Department of Infectious Diseases and Microbiology, Centre for Molecular Microbiology and Infection, Imperial College of Science Technology and Medicine, London SW7 2AZ, UK1
School of Crystallography, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK2
Department of Medical Microbiology, St Georges Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK3
Author for correspondence: Graham Stewart. Tel: +44 207 594 3090. Fax: +44 207 594 3095. e-mail: g.stewart{at}ic.ac.uk
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ABSTRACT |
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Keywords: HspR, HrcA, Hsp70, Hsp60, transcriptional regulator
Abbreviations: ADC, albumin/dextrose (glucose)/catalase; CIRCE, controlling inverted repeat of chaperone expression; HAIR, HspR-associated inverted repeat; OADC, oleic acid/dextrose (glucose)/albumin/catalase
a A list of the 100 ORFs most highly induced by heat shock is provided as supplementary data with the online version of this paper (http://mic.sgmjournals.org).
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INTRODUCTION |
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We have previously demonstrated that the regulation of the 70 kDa heat-shock protein, Hsp70 or DnaK, is mediated, as in Bacillus subtilis, by release of transcriptional repression (Stewart et al., 2001 ). In addition, positive regulation of Hsp70 via an alternative sigma factor, as in Escherichia coli (Grossman et al., 1984
), has also recently been demonstrated (Raman et al., 2001
). In B. subtilis, a single repressor, HrcA, coregulates the Hsp70 heat-shock genes together with those encoding the Hsp60 GroE family (Hecker et al., 1996
). In contrast, deletion of the Hsp70 regulator, HspR, in M. tuberculosis had no detectable effect on expression of Hsp60 proteins (Stewart et al., 2001
). This suggests that a second repressor which has similarity to HrcA may play a role in the mycobacterial heat-shock response. To test this hypothesis, we have generated mutant strains of M. tuberculosis with deletions affecting both of the repressors and characterized the resulting effects on patterns of gene expression.
In order to study transcriptional regulation at a global level, we have used microarrays prepared by spotting DNA sequences representing 3924 of the ORFs identified in the M. tuberculosis H37Rv genome sequence (Cole et al., 1998 ). Competitive hybridization to the microarray using RNA/cDNA prepared from different strains of M. tuberculosis or from bacteria exposed to different conditions provides a window on the overall pattern of transcriptional changes. By combining whole-genome analysis with targeted mutagenesis, we have been able to distinguish the contribution of two regulatory circuits to the overall heat-shock response and to reveal a number of novel heat-shock genes.
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METHODS |
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Deletion of hspR, hrcA in M. tuberculosis.
The gene replacement of hspR with the hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus has been described previously (Stewart et al., 2001 ). The sequential deletion of hrcA to generate a double hspR hrcA mutant strain was achieved using a similar suicide delivery strategy but replacing the target gene, hrcA, with the kanamycin-resistance gene (aph) from Tn903. Briefly, 1·5 kb regions of DNA up- and downstream of hrcA were cloned around the aph gene in the mycobacterial suicide plasmid pSMT99 to make pSMT163. This plasmid cannot replicate in mycobacteria and carries sacB for counterselection against single crossover and illegitimate integration of the plasmid. One microgram of plasmid was irradiated with 100 mJ cm-2 UV (Hinds et al., 1999
) and electroporated into M. tuberculosis or M. tuberculosis
hspR (Wards & Collins, 1996
). Following overnight recovery of the cells in 7H9/ADC, gene replacement transformants were directly selected on 7H11/OADC containing hygromycin, kanamycin and sucrose. Deletion of hrcA was confirmed by Southern blotting of KpnI digested genomic DNA using the 1·5 kb upstream hrcA fragment as hybridization probe.
Complementation of M. tuberculosis hspR.
pKinta is a ColE1-based E. coli plasmid which carries the aph kanamycin-resistance gene and the int gene and attP site from the L5 mycobacteriophage (Stover et al., 1992 ). This plasmid integrates into the chromosome in a single copy by site-specific recombination at the attB site. The Hsp70 operon promoter containing the two HspR-associated inverted repeat (HAIR)-regulated promoter regions (Stewart et al., 2001
) was amplified by PCR using the primers Hsp701 (tcggtcaagctggcggactga) and Hsp702 (agccatggtgaatcctcctg) and cloned into the SacI site of pKinta. The hspR ORF was then amplified and cloned downstream of the hsp70 promoter so as to transcriptionally fuse the ORF with its own promoter albeit without the intervening hsp70, grpE and dnaJ sequence. The resultant plasmid, pSMT168, was introduced into M. tuberculosis
hspR by electroporation.
RNA extraction and cDNA labelling.
Ten millilitres of broth culture in late exponential phase was added directly to 40 ml GTC solution containing 5 M guanidinium thiocyanate, 0·5% sodium N-lauryl sarcosine, 0·1 M ß-mercaptoethanol and 0·5% Tween 80. The bacteria were pelleted by centrifugation and resuspended in 1·2 ml Trizol (Life Technologies) with 0·5 ml of 0·1 mm silica/ceramic beads and processed at 6·5 W for 45 s in a Ribolyser (reciprocal shaker: Hybaid). The samples were centrifuged at 13000 r.p.m. for 15 min in a microfuge to remove bacterial debris and the supernatant removed to a fresh tube. The phases were separated by the addition of 0·6 ml chloroform, mixing and centrifugation. The aqueous phase was re-extracted with chloroform and the RNA precipitated with propan-2-ol, washed in 70% ethanol and dissolved in RNase-free water. The RNA was treated with amplification-grade DNase I (Life Technologies) and cleaned up by RNeasy purification (Qiagen).
cDNA was labelled by incorporation of Cy3 or Cy5 dCTP (Amersham) during reverse transcription of RNA. RNA (210 µg) was mixed with 3 µg random hexamer oligonucleotides in 11 µl water, heated to 95 °C and snap-cooled. In a total volume of 25 µl, the labelling reaction was initiated by the addition of 5 µl First Strand Buffer, 25 mM DTT, 1 mM each dATP, dGTP and dTTP, 0·4 mM dCTP, 2 nmol Cy3- or Cy5-dCTP and 500 U Superscript II reverse transcriptase (Life Technologies). The reaction was incubated in the dark at 25 °C for 10 min and then at 42 °C for 90 min. The relevant pairs of Cy3- (wild-type H37Rv) and Cy5- (mutant strain or heat-shocked cells) labelled cDNA were mixed and purified using a Qiagen MinElute kit, eluting in water.
Microarrays and hybridizations.
Whole genome microarrays were constructed by robotic spotting on to poly-L-lysine-coated glass microscope slides (MicroGrid II, BioRobotics, UK) of PCR amplicons (size range 601000 bp; mean 517 bp) derived from portions of each of the 3924 predicted ORFs of the sequenced strain of M. tuberculosis H37Rv. Primer pairs for each ORF were designed with Primer 3 software and selected by BLAST analysis to have minimal cross-homology with all other ORFs. All procedures used, including post-processing of deposited arrays, were as described by others (Wilson et al., 1999 , 2001
).
The microarray was incubated in prehybridization solution (3·5xSSC, 0·1% SDS and 10 mg BSA ml-1) at 65 °C for 20 min. The slide was rinsed in water for 1 min and propan-2-ol for 1 min before drying by centrifugation at 1500 r.p.m. for 5 min.
The purified Cy3/Cy5-labelled cDNA was adjusted to 16 µl in 4x SSC and 0·3% SDS. This hybridization solution was heated to 95 °C for 2 min, briefly centrifuged and applied to the array under a cover slip. The slide was sealed in a humid hybridization cassette and incubated at 65 °C in the dark for 1620 h. The slide was washed for 2 min at 65 °C in 1x SSC/0·05% SDS, for 2 min at room temperature in 0·06x SSC and then dried by centrifugation. The hybridized microarrays were scanned with an Affymetrix 428 scanner. The scanned images were analysed with ImaGene4.1 and the median spot intensities calculated.
Data processing and statistical analysis.
For each strain or condition, three or four independent RNA preparations were analysed. Background values were subtracted from signal values. In cases where this resulted in negative values, a small positive constant was assigned to prevent numerical problems when forming ratios or taking logarithms. Data were normalized to median signal intensities. All values were log2-transformed for further analysis.
Significance values were calculated for each ORF in the mutant:wild-type comparisons through an ANOVA analysis. Each of the three datasets (wild-type versus hspR; wild-type versus
hspR pSMT168; wild-type versus
hspRhrcA) forms a balanced factorial design. Three main effects were taken into account: the array effect A for each array, the gene effect G for each gene and the variety effect V for the two varieties, mutant and control. In addition, the three pairwise interactions between the main effects, that is, interactions A:V, A:G and V:G, have been accounted for. The resulting residuals stem from the A:V:G interaction of all three main effects which were used to estimate the standard error. One problem is that the residual or error variance is much higher for low expression values, which is not unexpected considering the higher uncertainty in these values. Hence, using one standard error estimate for all genes does not seem appropriate. Instead, we resampled from the residuals, redistributed them over the expected response values and fitted new models to these bootstrap replicates. The multitude of models allowed us to calculate confidence intervals for the estimates of the effects. The final value is based on the difference in estimated V:G effects, which represents the influence of variety, that is, mutant or control, on gene expression. Confidence intervals for these effects are calculated through the resampling procedure as above. Final P-values are obtained from confidence intervals by Bonferroni correction for multiple testing, that is, all raw P-values are multiplied by the number of genes resulting in the final adjusted P-values.
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RESULTS |
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We have previously described unsuccessful attempts to complement the M. tuberculosis hspR strain (Stewart et al., 2001
). Reintroduction of the gene with a constitutive promoter or even gently induced expression from the acetamidase promoter (Parish & Stoker, 1997
) rendered the bacteria non-viable. These findings suggest that expression of reintroduced hspR would have to be appropriately regulated so as to closely match wild-type expression dynamics. To achieve this, the hspR gene was cloned under the control of the natural promoter of the hsp70 operon, which includes two HAIR sequences. A single copy of this construct was inserted at the attB phage integration site in the chromosome of M. tuberculosis
hspR. In contrast to previous attempts at complementation, this strain was fully viable. Whole-genome expression profiling of the complemented mutant showed a pattern largely similar to the original wild-type strain (Fig. 1c
). The reintroduced hspR gene was approximately twofold overexpressed, demonstrating that the complementing construct did not express hspR identically to wild-type. This may be due to the changed position of hspR in relation to its promoter, or perhaps reflects some stoichiometric relationship between hspR expression and the number of HAIR sites. However, all the genes overexpressed in the
hspR strain showed a complete or substantial reduction of overexpression in the complemented strain (Table 1
). This demonstrates that the altered transcriptome of the mutant was specifically due to the absence of hspR and not to polar effects on neighbouring genes or to an inadvertently selected mutation.
The HrcA regulon
ORF Rv2374c in the M. tuberculosis genome shares sequence homology with the family of heat-shock repressors related to the hrcA gene of B. subtilis. To test whether this ORF is similarly involved in heat-shock regulation in M. tuberculosis, we undertook a deletion strategy analogous to that used to generate the hspR mutant, replacing hrcA with a kanamycin-resistance gene. We were unable to generate
hrcA mutants in wild-type M. tuberculosis, yet were successful at introducing the mutation into M. tuberculosis
hspR (Fig. 4a
). SDSPAGE analysis of the total protein profile of the double knock-out M. tuberculosis
hspR
hrcA demonstrated constitutive overexpression of proteins consistent in size with Hsp70, Hsp60 (GroEL) and GroES, as well as an additional band at approximately 20 kDa (Fig. 4b
).
