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Journal of Bacteriology, November 2008, p. 7068-7078, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00712-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Mycobacterium tuberculosis phoPR Operon Is Positively Autoregulated in the Virulent Strain H37Rv ^,

Jesús Gonzalo-Asensio,^1,6 Carlos Y. Soto,² Ainhoa Arbués,^1,6 Javier Sancho,³ María del Carmen Menéndez,⁴ María J. García,⁴ Brigitte Gicquel,⁵ and Carlos Martín^1,6*

Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, Zaragoza, Spain,¹ Departamento de Química, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia,² Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Institute for Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, Zaragoza, Spain,³ Departamento de Medicina Preventiva, Universidad Autónoma de Madrid, Madrid, Spain,⁴ Unité Génétique Mycobactérienne, Institut Pasteur, Paris, France,⁵ CIBER Enfermedades Respiratorias, Mallorca, Spain⁶

Received 21 May 2008/ Accepted 16 August 2008

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The attenuated Mycobacterium tuberculosis H37Ra strain is an isogenic counterpart of the virulent paradigm strain H37Rv. Recently, a link between a point mutation in the PhoP transcriptional regulator and avirulence of H37Ra was established. Remarkably, a previous study demonstrated negative autoregulation of the phoP gene in H37Ra. These findings led us to study the transcriptional autoregulation of PhoP in the virulent H37Rv strain. In contrast to the negative autoregulation of PhoP previously published for H37Ra, our experiments using a phoP promoter-lacZ fusion showed that PhoP is positively autoregulated in both H37Rv and H37Ra compared with an H37Rv phoP deletion mutant constructed in this study. Using quantitative reverse transcription-PCR (RT-PCR) analysis, we showed that the phoP gene is transcribed at similar levels in H37Rv and H37Ra. Gel mobility shift and DNase I footprinting assays allowed us to identify the precise binding region of PhoP from H37Rv to the phoP promoter. We also carried out RT-PCR studies to demonstrate that phoP is transcribed together with the adjacent gene phoR, which codes for the cognate histidine kinase of the phoPR two-component system. In addition, quantitative RT-PCR studies showed that phoR is independently transcribed from a promoter possibly regulated by PhoP. Finally, we discuss the possible role in virulence of a single point mutation found in the phoP gene from the attenuated H37Ra strain but not in virulent members of the M. tuberculosis complex.

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The role of the phoP gene in Mycobacterium tuberculosis virulence has been characterized extensively in various in vitro cultures and animal models. The inactivation of phoP in a fully virulent M. tuberculosis clinical isolate results in impaired growth in cultured macrophages and no bacillary multiplication in mouse organs (32). Vaccination with the phoP mutant has been shown to protect mice and guinea pigs against tuberculosis (1, 27), suggesting that attenuated phoP mutants could potentially be used as live antituberculosis vaccine candidates (2).

Both the attenuated phenotype and protective efficacy against tuberculosis of the phoP mutant can be accounted for by the mechanism of action of PhoP, the response regulator (RR) of the two-component system (2CS) PhoPR. 2CSs are signal transduction pathways enabling bacteria to detect and respond to environmental stimuli, resulting in adaptation. Generally, 2CSs consist of two proteins, a membrane-associated histidine kinase (HK) which senses an environmental signal(s) and an RR which is phosphorylated by its cognate HK. These changes in the phosphorylation state of the RR result in transcriptional adaptation (14, 33). The M. tuberculosis genome encodes only 11 2CSs (11, 23), far fewer than the numbers found in many other bacteria. The small number of 2CSs present could be the result of M. tuberculosis adaptation to an intracellular lifestyle. Of the 11 2CSs that M. tuberculosis possesses, the phoPR 2CS has been demonstrated to be essential for the virulence of the tubercle bacillus (32).

Biochemical analyses of M. tuberculosis phoP mutants have demonstrated that PhoP positively regulates the biosynthesis of virulence-associated lipids, such as sulfolipid (SL), diacyltrehaloses (DAT), and polyacyltrehaloses (PAT) (19). Consistent with these findings, the PhoP regulon identified in the M. tuberculosis H37Rv strain contains a number of genes involved in the synthesis of these complex lipids along with several genes implicated in virulence (49).

The relationship between virulence and lipid composition was demonstrated in analyses comparing the virulent H37Rv strain with the attenuated H37Ra strain, which, similar to M. tuberculosis phoP mutants, has been shown to lack SL, DAT, and PAT (8, 17). Conversely, a recent study identified a point mutation in PhoP from H37Ra with respect to that from H37Rv resulting in the amino acid substitution Ser219Leu, which probably affects the functionality of the DNA-binding domain. It was clearly demonstrated that this point mutation is responsible for the absence of SL, DAT, and PAT in H37Ra (10). This finding may explain some striking similarities between H37Ra and M. tuberculosis phoP mutants, as many of the phenotypes of the H37Ra strain, including virulence attenuation (9, 37), formation of smaller colonies on agar plates, loss of acid fastness (44), lack of reactivity with neutral red (13, 29, 41), and cording defects (17, 30), are correlated with the phenotypes of phoP mutants constructed from virulent M. tuberculosis strains (19, 32, 49).

