 8
). Four of these genes are significantly up-regulated in the mutant. Rv0096 encodes a member of the PE/PPE family whose function is unclear, although these proteins have been proposed to be involved in antigenic variation (Banu et al., 2002
), and other members of this family have been shown to be up-regulated in the frog model of mycobacterial-induced granuloma formation (Ramakrishnan et al., 2000
), suggesting a role in pathogenicity. Rv0097 encodes a possible oxidoreductase; Rv0098 and Rv0099 both encode conserved hypothetical proteins of unknown function. FadD10 is one of a large number of fatty acid CoA-ligases proposed to be involved in lipid metabolism. The nrp gene encodes a non-ribosomal peptide synthase whose biological role is yet to be firmly elucidated, but it has been proposed to be involved in lipid metabolism due to its location in this operon (Cole et al., 1998
).
|
Of the other genes that show increased expression, citrate synthase 3 is involved in energy metabolism (tricarboxylic acid cycle) at an important control point of the cycle, Rv0103c encodes a probable copper cation transporter, three others are insertion sequence or phage elements (Rv2424c, Rv2647, Rv3750c), one is a bacteriocin-like protein (Rv3660c) and the remainder encode conserved hypotheticals or unknowns.
Taken together, these differences seem to suggest a subtle change in the normal metabolism (and growth) of the mutant bacteria and also a change in the type of intracellular stress that the bacteria are facing. A secondary effect of the mutation may be to reduce the growth rate, but it is not clear why.
Comparison of expression data
Several genes that were down-regulated appeared to be involved in normal growth; for example, several ribosomal proteins and dnaB. Several genes identified as stress proteins were also differentially expressed, either down-regulated (grpE, ahpC and ahpD) or up-regulated (sodA). This fits in with our previous observation that the mutant had a slight growth defect and indicated that the deletion of the system is probably causing some type of stress within the cells. It is possible that these genes are not directly controlled by RegX3 itself, but are down-regulated as a secondary effect of the mutation. To refine the list of potential regulon members, we conducted a meta-analysis of previously published data to determine if there were any significant patterns of expression relating to stress.
By comparing genes that were differentially expressed under various stress conditions, we looked for patterns of expression which would indicate that certain groups of genes are being co-ordinately regulated in response to any type of stress. We looked at their expression patterns in other published array data representing several different types of stress: heat shock (Stewart et al., 2002), carbon limitation (Betts et al., 2002
), SDS treatment (Manganelli et al., 2001
), diamide treatment (Manganelli et al., 2002
), low oxygen tension (Sherman et al., 2001
) and low iron (Rodriguez et al., 2002
). Cluster analysis was used to group genes with similar patterns of expression. Genes which showed the same pattern of expression in more than one stress condition were in the same cluster and we considered them to be part of a general stress response. This made them unlikely to be directly controlled by RegX3, so we excluded them from our list. The remaining genes fell into four clusters.
Groups 1 and 2 were the up-regulated genes and groups 3 and 4 were the down-regulated genes. Two groups represented genes that only changed in the senX3regX3 mutant (1 and 3). The only condition in which senX3 or regX3 had been seen to change significantly was carbon starvation, where senX3 was down-regulated by 2·2-fold, and one group (2) contained the genes that went up in both conditions. Group 4 contained those genes that were down-regulated in the mutant, but up-regulated under the other stress conditions (indicating that the change in expression was not due to stress alone). Of the 98 genes originally identified, we narrowed our list down to 50 (Table 3).
|
Motif analysis
We further analysed the genes from Table 3 to see if there were any common motifs present in the regions immediately upstream of the genes that might be good candidates for a DNA-binding region for RegX3. We looked at a subset of those genes which showed the greatest-fold difference in expression. Sequences were analysed for the presence of tandem and inverted repeats and multiple alignments were carried out, but no significant patterns emerged. The sequences did not show any significant similarities with the previously identified binding site in the RegX3 promoter region (Himpens et al., 2000
). It may be that there are different DNA-binding recognition sites for RegX3 in alternative conformational states. Alternatively, it may be that these genes are controlled by an indirect effect and we would not expect to see RegX3 binding in that case.