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DISCUSSION |
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Heat shock is both a convenient and robust laboratory model for studying transcriptional regulation as well as an important element in the pathogenesis of M. tuberculosis. Heat-shock proteins are induced following uptake of M. tuberculosis by host cells (Lee & Horwitz, 1995 ; Monahan et al., 2001
), presumably contributing to bacterial survival in the phagocytic environment. However, they are also used by the host cell as peptide chaperones to enhance MHC class I presentation (Castellino et al., 2000
; Suto & Srivastava, 1995
) and as a signal to the immune system (Asea et al., 2000
). Directly because of these immunological roles of heat-shock proteins, the constitutive overexpression of bacterial Hsp70 proteins results in reduced survival of M. tuberculosis during prolonged infection (Stewart et al., 2001
). By understanding the regulation of the heat-shock response, we may be able to design interventions capable of enhancing resistance to persistent infection with tuberculosis.
Microarray analysis of an hspR deletion mutant of M. tuberculosis confirms and extends previous studies of Hsp70 regulation. HspR is a DNA-binding protein related to the MerR family. It recognizes either of two inverted repeat sequences (HAIR) in the promoter region of the hsp70 operon, reducing the level of transcription in unstressed conditions. The HspR protein interacts tightly with Hsp70 in vitro (Bucca et al., 2000 ; Stewart et al., 2001
). A system where this heterodimer forms the functional repressor unit with feedback achieved by titration of Hsp70 away from the HspR complex in the presence of unfolded polypeptides represents an attractive model for regulation (Bucca et al., 2000
; Narberhaus, 1999
). As predicted, we show that in the absence of HspR, there is release of transcriptional repression, and the genes of the Hsp70 operon are upregulated. There were also a further 46 genes with significantly elevated transcription. Of these, only three genes (Rv0249cRv0251c), arranged consecutively in an apparent operon, were associated with a HAIR-like sequence. Interestingly, the lead gene Rv0251c has also been shown to be under the control of the heat-shock responsive ECF sigma factor,
E, and is also prominent in response to treatment with SDS (Manganelli et al., 2001
). This dual control mechanism may account for the relatively modest elevation of Rv0251c transcription in the
hspR mutant compared with that observed under heat-shock conditions in the wild-type. Rv0251c encodes a 159 amino acid protein belonging to the small heat-shock protein family, termed Hsp20, or the
-crystallin family. Its predicted size is consistent with the approximately 20 kDa protein found by SDS-PAGE to be upregulated in the
hspR
hrcA mutant (Fig. 3b
). The small heat-shock proteins, like the larger heat-shock protein families, are found widely in bacterial and eukaryotic cells, and appear to function as molecular chaperones at least in vitro (Chang et al., 1996
; Yang et al., 1999
). There are two members of this family in M. tuberculosis. The other family member was originally identified as a prominent antigen and is variously referred to as the 14 kDa antigen, 16 kDa antigen, Hsp16·3,
-crystallin (Acr) or HspX. This gene is not induced by heat shock but is upregulated in stationary-phase cultures and during the hypoxic response (Cunningham & Spreadbury, 1998
; Sherman et al., 2001
; Yuan et al., 1996
, 1998
). It is possible that the different Acr homologues fulfil analogous functional roles in response to different stresses. The Acr gene is induced following phagocytosis of M. tuberculosis (Monahan et al., 2001
) and is required for growth in macrophages (Yuan et al., 1998
). It will be of interest to determine whether the protein encoded by Rv0251c, which we term Acr2, also plays a role during infection.
Within the hspR-upregulated ORF set, the Hsp70 and Acr2 operon genes were upregulated during heat shock along with bfrB, groES and Rv3654c. The bacterioferritin gene, bfrB, and Rv3654c, encoding an 8 kDa protein with unknown function, are not preceded by obvious HspR binding sites, but their coregulation with HAIR-associated genes in both heat shock and the mutant suggest an indirect link to HspR. The majority of genes upregulated in the mutant were neither associated with HAIR sequences nor upregulated during heat shock. We conclude that the induction of these genes is a consequence of the physiological changes associated with overexpression of the HspR-regulated proteins and may not be directly relevant to the normal heat-shock response. An interesting example of this was the trend for upregulation of ribosomal protein expression, which was also mirrored in the
hspR
hrcA strain.