Another remarkable example of the impact of the phoP mutation is the lack of secretion of the major T-cell antigen ESAT-6 and its binding partner, the 10-kDa culture filtrate protein CFP-10, in both H37Ra and an M. tuberculosis phoP mutant (16). The study established an interesting link between PhoP and ESAT-6/CFP-10 secretion, providing important information about PhoP virulence regulation in M. tuberculosis.

In a previous work, Gupta et al. demonstrated transcriptional autorepression of PhoP due to a sequence-specific interaction with its promoter when they used the H37Ra version of PhoP (21). Here we study many aspects of PhoP autoregulation in the virulent strain H37Rv, such as transcriptional organization, expression, and DNA-binding properties. Our results indicate that PhoP is positively autoregulated in the H37Rv and H37Ra strains of M. tuberculosis. We also describe the organization of the phoPR operon in H37Rv and address the importance of the PhoP mutation in the loss of virulence of H37Ra.

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Bacterial strains, media, and growth conditions. M. tuberculosis H37Rv (11), H37Ra (ATCC 25177), and MT103 (32), Mycobacterium smegmatis mc²155 (40), and their derivatives were cultured at 37°C in Middlebrook 7H9 medium supplemented with ADC (0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl, 0.0003% beef catalase) (Difco) and 0.05% Tween 80. Escherichia coli BL21(DE3)/pLysS and its derivatives were grown in Luria-Bertani (LB) medium at the appropriate temperature. When required, ampicillin (100 µg/ml), kanamycin (20 µg/ml), hygromycin (20 µg/ml), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; 50 µg/ml), and isopropyl-β-D-thiogalactopyranoside (IPTG; 1 mM) were used.

Construction of plasmids. The vectors and oligonucleotides used are shown in Table 1. For the overproduction of PhoP in E. coli, the phoP gene was amplified from M. tuberculosis genomic DNA by PCR, using the primers PhoPexp and PhoPR, and the PCR fragment was inserted into the pGEM-T Easy vector to obtain pMT1. The insert was then excised by digestion with NdeI and XhoI and ligated into pET15b to give pTEX1. This recombinant plasmid contains the codons for six histidine residues at the 5' end of phoP and therefore generates a protein with a His₆ motif at the N terminus (His₆-PhoP). The insert in pTEX1 was sequenced and confirmed to be identical to the phoP gene from the H37Rv strain. To construct the phoP promoter-lacZ fusion plasmid, a 238-bp fragment from positions –226 to +14 with respect to the phoP start codon was PCR amplified from M. tuberculosis MT103 genomic DNA by use of the primers BCG2B and PhoPro. The PCR product was cloned into pGEM-T Easy to obtain pTBLAC. The phoP promoter region was released from pTBLAC by digestion with ApaI and KpnI and then inserted into the pJEM14 E. coli-mycobacterial shuttle plasmid to give pTBGAL. The sequence of the phoP promoter cloned in pTBGAL was identical to that in the H37Rv strain.

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TABLE 1. Plasmids and primers used in this study

Quantification of β-galactosidase activity. Mycobacterial strains grown to stationary phase (optical density at 600 nm [OD₆₀₀] 2) were used to inoculate 50 ml of fresh medium at a ratio of 1:50. Aliquots were taken from this culture at the indicated times and centrifuged, and the pellet was washed with phosphate-buffered saline and stored at –80°C. Cells were resuspended in 1 ml Z buffer (0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 0.01 M KCl, 1 mM MgSO₄, and 0.05 M β-mercaptoethanol [pH 7 at 25°C]) and sonicated to generate a cell extract. The chromogenic substrate o-nitrophenyl-D-galactoside was added to cell extracts at a final concentration of 0.66 mg/ml. The mixtures were incubated at 28°C for 1 h, and the enzymatic reaction was stopped by adding 0.29 M Na₂CO₃. The A₄₂₀ of the supernatant was determined, and β-galactosidase activity was calculated in Miller units, using the following formula: β-galactosidase activity = (1,000 x A₄₂₀)/(time [min] x aliquot volume [ml] x A₆₀₀).

Overproduction and purification of PhoP. The PhoP protein from H37Rv was overproduced from pTEX1 in E. coli BL21(DE3)/pLysS and purified using a HisTrap affinity column (Amersham Pharmacia Biotech) as previously described (10).

EMSA. DNA probes for electrophoretic mobility shift assays (EMSAs) were obtained by PCR amplification from M. tuberculosis MT103 genomic DNA. Amplification products were purified with a GFX PCR DNA purification kit (Amersham Biosciences). Purified DNA fragments were 3' end labeled with a biotin 3'-end-labeling kit (Pierce) for use in gel shift assays. In each 20-µl binding reaction mix, 0.02 pmol of labeled fragment was incubated for 30 min at room temperature with 100 ng poly(dI-dC) and increasing amounts of PhoP in binding buffer (50 mM HEPES [pH 7.5], 50 mM KCl, 1 mM dithiothreitol, and 5% glycerol). Samples were loaded on a 6% nondenaturing polyacrylamide gel containing 1x Tris-borate-EDTA buffer (36). Electrophoresis was performed at 100 V and 7 mA at 4°C until the bromophenol blue reached the bottom of the gel. The DNA-protein complexes were electroblotted onto a positively charged Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) and detected by enhanced chemiluminescence, using a Lightshift chemiluminescence EMSA kit (Pierce) and Hyperfilm ECL (Amersham Pharmacia Biotech).

DNase I footprinting. DNase I footprinting assays were performed by the fluorescence labeling procedure (34). The fluorescently labeled DNA was amplified by PCR, using pJUZ1 (19) as a template, with the Cy5-labeled BCG2B primer (Sigma) and unlabeled BCG2A primer for the phoP coding strand and the unlabeled BCG2B primer and Cy5-labeled BCG2A primer for the noncoding strand. In each case, the same labeled primer was used to prime the sequencing reaction for the determination of molecular size. Labeled DNA fragment (100 ng) and 1 µg of PhoP were included in each 20-µl binding reaction mix, as described above for EMSA. The mixture was incubated for 30 min at room temperature, and DNase I (0.1 unit; Roche) was then added and allowed to act for 3 min at 30°C. The nuclease digestion was stopped by adding 180 µl of stop solution (10 mM Tris-HCl [pH 8], 40 mM EDTA). After chloroform extraction and ethanol precipitation, samples were loaded and analyzed on an ALF sequencer, as previously described (7).

Isolation of RNA from mycobacteria. The M. tuberculosis wild-type, phoP mutant, and complemented strains were grown at 37°C until they reached the desired OD₆₀₀ under aerobic conditions. Cells were harvested and total RNAs were isolated using a Fast RNA Pro Blue kit (Qbiogene) according to the manufacturer's recommendations. The extracted RNAs were treated with RNase-free DNase (Ambion), and the RNAs were then further purified using an RNeasy kit (Qiagen). The integrity of the RNAs was checked by gel electrophoresis on a 1% agarose gel.

RT-PCR. Reverse transcription-PCR (RT-PCR) was carried out in two steps. RT was carried out with Expand reverse transcriptase (Roche), using 1 µg RNA as the template and the appropriate reverse primer. Reaction mixtures were incubated at 43°C for 90 min. RT products were then subjected to PCR amplification, using TaqGold DNA polymerase (Roche) and the appropriate primers. PCR amplification involved initial denaturation for 10 min at 94°C, followed by 40 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 3 min. Samples were analyzed by electrophoresis on a 1% agarose gel.

qRT-PCR. cDNA libraries from M. tuberculosis wild-type, mutant, and complemented strains were constructed as follows. One microgram of RNA was mixed with 25 pmol of random hexanucleotide primers (Sigma) and 50 units of Expand reverse transcriptase (Roche) in a final volume of 20 µl. Reaction mixtures were incubated at 30°C for 10 min and then at 43°C for 90 min. Expression of phoP and phoR mRNAs was measured and normalized with respect to the level of sigA mRNA by quantitative real-time RT-PCR (qRT-PCR). qRT-PCR was carried out in a StepOne Plus (Applied Biosystems) instrument, using the cDNA generated by RT from 25 ng of RNA as a template, 1x Power SYBR green PCR master mix (Applied Biosystems), and the appropriate primers (Table 1), each at a concentration of 250 nM. The PCR program involved an initial denaturation step for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The specificity of the PCR products was confirmed by the loss of fluorescence at a single temperature, when the double-stranded DNA melted to single-stranded DNA.

Bioinformatic analyses and homology modeling. The full genome sequences of Mycobacterium bovis strains BCG and AF2122/97 and M. tuberculosis strains Haarlem, CDC1551, F11, C, H37Rv, and H37Ra were obtained from the NCBI website (http://www.ncbi.nlm.nih.gov/sites/entrez). We also sequenced the coding and promoter regions of the phoP gene from M. tuberculosis MT103 and included them in the sequence alignment. The ExPASy proteomic server (http://au.expasy.org/) was used to predict the organization of the PhoP domain.

From the structure of the DNA-binding domain of PhoP from M. tuberculosis (50) (Protein Data Bank [PDB] code 2pmu), the Ser219Leu mutation was modeled using Deepview (20). On the other hand, superpositioning of the wild-type and mutant PhoP structures was done with Deepview on the structure of the complex between the structurally homologous effector domain of E. coli PhoB and Pho box DNA (4) (PDB code 1gxp).

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phoP promoter activity in the presence of PhoP from M. tuberculosis H37Rv. It has been reported that PhoP from the H37Ra strain represses its own transcription if M. smegmatis is used as a surrogate host (21). We investigated whether this autoregulation was affected by the Ser219Leu mutation in PhoP by carrying out a similar experiment with PhoP from the H37Rv strain. A transcriptional fusion between the phoP promoter and a promoterless lacZ gene was constructed and inserted into pJEM14 (47), an E. coli-mycobacterial shuttle plasmid, yielding pTBGAL (Table 1). This plasmid was used to transform M. smegmatis mc²155. The resulting strain was cotransformed with pSO5 (32), which contains both the promoter region and the entire phoP coding region in the multicopy plasmid pNBV1 (24). The cotransformed strain was grown at 37°C in 7H9 medium supplemented with hygromycin and kanamycin. Aliquots were collected at the indicated times, and β-galactosidase activities in the cell extract were evaluated to determine levels of transcription from the phoP promoter in the presence of PhoP. β-Galactosidase activity levels were two to three times higher in M. smegmatis mc²155 cotransformed with pTBGAL and pSO5 than in the control strain cotransformed with pTBGAL and pNBV1 (Fig. 1). Thus, phoP expression seems to be positively autoregulated by the H37Rv version of PhoP when M. smegmatis is used as a surrogate host.

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FIG. 1. phoP promoter activity in M. smegmatis expressing the H37Rv variant of PhoP. The phoP promoter-lacZ fusion plasmid pTBGAL was introduced into M. smegmatis carrying either a PhoP expression vector (pSO5) or the expression plasmid lacking an insert (pNBV1) and cultured for the indicated times. White bars indicate β-galactosidase activity of M. smegmatis carrying pTBGAL and pNBV1, and black bars represent the results for the strain cotransformed with pTBGAL and pSO5. Results shown are the means and standard errors from two independent experiments.

Positive autoregulation of PhoP in M. tuberculosis H37Rv and H37Ra. In order to corroborate the previous finding and to rule out differences between the transcriptional machineries of the fast-growing M. smegmatis strain and the slow-growing M. tuberculosis strain, we attempted to study the expression of the phoP promoter in H37Rv, H37Ra, and a defective phoP mutant. Firstly, we constructed and characterized an H37Rv phoP::hyg mutant (see the supplemental material). M. tuberculosis H37Rv, H37Ra, and H37Rv phoP::hyg were transformed with either pJEM14 or pTBGAL and grown at 37°C in 7H9 medium supplemented with kanamycin. Aliquots were collected along the logarithmic growth phase (OD₆₀₀ 0.3, 0.6, and 1), and β-galactosidase activities in the cell extract were calculated. Our results indicate that the lacZ gene is transcribed from the phoP promoter at similar levels in H37Rv and H37Ra (Fig. 2). Moreover, we also observed that transcription of phoP is about twofold higher in H37Rv and H37Ra than in the H37Rv defective phoP mutant at early- and late-logarithmic growth phases, while no significant differences exist at mid-log phase (Fig. 2). In addition, the H37Rv and H37Ra strains transformed with either pJEM14 or pTBGAL were plated on 7H10-oleic acid-albumin-dextrose-catalase agar containing the β-galactosidase substrate X-Gal. It was observed that H37Rv and H37Ra transformed with pTBGAL developed blue coloration, while the pJEM14 transformants remained uncolored in the presence of X-Gal (data not shown). Taken together, these results indicate that phoP could be positively autoregulated in both M. tuberculosis H37Rv and H37Ra, and this effect was more noticeable at the time points described above.

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FIG. 2. phoP promoter activity in H37Rv and H37Ra. White, black, and hatched bars represent β-galactosidase activities from the H37Rv phoP::hyg, H37Rv, and H37Ra strains transformed with pTBGAL and cultured until the indicated OD600. Miller units for the aforementioned strains transformed with the empty vector (pJEM14) were below 10 and have been omitted for clarity. Results shown are the means and standard errors from two independent experiments.

EMSA with the phoP promoter and PhoP from H37Rv. Previous works have demonstrated that PhoP from H37Ra binds to its own promoter (21, 38). Here we studied whether PhoP from M. tuberculosis H37Rv specifically binds to its own promoter sequence, possibly mediating autoregulation of phoP expression. The same 238-bp fragment from the phoP promoter previously used in β-galactosidase experiments was used for gel shift assays. This fragment was amplified by PCR using the BCG2B and PhoPro primers (Table 1) and then labeled for use as a probe in mobility shift experiments. The labeled probe displayed a clear shift in electrophoretic mobility in the presence of PhoP, and larger amounts of protein resulted in a gradual decrease in probe mobility (Fig. 3, lanes 2 to 6). We confirmed the specificity of binding by using an unlabeled fragment as a competitor. PhoP binding to the labeled phoP promoter was reduced by adding a 100-fold excess of unlabeled probe (Fig. 3, lane 7). Further support for the specificity of the interaction with DNA was provided by experiments in which a nonspecific fragment corresponding to the sigA coding sequence was used, and as expected, no shift in DNA mobility was detected (data not shown). Similar experiments were performed in the presence of PhoP previously incubated with the phosphorylation reagent acetyl phosphate. Even if phosphorylation of PhoP did not appear to affect binding specificity, we cannot rule out differences in binding affinity due to phosphorylation (data not shown).

