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Department of Medicine, Johns Hopkins University, CRB2, Rm 1.08, 1550 Orleans Street, Baltimore, MD 21231-1044, USA
Correspondence
William R. Bishai
wbishai{at}jhmi.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Galactosidase gene fusion analysis revealed induction of the whiB1 promoter in M. tuberculosis on addition of exogenous dibutyric cAMP (a diffusible cAMP analogue) only when an intact CRP-binding site was present. These results indicate that M. tuberculosis whiB1 transcription is regulated in part by cAMP levels via direct binding of cAMP-activated CRPM to a consensus CRP-binding site in the whiB1 5'UTR.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). M. tuberculosis can survive in diverse surroundings ranging from the human host to droplet nuclei in the atmosphere. Adaptation to such diverse conditions requires controlled regulation of the expression of key genes that allow the bacillus to alter its physiology in response to changes in environmental stimuli. Sequencing of the M. tuberculosis genome has revealed the presence of more than 100 regulatory proteins, 13 sigma factors and 11 two-component systems (Cole et al., 1998), which provide the bacterium with a high degree of adaptability.
The Wbl (WhiB-like) family of proteins is present throughout the actinomycetes but absent from all other organisms evaluated so far (Molle et al., 2000; Soliveri et al., 2000). Due to the presence of a conserved helix–turn–helix motif, these proteins are believed to function as DNA-binding transcription regulators. The first of these proteins, WhiB, was identified in Streptomyces coelicolor, a Gram-positive sporulating bacterium closely related to M. tuberculosis. S. coelicolor whiB mutants produce abnormally long, tightly coiled aerial hyphae that are completely blocked in their ability to form sporulation septa (Chater, 1972; Davis & Chater, 1992; Flardh et al., 1999). Studies of whiB orthologues in mycobacteria have shown that the M. smegmatis whiB2 gene (also called whmD) is essential (Gomez & Bishai, 2000); M. tuberculosis whiB3 plays a role in virulence and its gene product may interact with a sigma factor of RNA polymerase (Steyn et al., 2002); whiB7 of M. tuberculosis is involved in multi-drug resistance (Morris et al., 2005). Each of the Wbl family of proteins contains four invariant cysteine residues, which are believed to be involved in binding a [4Fe–4S] cluster (Jakimowicz et al., 2005). The functional importance of this cluster is emphasized by the observation that none of the four whiD alleles carrying mutations at these cysteine residues was able to complement the whiD mutant phenotype in S. coelicolor (Jakimowicz et al., 2005).
It has recently been reported that the M. tuberculosis gene Rv3676 encoding a CRP-FNR family protein (hereafter referred to as CRPM) is defective in the vaccine strain Mycobacterium bovis BCG (Spreadbury et al., 2005). Moreover, an M. tuberculosis mutant lacking an intact Rv3676 gene is attenuated for virulence and shows reduced expression of several mycobacterial genes including resuscitation promoting factor, rpfA (Rickman et al., 2005). One of the genes that was dependent on the presence of Rv3676 for full expression was whiB1, suggesting the role of cAMP in the expression of whiB1 in M. tuberculosis.
In this study we show that whiB1 is transcribed by a class I-type CRP-dependent promoter. We provide evidence that whiB1 transcription is regulated by varying cAMP levels via a mechanism involving direct binding of cAMP-activated CRPM to a consensus site adjacent to the whiB1 promoter.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(F'/endA1 hsdR17 [
] glnV44 thi-1 recA1 gyrA [Nalr] relA1 
[lacIZYA–argF]U169 deoR [
80dlac(lacZ)M15]), from Stratagene, and BL21(DE3) (F– ompT hsdSB [
] gal dcm [DE3]), from Novagen. M. tuberculosis CDC1551 was obtained from Colorado State University, CO, USA. Plasmid pSD5B (mycobacteria–E. coli shuttle vector carrying promoterless lacZ gene, KmR; Jain et al., 1997) was kindly provided by Dr Anil Tyagi, University of Delhi South Campus, New Delhi, India, and pET-22a (expression vector with pBR322 origin of DNA replication and T7 promoter, ApR) was obtained from Novagen. Luria–Bertani (LB) broth and LB agar media were used for culturing E. coli, and 7H9 broth and 7H10 agar, both supplemented with 1x OADC were used for culturing mycobacteria. Both E. coli and mycobacteria were grown at 37 °C with shaking at 200 r.p.m. For cAMP treatment, the M. tuberculosis cells were cultured in 7H9 medium supplemented with 1x OADC (oleic acid/albumin/dextrose/catalase) and harvested at the desired OD600. The cell pellet was resuspended in 0.1 vol. 0.125 M potassium phosphate buffer, pH 7.4, and divided into two equivalent portions. One of these was incubated with a cell-diffusible form of cAMP, dibutyric cAMP (db-cAMP) (20 mM) and the other with an equivalent concentration of butyric acid (20 mM). After incubation for 2 h at 37 °C with shaking, the cell pellets were harvested and stored at –70 °C. Antibiotics were added when necessary: ampicillin (50 µg ml–1) and kanamycin (25 µg ml–1 for E. coli and 15 µg ml–1 for mycobacteria).