Identification of the stimulus
To identify the stimulus to which the senX3regX3 system responds, we tried two approaches. First, we assayed the ability of the mutant to survive different in vitro conditions and stresses. We looked at viability during exposure to extremes of pH (2 and 12), ability to survive extended stationary phase in standing culture and ability to withstand complete nutrient starvation. The mutant showed no significant difference compared to the wild-type strain (data not shown). To survey a larger number of conditions, we then looked at promoter activity from pIKL-R1 under a variety of conditions to determine if it was induced. Conditions tested included several antibiotics (kanamycin, tetracycline, gentamicin, isoniazid, ampicillin, rifampicin), lysozyme, HCl, NaOH, DMSO, SDS and H2O2, but no conditions were found to up-regulate promoter activity. Thus, the stimulus for this system stills remains unknown.
Conclusion
We have constructed a strain with a deletion of the senX3regX3 2CR. We used several different models to determine if the deletion of the senX3regX3 system had any effect on the virulence of the bacterium. The mutant showed significant attenuation in both activated and resting macrophages and in immunocompromised and immunocompetent mice. Thus, it seems that the mutant is less able to cause disease regardless of the involvement of the immune system. This attenuation was not as large as that seen previously for other mutants (e.g. the complete attenuation of the trpD mutant; Smith et al., 2001), but it was a significant reduction. We used whole-genome microarrays to identify genes that are differentially expressed in the mutant, and based on these data we have identified 50 potential members of the senX3regX3 regulon. The construction of further mutants in these genes should lead to the identification of the genes whose roles are required during infection.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barrett, J. F. & Hoch, J. A. (1998). Two-component signal transduction as a target for microbial anti-infective therapy. Antimicrob Agents Chemother 42, 15291536.
Belisle, J. T. & Sonnenberg, M. G. (1998). Isolation of genomic DNA from mycobacteria. In Mycobacteria Protocols, pp. 3144. Edited by T. Parish & N. G. Stoker. Totowa, NJ: Humana Press.
Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. (2002). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43, 717731.[CrossRef][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]
Dussurget, O., Timm, J., Gomez, M., Gold, B., Yu, S. W., Sabol, S. Z., Holmes, R. K., Jacobs, W. R. & Smith, I. (1999). Transcriptional control of the iron-responsive fxbA gene by the mycobacterial regulator IdeR. J Bacteriol 181, 34023408.
Dziejman, M. & Mekalanos, J. J. (1995). Two-component signal transduction and its role in the expression of bacterial virulence factors. In Two-component Signal Transduction, pp. 305317. Edited by J. A. Hoch & T. J. Silhavy. Washington, DC: American Society for Microbiology.
Ewann, F., Jackson, M., Pethe, K., Cooper, A., Mielcarek, N., Ensergueix, D., Gicquel, B., Locht, C. & Supply, P. (2002). Transient requirement of the PrrAPrrB two-component system for early intracellular multiplication of Mycobacterium tuberculosis. Infect Immun 70, 22562263.
Feng, Z. Y., Caceres, N. E., Sarath, G. & Barletta, R. G. (2002). Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth. J Bacteriol 184, 50015010.
Garcia Vescovi, E., Soncini, F. C. & Groisman, E. A. (1996). Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence. Cell 84, 165174.[Medline]
Groisman, E. A. & Heffron, F. (1995). Regulation of Salmonella virulence by two-component regulatory systems. In Two-component Signal Transduction, pp. 319332. Edited by J. A. Hoch & T. J. Silhavy. Washington, DC: American Society for Microbiology.
Himpens, S., Locht, C. & Supply, P. (2000). Molecular characterization of the mycobacterial SenX3RegX3 two-component system: evidence for autoregulation. Microbiology 146, 30913098.