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FIG. 3. Binding of PhoP to the phoP promoter. Electrophoretic mobilities of a 238-bp fragment containing the phoP promoter in the absence (lane 1) and presence of increasing amounts of recombinant PhoP (180 µM [lane 2], 360 µM [lane 3], 720 µM [lane 4], 1.08 mM [lane 5], and 1.44 mM [lane 6]). Lane 7 represents the electrophoretic mobility of the phoP promoter in the presence of 360 µM PhoP and a 100-fold excess of unlabeled probe.

DNase I protection of the phoP promoter with PhoP. We attempted to compare the PhoP binding region in H37Rv with that previously reported for H37Ra (21). The sequence to which PhoP binds was mapped by DNase I footprinting assays with a 463-bp fragment from positions –226 to +237 relative to the phoP start codon. The regions protected by PhoP from degradation by DNase I were located between positions –66 and –18 on the noncoding strand and –38 and +1 on the coding strand, resulting in a 67-bp binding region, from positions –66 to +1 (Fig. 4). The degree of protection was higher for the coding strand than for the noncoding strand (Fig. 4). Similar results were obtained in other footprinting assays using radiolabeled primers (data not shown). Our results indicate that the PhoP binding site described here, although similar, is shorter to that previously described for the H37Ra strain, which is located between positions –79 and +9 relative to the phoP start codon (21).

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FIG. 4. DNase I footprinting assay of the phoP promoter. (A) Fluorograms indicate fluorescence intensities after DNase I digestion of fragments containing Cy5-labeled coding and noncoding strands in the absence (–PhoP) and presence (+PhoP) of recombinant PhoP. Sequencing reaction mixtures with Cy5-labeled primers were included in the gel (data not shown). Sites protected by PhoP are indicated by boxes. The ATG translation initiation triplet is indicated by asterisks. (B) PhoP binding region. The nucleotides to which PhoP binds are indicated in a box. The ATG translation initiation triplet is indicated by asterisks.

Transcriptional organization of the M. tuberculosis phoPR operon. In the M. tuberculosis H37Rv chromosome, the phoP gene maps upstream of the phoR gene, coding for the HK of the 2CS PhoPR, and the stop codon of phoP is separated from the start codon of phoR by a 47-bp intergenic region (Fig. 5), suggesting that both genes may be transcribed in an operon. RT-PCR was used to determine whether phoP and phoR were transcribed to give a single mRNA. We obtained amplification products for the 5' end of phoP and the 3' end of phoR and a 393-bp amplification product for the phoPR intergenic region (Fig. 5), indicating that these two genes are cotranscribed. For confirmation of the cotranscription of phoPR, a larger region, from positions –52 to +1900 with respect to the phoP start codon, was amplified and the expected cotranscribed fragment obtained (Fig. 5). We checked that the RNA samples were not contaminated with genomic DNA by carrying out RT-PCRs without reverse transcriptase. The lack of amplification products for all of the fragments described above demonstrates clearly that the RT-PCR products obtained were amplified from RNA that had been reverse transcribed into cDNA (Fig. 5). Our results indicate that the two genes are cotranscribed in an operon, as described for other 2CSs (22), but these results do not exclude the possibility of independent promoters for phoP and phoR.

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FIG. 5. RT-PCR analysis of phoPR from M. tuberculosis. (A) Schematic diagram of M. tuberculosis phoPR and the gene-specific primers used for RT-PCR (Table 1). The direction of transcription for phoP and phoR is indicated by arrows. Primers used for RT-PCR and the sizes of the fragments obtained with each pair of primers are indicated. (B) RT-PCR of phoPR. The combination of primers is indicated above each set of reactions. Amplification products for the 5' end of phoP (436 bp), the intergenic region (393 bp), and the 5' end of phoR (464 bp) are shown. A larger fragment, from the phoP promoter region to the 3' end of phoR (1,952 bp), was also amplified. The positions of the standard DNA size markers are indicated on the right. Each set of three reactions consists of a positive control PCR assay with genomic DNA as the template (+), an RT-PCR (*), and a negative control assay without reverse transcriptase (–).

Quantification of phoP and phoR expression by qRT-PCR. M. tuberculosis H37Rv and H37Ra were cultured to mid-exponential growth phase. RNAs were extracted and reverse transcribed to generate cDNAs, which were then amplified in qRT-PCR experiments. The expression of the phoP gene was measured by qRT-PCR and normalized to sigA expression levels. Transcription rates for the phoP gene were similar in H37Rv and H37Ra (Fig. 6A), in concordance with our previous results from the β-galactosidase activity assays. Once we demonstrated that phoP is similarly transcribed in H37Rv and H37Ra, we sought to compare phoR expression levels in both strains. qRT-PCR experiments using phoR-specific primers (Table 1) indicated that phoR is transcribed about 10-fold more in H37Rv than in H37Ra (Fig. 6A), suggesting that the phoP mutation could affect phoR transcription. To assess this possibility, we compared phoR expression between an M. tuberculosis clinical isolate MT103 wild-type strain and an MT103 defective phoP mutant (19). Similar to that observed in H37Ra, phoR expression was highly reduced in the phoP mutant of M. tuberculosis (Fig. 6B). To rule out the possibility that phoR transcription was reduced as a consequence of a polar effect of the phoP mutation on the transcription of the entire phoPR operon, we studied phoR expression in the phoP mutant complemented with pSO5K (19), a multicopy plasmid carrying the phoP gene from H37Rv. We observed that phoR transcription was restored in the complemented strain (Fig. 6B). Moreover, the levels of phoR transcripts seemed to be correlated directly with PhoP production, since overexpression of PhoP from the multicopy plasmid resulted in a significant increase in the level of phoR with respect to that in the wild-type strain (Fig. 6B). Altogether, these results indicate that the phoR gene could be transcribed from a PhoP-regulated promoter.

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FIG. 6. Quantification of phoP and phoR expression by qRT-PCR. (A) Quantification of phoP and phoR expression in H37Ra relative to that in H37Rv. (B) Relative quantification of phoR expression in MT103 phoP::hyg and the complemented strain MT103 phoP::hyg-pSO5K with respect to phoR expression in the MT103 wild-type strain. Results are the means for two independent experiments; error bars indicate the standard deviations of the means.

Characterization of the Ser219Leu mutation in PhoP. The M. tuberculosis PhoPR system is highly conserved among slow-growing mycobacteria. Sequence alignment indicated full conservation of the phoP gene in all eight M. tuberculosis complex genomes annotated to date, with the remarkable exception of the H37Ra strain (Fig. 7A). The phoP gene from M. tuberculosis H37Ra has acquired a point mutation in codon 219 (TCGTTG), resulting in the replacement of a serine by a leucine residue (Fig. 7A). Bioinformatic approaches based on sequence alignment led to the identification of two putative distinct domains in PhoP. The PhoP N-terminal domain is involved in the phosphotransfer reaction through the conserved residue Asp71, whereas the C-terminal domain is involved in DNA binding (data not shown). The recent structure of the PhoP DNA-binding domain (50) situates the above-mentioned missense mutation in the DNA recognition helix (Fig. 7B), potentially affecting DNA interactions and consequently the role of this protein in transcriptional regulation.

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FIG. 7. Characterization of the point mutation in PhoP from H37Ra. (A) Domain organization of PhoP. The N-terminal domain of PhoP (blue) contains the Asp71 residue involved in the phosphotransfer reaction. The C-terminal domain (red) interacts with target DNA molecules, modulating gene expression. Alignment of the phoP gene sequences from the annotated genomes of the M. tuberculosis complex shows a point mutation responsible for the Ser219Leu substitution in the H37Ra strain, indicated by an asterisk. (B) DNA-binding domain of PhoP (blue ribbon) (see text for details) superimposed on the structure of a PhoB-DNA complex (gray ribbon, protein; sticks in CPK color, DNA). Solid spheres show the wild-type serine 219 residue (left) or the leucine residue that appears in the mutant protein (right). The mutant leucine residue is expected to interfere with DNA binding and/or recognition.

A model of the three-dimensional structure of the PhoP Ser219Leu mutant was constructed, and both the wild-type and mutant structures were superimposed on the structure of the PhoB-DNA complex from E. coli (Fig. 7B). The mutation is located in the central part of the C-terminal -helix (₃) of the three-helix bundle of the effector domain (Fig. 7B). The helix is amphipathic, with the wild-type serine residue facing the solvent. Replacement of a solvent-exposed serine residue by a leucine residue is unlikely to significantly reduce the conformational stability of the protein. On the other hand, the mutant leucine residue cannot easily favor any aggregation of the protein because the region is highly charged. Apparently, the Ser219Leu mutation should give rise to a protein with stability and solubility similar to those of the wild-type protein. In contrast, the mutation is clearly expected to reduce the affinity and/or specificity of PhoP for its DNA-binding region. As the superposition of the model structure of PhoP with the PhoB-DNA complex indicates, the helix bearing the mutation (₃) is the DNA recognition helix of a modified helix-turn-helix DNA-binding motif where loop 2-3 replaces the turn (4). The mutation therefore takes places right at the DNA-binding site (Fig. 7B). Given the capability of serine residues to establish hydrogen bonds, Ser219 could be important for the specificity of the binding to DNA. But even if the contribution of Ser219 to specificity and/or binding affinity is small, its replacement by a bulky leucine residue is expected to interfere with a tight protein-DNA interaction and therefore to reduce the affinity of the complex.

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In 1934, Steenken et al. observed that subculture of the H37 strain by serial passages resulted in two different colony morphologies, with one of them being highly attenuated for guinea pigs. The passages continued until two stable strains were obtained, the virulent H37Rv strain and the attenuated H37Ra strain (28, 43-45). Since then, both strains have been used extensively worldwide; indeed, H37Rv is the most widely used virulent M. tuberculosis laboratory reference strain today.

The H37Rv genome was the first mycobacterial genome to be sequenced (11) and provided general insight into the biology of M. tuberculosis. However, the genetic basis for H37Ra attenuation has remained unclear. H37Ra is likely to have acquired multiple point mutations, deletions, and/or genomic rearrangements during in vitro passage. Somewhat analogously to the attenuation process for BCG, the current antituberculosis vaccine was derived by serial passage of M. bovis in the laboratory over a period of 13 years (18), resulting in the loss of more than 100 genes (3).

Even if the presence of genomic variations between H37Rv and H37Ra has been confirmed, their role in H37Ra attenuation remains unclear (5). Pioneering studies in mycobacterial genetics sought to restore virulence to the H37Ra strain by in vivo complementation with an H37Rv cosmid library in an attempt to identify genomic fragments associated with virulence. Despite allowing the identification of a genomic fragment which conferred an enhanced in vivo growth rate, this study failed to completely restore virulence to the H37Ra strain (31).

The recent release of the H37Ra genome sequence available from the NCBI website (NC_009525) should increase our understanding of the mechanism of attenuation of H37Ra. Recent studies have identified a number of nucleotide polymorphisms between H37Rv and H37Ra (16, 25), making it possible to evaluate the role of discrete regions of the genome in M. tuberculosis virulence.

In this study, we focused on the phoP gene, which has been shown to play an important role in virulence (19, 27, 32). PhoP is fully conserved in all of the annotated genomes of the M. tuberculosis complex except that of H37Ra. The phoP gene from H37Ra contains a point mutation that results in the replacement of the polar residue Ser219 by the nonpolar residue Leu in the DNA-binding domain of PhoP.

Apparently, this mutation should not compromise either the stability or the aggregation state of the protein. Indeed, circular dichroism studies indicate that the point mutation does not affect the global secondary structure of PhoP (10). However, homology modeling of both variants of PhoP clearly indicates that the replacement of the polar residue Ser by the nonpolar residue Leu lies within the DNA recognition helix (50), and this may result in a lower affinity for DNA binding as a consequence of both the impaired ability of the nonpolar residue Leu to establish hydrogen bonds with the bases of the DNA helix and the steric difficulties imposed by the bulky Leu residue in establishing a tight PhoP-DNA interaction (Fig. 7B).

We found that PhoP from H37Rv binds to its own promoter (Fig. 3), like PhoP from H37Ra (21, 38). However, a recent work demonstrated that PhoP from H37Rv, but not PhoP from H37Ra, binds a 40-mer promoter region (10). In this study, we used a 238-bp fragment containing the promoter region of phoP to demonstrate that the point mutation in H37Ra appears to diminish the DNA-binding affinity of PhoP (data not shown). The results presented in this work are in agreement with a recent study which demonstrated that the H37Ra version of PhoP displays a decreased ability for DNA binding to the PhoP binding motif (25).

The PhoP binding site in the phoP promoter extends from nucleotides –79 to +9 relative to the phoP start codon in H37Ra (21) and from nucleotides –66 to +1 in the H37Rv strain (Fig. 4). Even if the Ser219Leu substitution may be responsible for these subtle variations, the differences in the methods used should also be taken into account.

PhoP negative autoregulation has been characterized using the protein from M. tuberculosis H37Ra (21). Here we carried out similar experiments with PhoP from H37Rv and found that unlike that reported for H37Ra, the phoP gene from H37Rv is positively autoregulated with M. smegmatis as the expression host (Fig. 1). Furthermore, given that in M. tuberculosis phoP is transcribed at similar levels in H37Rv and H37Ra, with transcription being reduced in the H37Rv phoP mutant at early- and late-logarithmic phases (Fig. 2A), we consider that phoP is positively autoregulated in both H37Rv and H37Ra. We and others (25) have demonstrated that the Ser219Leu mutation in H37Ra decreases the DNA-binding affinity of PhoP. However, the remaining DNA-binding activity may be sufficient to properly autoregulate phoP expression. Consequently, this point mutation might not affect the autoregulatory capacity of PhoP.

Transcription of the phoP promoter in the H37Rv phoP null mutant indicates that in M. tuberculosis other transcriptional regulators influencing phoP transcription, apart from PhoP, may exist. This is particularly observed at the mid-logarithmic phase of growth, wherein phoP is transcribed at similar levels in the H37Rv phoP mutant, H37Rv, and H37Ra strains. It is also possible that PhoP could be tightly autoregulated only in the presence of an as yet unknown PhoR-stimulating signal that would control PhoP phosphorylation and consequently the expression of PhoP-regulated genes, including phoP itself.

Even though the Ser219Leu substitution does not appear to affect the autoregulatory mechanism of PhoP, it seems to have important implications in the virulence regulation of M. tuberculosis. A recent study demonstrated that even if H37Ra and an M. tuberculosis phoP mutant produce the major antigen ESAT-6/CFP-10, neither of these strains secretes the antigen into the supernatant, resulting in decreased T-cell responses against both ESAT-6 and CFP-10 (16). A plausible explanation comes from the observation that the Rv3614c-to-Rv3616c (espA) gene cluster essential for ESAT-6/CFP-10 secretion (15, 26) appears to be downregulated in both H37Ra and an H37Rv phoP mutant (16, 17, 49).

Results for the PhoP regulon in M. tuberculosis H37Rv and the transcriptome comparison between H37Rv and H37Ra further support the implications of the PhoP mutation in H37Ra avirulence. The Rv1184c, fadD21, and papA1 genes, encoding proteins involved in the biosynthesis of the virulence-associated lipids SL, DAT, and PAT (12, 39), are much less expressed in H37Ra than in H37Rv (17), which probably accounts for the absence of these lipids in H37Ra. Other genes downregulated in H37Ra (Rv1639c, cdh, narK1, Rv2376c, nirA, fadD9, Rv3312A, Rv3479, lipF, Rv3686c, and Rv3822) (17) have also been shown to belong to the PhoP regulon identified in H37Rv (49). Altogether, these observations indicate that 14 of the 22 genes differentially expressed between H37Rv and H37Ra are under the control of PhoP. This strongly suggests that the mutated version of PhoP contributes to M. tuberculosis H37Ra attenuation by leading to major global changes in the gene expression profile of this avirulent strain. Downregulation of PhoP-regulated genes in H37Ra could be a consequence of the decreased ability of PhoP for DNA binding and/or the inadequate phosphorylation of the protein as a result of decreased expression of PhoR in H37Ra.

Despite these important contributions of the PhoP mutation to the attenuation process of H37Ra, complementation of this strain with the H37Rv version of PhoP only partially restored the virulence of H37Ra (16, 25). This is probably due to the existence of other polymorphisms affecting the expression of virulence factors. Supporting this hypothesis, it has been found that the production of phthiocerol dimycocerosates—a family of lipids implicated in virulence (6, 35)—is abrogated in the H37Ra strain and that their synthesis is independent of the PhoP mutation. Alternatively, given that phoR is expressed much less in H37Ra than in H37Rv, which may be a consequence of the phoP mutation in the former strain (Fig. 6), another plausible explanation for the partial virulence complementation of H37Ra is that decreased expression of PhoR in this strain could result in inadequate phosphorylation-mediated activation of PhoP. Accordingly, complementation of H37Ra with the whole H37Rv phoPR operon could restore virulence more proficiently than complementation with only the phoP gene.

Naturally occurring mutations causing attenuation of bacterial pathogens have already been described. Sequencing of low-virulence field strains of Listeria monocytogenes recently showed multiple point mutations affecting the virulence of this intracellular pathogen (46). Remarkably, like the Ser219Leu mutation in PhoP, the naturally occurring mutation Lys220Thr in the transcriptional regulator PrfA from L. monocytogenes results in abrogated DNA binding, no expression of PrfA-regulated proteins, and attenuated virulence (48).

Our study also suggests that differences between M. tuberculosis strains should be taken into account in genetic studies. Continuous passages in synthetic laboratory media may well result in genetic polymorphisms and, consequently, in substrain variability. H37Rv is a highly pathogenic strain and can be manipulated only in biosafety level 3 containment laboratories. For this reason, many genetic studies have made use of the attenuated counterpart, H37Ra, based on the assumption that the results obtained could be extrapolated to all M. tuberculosis complex strains. However, different works, including this one, clearly demonstrate the important implications of a single mutation in virulence regulation.

 
We thank Jordi Barbé for scientific advice about the nonradioactive footprinting assay, Stewart Cole for kindly providing the H37Rv strain, Juan Carlos Ramírez for bioinformatic studies, and Alberto Cebollada for statistical analysis.

This work was supported by the Spanish MEC (BIO2005-07949-C02-01), EU FP6 TB-VAC (LSHP-CT203-503367), INCO (ICA4-CT-2002-10063), BFU2007-61476/BMC, and PM076/2006 projects.

 
* Corresponding author. Mailing address: Departamento de Microbiología, Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad de Zaragoza, C/Domingo Miral s/n, 50009 Zaragoza, Spain. Phone: 34 976 76 17 59. Fax: 34 976 76 16 64. E-mail: carlos{at}unizar.es

Published ahead of print on 29 August 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, November 2008, p. 7068-7078, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00712-08
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