A surprising omission from the hspR upregulated list was clpB, which encodes another probable molecular chaperone. We have previously shown the elevation of ClpB expression in the mutant by proteomic analysis (Stewart et al., 2001
), which suggests that the clpB mRNA is of a sufficiently short half-life to preclude detection of the
hspR-associated transcriptional increase. The detection of substantially increased clpB mRNA in the wild-type after heat shock at 45 °C is explained by upregulation of clpB transcription by the heat-inducible sigma factor,
H, as well as release of HspR repression (Raman et al., 2001
).
We were able to delete the proposed hrcA gene in the hspR mutant, but the same approach has been unsuccessful with wild-type M. tuberculosis. This may reflect some technical problem, but it is also possible that overexpression of Hsp70 proteins compensates in some way for a deleterious effect of hrcA deletion. Upregulation of the major HspR-regulated genes was preserved in the double mutant, alongside upregulation of the HrcA regulon, which included the Hsp60 family genes, groES, groEL1 and groEL2. GroES is functionally related to GroEL, and its gene is situated immediately upstream of groEL1. While the expression of groES was enhanced in the
hspR mutant, its upregulation in the
hspR
hrcA strain was much greater. The M. tuberculosis HrcA protein has yet to be analysed for DNA binding in vitro, but it has a strong sequence similarity to B. subtilis HrcA, and analogous CIRCE-like structures are present in the groES/groEL1 and groEL2 promoter regions. Thus, we can conclude that the HrcA repressor acts as the main transcriptional controller of the Hsp60/GroE family heat-shock response, with some cross-talk between the Hsp60 and Hsp70 responses demonstrated by the induction of GroES expression in the hspR deleted strain. The mechanism for this cross-talk is unclear, although a weak match for the HspR binding site, HAIR, is present at the beginning of the GroES ORF. Interaction of HspR with this inverted repeat could conceivably have a more subtle effect on transcription than that observed with HAIR sequences that directly overlap the RNA polymerase footprint.
A good match for the CIRCE sequence is found upstream of another hspR
hrcA upregulated gene, Rv0991c, which encodes a conserved hypothetical protein with unknown function. Expression of both Rv0991c and the adjacent downstream ORF, Rv0990c, was elevated during heat shock, but Rv0990c was not significantly upregulated in the mutant. Whether the two genes are transcribed as a bicistronic message or are separately regulated and transcribed remains to be conclusively determined. Thus, it is clear that HrcA regulates not just the Hsp60 heat-shock response but also Rv0991c and probably Rv0990c. In light of the effect of the
hspR mutation on the virulence of M. tuberculosis (Stewart et al., 2001
), it will be of considerable interest to study the double mutant in infection models.
It is obvious from the 45 °C transcriptional snapshot that the HspR and HrcA regulons, which dominate the heat-shock proteome, constitute only a part of the overall adaptive response. Genes regulated by H and
E are prominent in the 45 °C response, and upregulation of the
B gene suggests overlap with the general stress response. These different regulatory layers are interlinked, with hsp70 and clpB under dual HspR and
H control, and acr2 under dual HspR and
E control. Moreover, the heat-inducible expression of
B and
E is dependent on
H, which autoregulates its own expression (Raman et al., 2001
). In addition, it is probable that the functional activity of the sigma factors is subject to post-translational control by anti-sigma factor pathways (Helmann, 1999
). Detailed analysis of bacteria exposed to different temperatures for different time periods will be important in further dissection of this complex pattern of regulatory circuits.
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ACKNOWLEDGEMENTS |
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Received 16 April 2002;
revised 1 July 2002;
accepted 4 July 2002.