DNA techniques.
Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs and Taq polymerase was purchased from Invitrogen. Protocols for DNA manipulations, including plasmid DNA preparation, restriction endonuclease digestion, agarose gel electrophoresis, and isolation and ligation of DNA fragments were as described by Sambrook et al. (1989). E. coli DH5 was transformed by the standard protocol (Sambrook et al., 1989) and M. tuberculosis by electroporation. PCR amplifications were carried out according to the manufacturer's specifications (Applied Biosystems). Each of the 30 cycles was carried out at 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, followed by final extension at 72 °C for 10 min. DNA fragments used for cloning and labelling reactions were purified by using the Qiagen gel extraction kit according to the manufacturer's specifications.
Mapping the 5' end of the whiB1 mRNA.
The 5'-RACE (rapid amplification of cDNA ends) technique (Frohman, 1993) was used to determine the transcription initiation site with a kit purchased from Invitrogen. Total RNA was isolated from M. tuberculosis (mid-exponential phase) by using the Trizol method according to the instructions provided by the supplier (Invitrogen). The abridged anchor primer (AAP) and abridged universal amplification primers (AUAP) were used in combination with the gene-specific primers. The gene-specific primers used for RT-PCR, nested PCR1 and nested PCR2 were whiB1/RACE1 and whiB1/RACE2, respectively (see Fig. 1b). The PCR products were subsequently sequenced by using the gene-specific primer whiB1/RACE2.
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-galactosidase activity.
250 bp in length containing the whiB1 promoter sequence from positions –214 to +33 with respect to the transcriptional start point was PCR-amplified from chromosomal DNA of M. tuberculosis using the primers PwhiB1(F) (carrying an XbaI site) and PwhiB1(R) (carrying an SphI site) (Fig. 1b
). A transcriptional fusion construct was generated by cloning the PCR amplicon in pSD5B at the XbaI and SphI sites, and the recombinant vector was introduced into M. tuberculosis by electroporation. Promoter activity was determined by -galactosidase assays using cell lysates of the cultures harvested at their exponential phase of growth. Assays were performed as described by Miller (1972); the values presented are means of three separate assays.
Generation of point mutations.
DNA fragments carrying the mutations in promoter sequences were constructed by two-step PCR amplification using overlapping modified primers, as described earlier (Good & Nazar, 1992). After cleavage with XbaI/SphI, these fragments were cloned in pSD5B, the recombinant vectors were used to transform the M. tuberculosis CDC1551 strain, and the transformants were grown on 7H10 agar plates containing kanamycin (25 µg ml–1) and X-Gal (60 µg ml–1).
Purification of CRPM of M. tuberculosis.
The gene encoding the CRPM of M. tuberculosis was PCR-amplified from M. tuberculosis chromosomal DNA using gene-specific primers, Pr3676(F) (5'-GGGGCATATGGACGAGATCCTGGCCAGGG-3') and Pr3676(R) (5'-GGGGCTCGAGCCTCGCTCGGCGGGCCAGTCT-3'). These primers were selected such that the amplicons carried the sequence of the complete ORF excluding the termination codon. The amplicons were cloned into the expression vector pET-22a at the NdeI and XhoI sites. The resulting construct pETcrp was used to transform E. coli BL21(DE3). CRP with histidine tags at the C-terminus was purified from the transformants by Ni-NTA chromatography according to the manufacturer's protocol (Qiagen). Purity of the protein was analysed by SDS-PAGE.
Electrophoretic mobility shift assay.
DNA probes used for electrophoretic mobility shift assay (EMSA) analysis were prepared by PCR amplification of the desired regions of the whiB1 promoter, using 5'-end biotin-labelled synthetic oligonucleotides as the primers. The amplicons were purified from agarose gels and used for gel-shift experiments. The EMSA reaction mixture (10 µl) contained 20 fmol biotin-labelled probe and CRPM in binding buffer (40 mM HEPES/NaOH pH 7.5, 50 mM KCl, 2 mM MgCl2, 0.01 mM EDTA, 1 mM DTT and 1 mg BSA ml–1) containing 1 µg nonspecific competitor DNA poly(dI-dC) (Amersham Pharmacia Biotech) and 1 mM cAMP. Following incubation at 37 °C for 15 min, the DNA–protein complexes were resolved in a native 5 % polyacrylamide gel (acrylamide : bisacrylamide, 30 : 1, w/w) in 0.5x TBE buffer (45 mM Tris base, 45 mM boric acid and 1 mM EDTA, pH 8.0). Samples were electrophoresed at 100 V, and transferred to positively charged nylon membranes (Amersham Pharmacia Biotech). After UV cross-linking (100 µJ cm–2 for 2 min), the biotinylated probes in the membrane were detected using the LightShift Chemiluminescent EMSA kit (Pierce). Each EMSA was performed three times to confirm CRPM-DNA binding.
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RESULTS |
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). In order to define the promoter region, we determined the transcription start point (TSP) of the whiB1 transcript by the 5'-RACE technique, as described in Methods. Total RNA was isolated from M. tuberculosis in mid-exponential phase (OD600 0.6), and final amplification of dCTP-tailed cDNA was performed using primers AUAP and whiB1/RACE2 (Fig. 1b). As shown in Fig. 1(a), PCR amplification of the RACE cDNA products revealed a major band (>200 bp in size) which upon DNA sequencing corresponded to a whiB1 TSP from a purine base, G, located 109 bp upstream of the translation start codon (Fig. 1b). Analysis of the sequence upstream of this TSP revealed the presence of promoter-like sequences at the –35 (TTGACA) and –10 (AACGAT) positions. The minor band (<100 bp in size) observed in Fig. 1(a) proved by sequence analysis to be an artefactual primer-dimer amplification product.
The CRP-binding consensus sequence in E. coli (5'-TGTGA-TCTAGA-TCACA-3') exhibits perfect twofold sequence symmetry, with the bold-faced bases representing the left and right arms for binding of the active CRP dimer (Berg & von Hippel, 1988; de Crombrugghe et al., 1984). The putative CRP-binding site in the whiB1 5'UTR (5'-AGTGAGATAGCCCACG-3') is located between positions –51 and –66 bp (centred at –58.5 bp) upstream of the TSP, and contains the critical residues, 5'-GTG-3' in the left arm and 5'-CAC-3' in the right arm (Fig. 1b). The left arm contains a near-perfect sequence (AGTGA) and the right arm contains 3/5 matching residues (CCACG).
Transcription of whiB1 is constitutive and positively regulated by CRPM
In order to assess the expression of the whiB1 promoter in M. tuberculosis, a DNA fragment carrying the wild-type whiB1 5'UTR (spanning positions –214 to +33) was cloned upstream of a promoterless lacZ gene in pSD5B (a mycobacterial promoter probe vector), and the recombinant plasmid (pSD5B-wtwhiB1) was used to transform M. tuberculosis. Promoter activity was determined by -galactosidase assays with cell lysates of bacilli harvested during different phases of growth (OD600 0.4, 0.8, 2.0 and 3.5, respectively). As shown in Fig. 2(b), the -galactosidase specific activity resulting from whiB1 promoter expression at different growth phases remained constant, thus indicating that whiB1 is expressed constitutively in M. tuberculosis. Recently, a similar pattern of whiB1 expression was observed in M. tuberculosis by Geiman et al. (2006) by using the RT-PCR method.
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), known to be critical for CRP binding in E. coli (Jansen et al., 1987), were replaced, altering the wild-type left arm consensus sequence from AGTGA to AGGTA (Fig. 2a). The mutant promoter DNA fragment was cloned in pSD5B (resulting in the construct pSD5B-mtwhiB1), and the activities of both the wild-type and mutant whiB1 promoters were compared in M. tuberculosis at OD600 0.4, 0.8, 2.0 and 3.5, as described above. As shown in Fig. 2(b), the whiB1 promoter with a wild-type CRP-binding site exhibited -galactosidase activities of 7500 nmol min–1 mg–1 in M. tuberculosis. Point mutations in the CRP-binding site resulted in a 3–4-fold reduction in -galactosidase specific activity to 2000 nmol min–1 mg–1 at each OD600 tested (Fig. 2b). These observations indicate that the putative CRP-binding site is required in its intact form for full-level whiB1 expression in M. tuberculosis.