Hoch, J. A. (2000). Two-component and phosphorelay signal transduction. Curr Opin Microbiol 3, 165170.[CrossRef][Medline]
Lee, T. I., Rinaldi, N. J., Robert, F. & 18 other authors (2002). Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799804.
Lukey, P. T. (2001). Macrophage virulence assays. In Mycobacterium tuberculosis Protocols, pp. 271280. Edited by T. Parish & N. G. Stoker. Totowa, NJ: Humana Press.
Manganelli, R., Voskuil, M. I., Schoolnik, G. K. & Smith, I. (2001). The Mycobacterium tuberculosis ECF sigma factor sigma(E): role in global gene expression and survival in macrophages. Mol Microbiol 41, 423437.[CrossRef][Medline]
Manganelli, R., Voskuil, M. I., Schoolnik, G. K., Dubnau, E., Gomez, M. & Smith, I. (2002). Role of the extracytoplasmic-function sigma factor sigma(H) in Mycobacterium tuberculosis global gene expression. Mol Microbiol 45, 365374.[CrossRef][Medline]
Movahedzadeh, F., Gonzalez-Y-Merchand, J. A. & Cox, R. A. (2001). Transcription start site mapping. In Mycobacterium tuberculosis Protocols, pp. 105124. Edited by T. Parish & N. G. Stoker. Totowa, NJ: Humana Press.
Parish, T. & Stoker, N. G. (2000). Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146, 19691975.
Parish, T., Turner, J. & Stoker, N. G. (2001). amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis. BMC Microbiol 1, 19.[CrossRef][Medline]
Parish, T., Smith, D. A., Kendall, S., Casali, N., Bancroft, G. J. & Stoker, N. G. (2003). Deletion of two-component regulatory systems increases virulence of Mycobacterium tuberculosis. Infect Immun 71, 11341140.
Perez, E., Samper, S., Bordas, Y., Guilhot, C., Gicquel, B. & Martin, C. (2001). An essential role for phoP in Mycobacterium tuberculosis virulence. Mol Microbiol 41, 179187.[CrossRef][Medline]
Ramakrishnan, L., Federspiel, N. A. & Falkow, S. (2000). Granuloma-specific expression of Mycobacterium virulence proteins from the glycine-rich PE-PGRS family. Science 288, 14361439.
Rodriguez, G. M., Voskuil, M. I., Gold, B., Schoolnik, G. K. & Smith, I. (2002). ideR, an essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect Immun 70, 33713381.
Sherman, D. R., Voskuil, M., Schnappinger, D., Liao, R. L., Harrell, M. I. & Schoolnik, G. K. (2001). Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha-crystallin. Proc Natl Acad Sci U S A 98, 75347539.
Smith, D. A., Parish, T., Stoker, N. G. & Bancroft, G. J. (2001). Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates. Infect Immun 69, 11421150.
Stewart, G. R., Wernisch, L., Stabler, R., Mangan, J. A., Hinds, J., Laing, K. G., Young, D. B. & Butcher, P. D. (2002). Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148, 31293138.
Urao, T., Yamaguchi-Shinozaki, K. & Shinozaki, K. (2000). Two-component systems in plant signal transduction. Trends Plant Sci 5, 6774.[CrossRef][Medline]
Wayne, L. G. & Sohaskey, C. D. (2001). Nonreplicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 55, 139163.[CrossRef][Medline]
Wernisch, L., Soneji, S., Wietzorrek, A., Parish, T., Hinds, J., Butcher, P. D. & Stoker, N. G. (2003). Analysis of whole-genome microarray replicates using mixed models. Bioinformatics 19, 5361.
Wren, B. W., Colby, S. M., Cubberley, R. R. & Pallen, M. J. (1992). Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria. FEMS Microbiol Lett 99, 287292.
Received 17 January 2003;
revised 6 February 2003;
accepted 20 February 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |