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Keywords:

  • Mycobacterium tuberculosis;
  • GroES;
  • GroEL;
  • promoter;
  • transcription and operon

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References

Heat shock promoters of mycobacteria are strong promoters that become rapidly upregulated during macrophage infection and thus serve as valuable candidates for expressing foreign antigens in recombinant BCG vaccine. In the present study, a new heat shock promoter controlling the expression of the groESL1 operon was identified and characterized. Mycobacterium tuberculosis groESL1 operon codes for the immunodominant 10 kDa (Rv3418c, GroES/Cpn10/Hsp10) and 60 kDa (Rv3417c, GroEL1/Cpn60.1/Hsp60) heat shock proteins. The basal promoter region was 115 bp, while enhanced activity was seen only with a 277-bp fragment. No promoter element was seen in the groESgroEL1 intergenic region. This operon codes for a bicistronic mRNA transcript as determined by reverse transcriptase-PCR and Northern blot analysis. Primer extension analysis identified two transcriptional start sites (TSSs) TSS1 (−236) and TSS2 (−171), out of which one (TSS2) was heat inducible. The groE promoter was more active than the groEL2 promoter in Mycobacterium smegmatis. Further, it was found to be differentially regulated under stress conditions, while the groEL2 promoter was constitutive.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References

Regulated expression of heat shock proteins (Hsps) during macrophage infection play an important role in the pathogenesis of Mycobacterium tuberculosis (Segal & Ron, 1998). Heat shock response is a ubiquitous adaptive pathway involved in the survival of cells exposed to a sudden increase in ambient temperature. It is characterized by global transcriptional changes including elevated expression of a set of highly conserved Hsps. The mycobacterial Hsp family includes proteins such as DnaK, DnaJ, GrpE, GroES, GroEL and other low-molecular-weight Hsps (Qamra et al., 2005). Most of these proteins have house keeping functions and are essential for survival. DnaK, DnaJ, GroES, GroEL1, GroEL2 and Acr1 proteins are well-characterized immunodominant antigens of mycobacteria and serve as excellent vaccine candidates (Walker et al., 2007). The mechanism of heat shock regulation in mycobacteria is not completely known. However, in general, two mechanisms have been characterized for heat shock regulation in bacteria: induction and repression (Duchene et al., 1994; Segal & Ron, 1996; Barreiro et al., 2004). In Escherichia coli, following heat shock, the alternate sigma factor, σ32, becomes activated and directs RNA polymerase to heat shock promoters, thereby bringing about induction of Hsps. In contrast, in Bacillus subtilis, the heat shock response is predominantly by transcriptional repression, wherein repressors such as HspR and HrcA bind to Hsp promoters and block transcription (Narberhaus, 1999). Upon heat shock, this block is removed by the inactivation of these repressor proteins. Inspection of M. tuberculosis genome indicates repression to be the most probable mechanism of heat shock regulation. Interestingly, in mycobacteria, all the sigma factors belong to the σ70 gene family and not σ32, indicating a more complicated heat shock circuit in mycobacteria (Rodrigue et al., 2006). A combination of targeted mutagenesis and whole-genome expression profiling has helped in the characterization of transcription factors responsible for the control of major Hsps of M. tuberculosis (Stewart et al., 2002a, b). Using this approach, two heat shock regulons were identified: the hsp70 (dnaK) regulon, which is under the control of HspR, and hsp60 (groESL) regulon, which is under the control of HrcA. Both dnaK and groESL operons are highly conserved in bacteria (Segal & Ron, 1998). However, the exact mechanisms of regulation of both hsp60 and hsp70 regulons are not known in mycobacteria.

Unlike other bacteria, mycobacteria possess two groEL (groEL1 and groEL2) genes, out of which the groEL1 is present downstream to that of the groES gene, while groEL2 is present at a different location and is monocistronic (Fig. 1) (Cole et al., 1998). The groESL1 operon contains one CIRCE (Controlling Inverted Repeat of Chaperone Expression) sequence while groEL2 is preceded by two such CIRCE sequences. The HcrA protein binds to these CIRCE sequences and induces repression in other bacteria (Zuber & Schumann, 1994; Roncarati et al., 2007). Apart from Hsps, sigB, sigC, sigE, sigH and sigM genes of M. tuberculosis and Mycobacterium smegmatis also become upregulated in response to heat shock, indicating another level of regulation of heat shock response (Stewart et al., 2002a, b). In the present study, we set out to characterize the groE promoter of M. tuberculosis by determining the promoter region, identifying the transcriptional start site (TSS) and exploring differential regulation under stress conditions.

image

Figure 1.  Genomic organization of the groESgroEL1 operon and the groEL2 gene. CIRCE; −35/−10, minus 10 and minus 35 sequences; TSS, transcription start site; RBS, ribosome-binding site; Ter, terminator sequence; groES, codes for the Cpn10 antigen (Hsp10); groEL1, codes for the Cpn60.1 antigen (Hsp60) and groEL2, codes for the Cpn60.2 antigen (Hsp65). Arrows at the top and bottom denote the direction and position of the various primers used in this study (Table 1). β-Galactosidase activities (in Miller Units) of the promoter plasmids used in this study in Escherichia coli (DH5α) and Mycobacterium smegmatis mc2155 are presented in the table. Briefly, E. coli and M. smegmatis cells harboring various plasmids were grown to the mid-logarithmic phase, harvested by centrifugation, lysed by sonication and the lysate was used for estimating the β-galactosidase activity. Growth rate-associated alterations in the total β-galactosidase values were normalized by dividing the total value by OD600 nm value of the cultures. The values represent mean±SE (Miller Units) of three independent experiments.

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Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References

Bacterial strains and culture conditions

Mycobacterium smegmatis mc2155 was grown in Luria–Bertani (LB) broth with 0.05% Tween-80 and plated in the same medium with 1.5% agar supplemented with kanamycin (25 μg mL−1) wherever appropriate. Escherichia coli DH5α and TOP10 cells (Invitrogen Inc.) were grown in LB medium without Tween-80 and with kanamycin (50 μg mL−1) wherever appropriate. Mycobacterium tuberculosis H37Ra was grown in Middlebrook 7H9 broth supplemented with oleic acid, albumin and dextrose complex (7H9-OADC) (Difco) plus 0.05% Tween 80 or on Middlebrook 7H10-OADC agar plates.

Cloning of upstream sequences in pJEM13 and pJEM15

The upstream promoter sequences of the groES gene were amplified by PCR from M. tuberculosis genomic DNA and cloned in the pCR2.1 vector (Table 1 and Fig. 1). All the forward primers had an ApaI restriction site and all the reverse primers had a KpnI restriction site at their 5′ overhang (Invitrogen Inc.). The promoter fragments were then sub-cloned into pJEM13 (Timm et al., 1994) following ApaIKpnI double digestion. The recombinant plasmids were labeled as pJUPS1, pJUPS2, pJUPS3 and pJUPS4 (Table 2). The full-length groES gene, along with a 300 bp upstream sequence and a 100 bp downstream sequence, was cloned in pCR2.1 to obtain the plasmid, pCpn10. The groES–groEL1 intergenic sequence, along with the first four codons of groEL1 gene, was then excised as a BamHI fragment from the plasmid pCpn10 and was cloned in pJEM13 and pJEM15 (Timm et al., 1994) in-frame with lacZ, to obtain pJGEL1 and pJ15GEL1, respectively (Table 2). Similarly, the groEL2 promoter was excised as a DraI/BamHI fragment from the E. coliMycobacterium expression vector pMV261 (Stover et al., 1991). It was subcloned at the ScaI/BamHI site in pJEM13, which resulted in an in-frame fusion of the first six codons of the groEL2 gene with the lacZ gene (pJGEL2) (Table 2). The sequences of all the cloned fragments were confirmed using big dye terminator cycle sequencing chemistry for ABI BioPrism (Applied Biosystems, California). Mycobacterium smegmatis mc2155 cells were electroporated with these plasmids as described previously (Wards & Collins, 1996).

Table 1.   Primers used in this study
No.PrimerSequencePosition
  • *

    Position of the 5′ end of the primer with respect to G(+1) of the GTG start codon of the groES ORF.

  • Position of the 5′ end of the primer with respect to A(+1) of the ATG start codon of groEL1 ORF.

  • − Sign denotes upstream location while +sign denotes downstream position with respect to the G/A of the GTG/ATG start codons, respectively.

1UPS15′-GGGCCCCCGAGTGTGTGGCGTTGTTG-3′−914*
2UPS25′-GGGCCCGCTTCCGTACCGCCGACATT-3′−598*
3UPS35′-GGGCCCCGGACATTGCACCTGGCGTA-3′−266*
4UPS45′-GGGCCCCCTGGTAATTCGGACGGTT-3′−102*
5UPSR5′-GGGGTACCCCTCACCTTCGCCACGATTGGAG-3′+13*
6groES4775′-ATCAGAGCCCGGGACGC-3′−40*
7groES5345′-TGTCCTCGAGTGGCTTGATG-3′+34*
8gel1R25′-CATCAGGCTCCTCTACGCAG-3′+3
9VA35′-GCTCCAATCGTGGCGAAGGT-3′−9*
10VA55′-GAACACGCTCTACTACTTGG-3′−82
Table 2.   Plasmids used in this study
No.PlasmidsDescriptionCloning site
1pCpn10groES ORF with 200 bp upstream and 100 bp downstream sequenceEcoRI
2pMV261Mycobacterial expression vector with Hsp65 promoter (Stover et al., 1991)
3pCR2.1Linear vector to clone PCR products (Invitrogen)
4pUPS1926 bp groES UPS in pCR2.1 
 (primers – UPS1 and UPSR)
5pUPS2611 bp groES UPS in pCR2.1 
 (primers – UPS2 and UPSR)
6pUPS3277 bp groES UPS in pCR2.1 
 (primers – UPS3 and UPSR)
7pUPS4115 bp groES UPS in pCR2.1 
 (primers – UPS4 and UPSR)
8pJEM13Mycobacterial promoter probe vector with β-galactosidase (transcriptional fusion/gene fusion) (Timm et al., 1994)
9pJUPS1926 bp groES UPS in pJEM13ApaI and KpnI
10pJUPS2611 bp groES UPS in pJEM13ApaI and KpnI
11pJUPS3277 bp groES UPS in pJEM13ApaI and KpnI
12pJUPS4115 bp groES UPS in pJEM13ApaI and KpnI
13pJGEL1groES-groESL1 intergenic sequence in pJEM13BamHI
14pJGEL2groEL2 promoter in pJEM13ScaI and BamHI
15pJEM15Mycobacterial promoter probe vector with β-galactosidase (translational fusion/operon fusion) (Timm et al., 1994)
16pJ15GEL1groES–groEL1 intergenic sequence in pJEM15BamHI

Stress treatment

Recombinant M. smegmatis harboring pJUPS3 were grown to the mid-logarithmic phase (an OD600 nm of 0.8) in LB broth with 0.05% Tween-80 and kanamycin (25 μg mL−1). Treatment of the bacteria under various stress conditions was as follows: an exponentially growing culture of M. smegmatis harboring pJUPS3 (groES promoter), pJGEL2 (groEL2 promoter) or pJEM13 (vector control) was divided into 10-mL aliquots, cells were centrifuged at 3000 g for 4 min at room temperature and each aliquot was resuspended in 5 mL of the appropriate stress medium (pre-equilibrated at the temperature to be used in the stress experiment), and were subjected to the stress conditions for 1 h. The stress conditions were 10 mM H2O2 (oxidative stress), pH 5 (acid stress), pH 9 (alkaline stress), 0.05% sodium dodecyl sulfate (SDS) (membrane damage stress), 0.1% ethanol (dehydration stress), 10% NaCl (hyperosmolar), 0.1% NaCl (hypo-osmolar), 4 °C in an ice bath (cold shock) and 42 °C in a water bath (heat shock) as described previously (Stover et al., 1991; Bagchi et al., 2003). With the exception of cold and heat shock, bacteria were incubated in 50-mL roller bottles during the exposure to the stress conditions at 37 °C. The control sample was the culture, which was grown at 37 °C and pH 7. After the stress treatment, cells were centrifuged at 3000 g for 3 min at room temperature, resuspended in 1 mL of ice-cold β-galactosidase lysis buffer (60 mM disodium hydrogen phosphate; 40 mM sodium dihydrogen phosphate; 10 mM potassium chloride; 1 mM magnesium sulfate; and 50 mM 2-mercapto-ethanol), lysed and used for β-galactosidase assay.

β-Galactosidase assay

β-Galactosidase assay was carried out for the lacZ-expressing promoter clones of E. coli and M. smegmatis following Miller's protocol with minor alterations as reported previously (Kamalakannan et al., 2002). The colonies were selected based on the production of blue color upon plating in X-Gal-containing plates. For E. coli, overnight grown cultures were used, while for M. smegmatis 3-day-old cultures were used. The OD600 nm value was measured and 1-mL aliquot of the cell suspension was taken for the assay. The OD600 nm value was 0.8 for all cultures. After harvesting, the cell suspension was sonicated at the maximum pulse for three cycles for M. smegmatis (a 30-s pulse at a 30-s interval) and once for E. coli. The lysate was immediately used for the β-galactosidase assay.

RNA isolation and primer extension analysis

Isolation of total RNA from M. tuberculosis and M. smegmatis was carried out as described previously (Roy et al., 2004). Briefly, cells were suspended in 1 mL of trizol (Invitrogen Inc.) and RNA was extracted by bead beating, followed by phenol–choloroform extraction. End-labeling of the primers (groES477, groES534 and GEL1R2) and primer extension analysis were carried out as described previously (Roy & Ajitkumar, 2005). MoMuLV RNase H reverse transcriptase (RT) (MBI Fermentas) was used for cDNA synthesis. For the heat shock model experiment, mid-logarithmic M. tuberculosis cultures grown at 37 °C were shifted to 42 °C for 1 h and an aliquot was taken for RNA extraction. The remaining culture was shifted back to 37 °C and left undisturbed for 1 h. A second aliquot was removed and the culture was again shifted to 42 °C for 1 h, after which the cells were pelleted out. All the cell pellets were immediately solubilized in Trizol, frozen in liquid nitrogen and stored at −80 °C before RNA extraction.

Northern blot

Northern blot analysis was carried out as described previously (Narayanan et al., 2000). RNA (50 μg) was fractionated on 1% formaldehyde gel at 70 V/cm. RNA was transferred to a Hybond N+nylon membrane using the Transvac vacuum-blotting apparatus (Hoefer scientific instruments) set at a vacuum suction of 25 psi. The groES ORF, along with its promoter and downstream sequence, was PCR amplified using primers VA3 and VA5 (Table 1 and Fig. 1), end-labeled with α-[32P]-dCTP using the Rediprime labeling kit (Invitrogen Inc.) and used for hybridization. Hybridization and autoradiography were performed as described previously (Narayanan et al., 2000).

RT-PCR

RT-PCR was performed as reported previously, with minor alterations (Narayanan et al., 2000). RNA (1 μg) was reverse transcribed into cDNA using the MoMuLV RNase H RT enzyme (MBI Fermentas) at 42 °C, for 1 h, using a random hexamer primer mix (Invitrogen Inc.). The cDNA was purified by the column purification method (Qiagen) and amplified by PCR using primers UPS3AP1-UPSRKP1, VA3-VA5, VA3-GEL1R2, UPS3AP1-VA5 and UPS3AP1-GEL1R2 (Table 1 and Fig. 1). The PCR conditions were as follows: 95 °C for 5 min; 25 cycles of 95 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min; and a final extension of 72 °C for 10 min. For negative control, RT was omitted in the reaction mixture. The RT-PCR product was subjected to electrophoresis on 1.5% agarose gel.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References

Characterization of groE promoter activity

Four different deletion fragments from the putative groE promoter region, when cloned in the promoter probe vector pJEM13 (pJUPS1/926 bp, pJUPS2/611 bp, pJUPS3/277 bp and pJUPS4/115 bp) (Table 2), yielded blue colonies in both E. coli and M. smegmatis, indicating promoter activity. Out of the four fragments, the smallest fragment (pJUPS4) showed the lowest activity in both species (Fig. 1). Thus, the basal region necessary for promoter activity was about 115 bp, while enhanced activity was seen only with the 277-bp fragment. Further, the basal activity of the groE promoter was much higher than that of the groEL2 promoter. Previously, Dellagostin et al. (1995) showed that, for the Mycobacterium leprae gene coding for the 18-kDa antigen, the basal promoter was about 136 bp, while the enhanced promoter activity was seen only with the 256-bp fragment. It is of interest to note that the 18-kDa antigen of M. leprae is also a Hsp and a homologue of the 16-kDa (Acr1) antigen of M. tuberculosis. The 18-kDa promoter, like the groE promoter, was active in E. coli, M. smegmatis and BCG. Kamalakannan et al. (2002) showed that the basal promoter region for the guaA gene was about 300 bp in M. smegmatis and about 280 bp for E. coli. Thus, it seems that, in mycobacteria even though a smaller fragment is sufficient enough to express the downstream gene, a larger fragment with additional cis-acting elements is needed for stabilizing the RNA polymerase complex and/or recruiting additional trans-acting factors to the promoter.

A comparative analysis of the groE promoter with the groEL2 promoter showed that, the former was much more active than the latter both in E. coli and in M. smegmatis (Fig. 1) in accordance with our previous observation (Aravindhan et al., 2006). It is of interest to note that the groE promoter contains one CIRCE element, while the groEL2 promoter contains two such sequences and might be under tight repression (Stewart et al., 2002a, b). HrcA is the putative transcriptional repressor that binds to this sequence and induces repression, as has been shown in other organisms (Schulz & Schumann, 1996). Mycobacterium tuberculosis contains a full-length hrcA gene. But whether the protein coded by this gene can actually block groE and groEL2 promoters is yet to be determined. Microarray analysis of the ΔHrcA M. tuberculosis mutant showed strong upregulation of groES, and groEL1 genes supporting co-ordinated regulation of these genes by the CIRCE–HcrA system (Stewart et al., 2002a, b). The groE basal promoter sequence was identical in M. tuberculosis, Mycobacterium bovis, M. leprae and M. smegmatis, but was different in E. coli. Both TSS1 and TSS2 and their surrounding sequence were highly conserved in these organisms. With respect to E. coli, the −35 sequence was identical, but not the −10 sequence. No CIRCE sequence could be identified in the E. coli promoter. In spite of these sequence differences, the mycobacterial groE promoter was well recognized by the E. coli transcription apparatus and a high level of reporter gene activity was observed (Fig. 1).

The groE promoter controls the expression of the groESL1 operon

In all mycobacterial species for which genome sequences are available (M. tuberculosis, M. bovis, M. leprae and M. smegmatis), the organization of groES and groEL1 is identical –groES is upstream to groEL1. Thus, it is tempting to speculate that as in other bacteria, groES and groEL1 may form an operon in mycobacteria. However, previously, Kong et al. (1993) had shown by primer extension analysis that the groEL1 gene is not cotranscribed with the upstream groES gene, and argued against the operon-like organization of these genes in mycobacteria. In order to test this possibility, the groES–groEL1 intergenic sequence was cloned in-frame with the lacZ gene in pJEM13 vector (pJGEL1). This construct yielded white colonies in both E. coli and M. smegmatis and showed only background activity in the β-galactosidase assay, indicating the absence of a promoter in the intergenic sequence (Fig. 1). Subcloning the same fragment in another promoter probe vector (pJEM15) showed a similar phenotype in M. smegmatis. It is important to note that pJEM13 is a ‘gene fusion’-based promoter probe vector, while pJEM15 is an ‘operon fusion’-based promoter probe vector. Thus, irrespective of the vector background, the groES–groEL1 intergenic sequence lacked promoter activity. Primer extension analyses using the groEL1-specific primer (GEL1R2) and M. tuberculosis RNA also did not show any transcription start site (TSS) in the groES–groEL1 intergenic region (data not shown). These observations confirmed the absence of promoter elements in the groESgroEL1 intergenic region, implying that the promoter upstream of groES might drive the expression of both groES and groEL1 genes as in other organisms.

We next performed Northern blot analysis to confirm the bicistronic nature of the groESL1 operon. The radio-labeled PCR product, encompassing the groE promoter, groES ORF and the groES–groEL1 intergenic spacer sequence, when used as the probe, identified a 2-kb transcript, which is consistent with the size predicted for the bicistronic groESL1 operon (Fig. 2a). RT-PCR experiments further confirmed the bicistronic nature of the groESL1 operon (Fig. 2b). It is possible that the TSS identified by Kong et al. (1993) could probably represent the 5′ end of the processed groESL1 mRNA molecule. In the present study, we have provided ample evidence to show that as in other organisms, even in mycobacteria, the groES and groEL1 genes form a bicistronic operon and are cotranscribed from the groE promoter.

image

Figure 2.  Transcriptional analysis of the groESL1 operon. (a) Northern blot analysis showing the 2 kb bicistronic mRNA transcript of the groESL1 operon. One microgram of Mycobacterium tuberculosis RNA was fractionated in 1% agarose gel, transferred to a nylon membrane and probed with a radio-labeled groES PCR product. M, 1 kb DNA ladder (NEB) and 1, M. tuberculosis RNA. (b) RT-PCR was performed on total RNA isolated from M. tuberculosis. Lane 1, 1 kb DNA ladder (NEB); Lane 2, RT negative control; Lane3, UPS3AP1-UPSRKP1 (300 bp); Lane 4, VA3-VA5 (312 bp); Lane 5, VA3-GEL1R2 (412 bp); Lane 6, UPS3AP1-GEL1R2 (712 bp); and Lane 7, 100 bp DNA ladder (NEB).

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Identification of TSSs in the groE promoter

We next sought to determine the TSS(s) for the groE promoter. Primer extension analysis of RNA from M. tuberculosis, using the UPSRKP1 primer, identified two such sites: TSS1 and TSS2 (Fig. 3a). Identical results were obtained with RNA obtained from M. smegmatis harboring pJUPS3 (results not shown). TSS1 maps at position −236, at C and TSS2 at −171, at T with respect to the GTG start codon, where G is taken as +1 (Fig. 3b). In fact, the groE promoter is one of the very few mycobacterial promoters that has GTG as a start codon, and not an ATG. Out of the two TSSs, TSS1 was upstream to that of the −35 sequence and TSS2 was a single nucleotide downstream to that of the CIRCE sequence. The results were reconfirmed by repeating the experiment using a second primer, groES477 (data not shown). The consensus −10 and −35 sequences can be identified upstream to that of TSS2, while no such sequence could be located upstream to that of TSS1. The groE promoter is not unique with its two TSSs because the M. smegmatis acetamidase promoter previously identified in our lab also had two TSSs (Narayanan et al., 2000). The newly characterized M. tuberculosis groE promoter shows several similarities to that of the Corneybacterium glutamicum groE promoter, which had two overlapping CIRCE elements, an identical −35 sequence, but a different −10 sequence (Barreiro et al., 2004). The newly identified TSS2 was identical to that identified in C. glutamicum with no corresponding TSS1. Interestingly, both TSS1 and TSS2 were absent in the 115 bp groE promoter fragment of the groE promoter, which showed basal promoter activity (pJUPS4) in E. coli and M. smegmatis. This fragment also lacked other cis-acting elements such as −10, −35, and CIRCE, except for the RBS, but was still able to drive the lacZ expression. This indicates that, apart from the identified TSSs, this promoter might have other cryptic TSS(s) sites that may become functional when the major TSSs are removed. The mycobacterial CIRCE was also unique because it has seven nucleotides in the spacer region, while C. glutamicum has nine nucleotides and R. capsulatus has five nucleotides (Barreiro et al., 2004; Jager et al., 2004). A search for a functional copy of the HrcA repressor in the available mycobacterial genome databases revealed its presence in M. tuberculosis, M. bovis, M. leprae and M. smegmatis species. Primer extension analysis carried out using a heat shock model showed upregulation of TSS2, while TSS1 was constitutive (Fig. 3c). Interestingly, TSS2 underwent rapid upregulation at 42 °C and downregulation (within an hour) upon returning to 37 °C. It again became upregulated following a second heat shock, indicating rapid fine tuning of this TSS following a heat shock. The groE promoter of C. glutamicum also showed a similar kinetics of induction in a heat shock model experiment (Barreiro et al., 2004). The close proximity of TSS2 to the CIRCE sequence indicates that, probably, this TSS might be under the control of the HcrA repressor, which disassociates from CIRCE during a heat shock, and might thus be rapidly regulated (Minder et al., 2000).

image

Figure 3.  Identification of the groE transcription start site (TSS) by primer extension analysis (PEA). (a) Primer extension product and DNA sequencing products were generated by end-labeling primer groES477 using Mycobacterium tuberculosis RNA as a template. The products were separated on a 6% urea-polyacrylamide sequencing gel and visualized by autoradiography. Lanes G, A, T and C denote nucleotide-specific sequencing ladders. Lane P denotes the primer extension products. TSS1 and TSS2 are the two TSSs identified. (b) Mycobacterium tuberculosis groE promoter sequence. The −10, −35, TSS1, TSS2, RBS and start codons are boxed and labeled. The CIRCE sequence is denoted by an arrow. The two TSS identified in this study are indicated with bold letters and a bent arrow. (c) Differential utilization of TSS2 during a heat shock response. Mycobacterium tuberculosis cells were left untreated at 37°C (lane 4), heat shocked at 42°C (lane 3), heat shocked at 42°C and returned to 37°C (lane 2) or heat shocked at 42°C, returned to 37°C and again heat shocked at 42°C (lane 1). RNA was extracted and PEA was performed.

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Differential regulation of groESL and groEL2 promoters under stress conditions

Heat shock promoters are the early promoters that undergo rapid upregulation under stress conditions. Previously, Stover et al. (1991) have shown upregulation of groEL2 promoter under heat (42 °C), acidic (pH 4) and oxidative (H2O2) stresses. Similarly, having demonstrated the heat-responsive property of the groE promoter at the transcript level (Fig. 3c), we next examined whether the promoter is responsive to other stress conditions. High activity of the groE promoter was observed under all the stress conditions studied, except for cold shock and SDS stress (Fig. 4). A high level of induction was seen during heat shock, followed by osmotic, dehydration and oxidative stress conditions. A moderate increase in the activity was observed under pH stress. On the other hand, no major difference was observed in the activity of the groEL2 promoter, under various stress conditions, except under hypo-osmolar stress, where the promoter activity was found to be reduced. In order to ensure that the differential activity of the two promoters studied was not due to cell death, a viability count was performed for all the cultures after the stress treatment. No difference in the cell viability were observed before and after the stress, indicating that the difference in the promoter activity was not due to loss of cell viability (data not shown). It is interesting to note that even though both groE and groEL2 promoters are heat shock promoters and are under the control of the CIRCE element, only the groE showed induction during heat shock, while the groEL2 was more constitutive. Even though these are purely artificial conditions and cannot be equated to the in vivo conditions, study of the differential regulation of heat shock promoters under these conditions provides us with a clue about the complex regulatory network that might operate under in vivo conditions. Thus, perhaps the groE promoter might be more active during infection while groEL2 might be more constitutive (Fossati et al., 2003). To conclude, in the present study, a new mycobacterial heat shock promoter (groE), which controls the expression of the bicistronic groESgroEL1 operon, was identified and characterized. This promoter was found to be much more active than the widely used groEL2 promoter under house keeping and stress conditions. Elucidation of the transcriptional regulation of this promoter might shed more light on the complexities of differential gene regulation in mycobacteria.

image

Figure 4.  Differential regulation of the groE and groEL2 promoters under various stress conditions. Recombinant Mycobacterium smegmatis cells harboring pJUPS3 (groE promoter; white bar) or pJGEL2 (groEL2 promoter; black bar) were grown in LBT medium till OD600 nm of 0.8. The cells were harvested, washed and subjected to various stress conditions for 1 h as detailed in Materials and methods. The β-galactosidase activity was estimated using the modified Miller's procedure. Significant upregulation was observed for the groE promoter under heat, pH, osmotic and dehydration stresses. Significant downregulation was observed for the groEL2 promoter under hypo-osmolar stress. Under other conditions, the groEL2 promoter showed constitutive expression. Each bar represents the mean±SE of three independent experiments.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References

V.A. and A.J.C. acknowledge the financial support from the Council of Scientific and Industrial Research (CSIR) and the Indian Council of Medical Research (ICMR), respectively. The DBT-supported phosphorimager facility at the Indian Institute of Science is acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
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