CRPM regulates expression of whiB1 by directly binding to the upstream CRP recognition site
Based on the whiB1 promoter activities in mycobacteria, we examined whether whiB1 expression was regulated by direct binding of CRPM to the putative CRP-recognition sequence adjacent to the whiB1 promoter. We performed EMSA analyses using purified recombinant CRPM and DNA comprising the whiB1 promoter region and putative CRP binding site. The target DNA region extended from base –214 to +33 relative to the TSP, and was PCR-amplified using forward primer PwhiB1(F) and biotin-labelled reverse primer PwhiB1(R) (Fig. 1b) as described in Methods. Recombinant CRPM protein was expressed in E. coli as a fusion protein with a His6 tag at its C-terminus and purified using metal affinity chromatography. Analysis of the purified protein by SDS-PAGE confirmed that the preparation was homogeneous, and CRPM had an apparent molecular mass of approximately 26 kDa, which is in good agreement with its predicted molecular mass (Fig. 2c).
As shown in Fig. 2(d), CRPM was able to bind the wild-type whiB1 5'UTR with a concentration of 1.5 µM per 2 nM template DNA fragment (Fig. 2d, lane 2). Addition of CRPM at lower concentrations did not result in sufficient complex formation to be detected by EMSA under these conditions (data not shown). In order to confirm that the binding was specific, the binding reactions were carried out in the presence of a 20 molar excess of unlabelled target DNA. In the presence of competitor, binding was completely abolished (Fig. 2d, lane 3), indicating that CRPM binds specifically to the whiB1 5'UTR. To verify that CRPM binds to its predicted CRP recognition site in the whiB1 5'UTR, we performed the EMSA experiment with a whiB1 5'UTR containing a defective CRP-binding site (substitution of the left arm consensus sequence from 5'-AGTGA-3' to 5'-AGGTA-3', Fig. 2a) as the template. As shown in Fig. 2(d), lane 5, CRPM is unable to bind to the mutant CRP-recognition site in whiB1 5'UTR, thus supporting the premise that CRPM directly regulates the expression of whiB1 in M. tuberculosis by specifically binding to the CRP-recognition site adjacent to the whiB1 promoter.
Regulation of the expression of whiB1 by CRPM requires cAMP
It is well established that both CRP and CRPM specifically bind to DNA at CRP recognition sites in the presence of cAMP (Bai et al., 2005; Botsford & Harman, 1992; Busby & Buc, 1987; Crasnier, 1996; Kolb et al., 1993; Lawson et al., 2004). To characterize the role of cAMP in CRPM binding to the whiB1 5'UTR, we carried out EMSA with CRPM and the whiB1 5'UTR in the presence of a gradient of cAMP concentrations varying from 0.1 µM to 1 mM (Fig. 3a). Since no complex was observed when cAMP was omitted from the binding reaction, our results demonstrate that the mycobacterial CRP requires cAMP for significant binding to the whiB1 5'UTR (Fig. 3a, lane 2). Although binding of CRPM to the whiB1 5'UTR was observed in the presence of cAMP at concentrations as low as 0.1 mM, maximal complex formation required 1 mM cAMP (Fig. 3a). In addition, CRPM was unable to bind to the mutant CRP-recognition site in the whiB1 5'UTR at any concentration of cAMP used, thus indicating that CRPM binds specifically to the CRP-recognition site in the whiB1 5'UTR (Fig. 3b). Our results therefore indicate that the ability of CRPM to bind the whiB1 5'UTR varied directly with the cAMP concentration.
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-galactosidase assays with cell lysates of bacilli harvested after treatment with either db-cAMP or butyric acid at different phases of growth (OD600 0.4, 0.8, 2.0 and 3.5). As shown in Fig. 3(c), at all OD600 levels, db-cAMP treatment resulted in a 2–2.5-fold increase in the activity of the wild-type whiB1 promoter, compared with its activity in bacilli grown in the presence of butyric acid. However, transcription by the mutant whiB1 promoter essentially remained constant under both these conditions (Fig. 3c). These results thus suggest that whiB1 expression in M. tuberculosis may be controlled by a molecular switch provided by the cellular cAMP levels. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In all probability the four cysteine residues, conserved in all but one of the Wbl proteins identified so far, are involved in binding the iron–sulfur cluster. As a consequence of this interaction, it has been postulated that the WhiD protein may play a role in maintaining the redox balance of the cell. The presence of a putative helix–turn–helix motif in the WhiB protein family implies that these proteins may perform a regulatory role as DNA-binding proteins. Recently, Kim et al. (2005) proposed that the orthologue of WhiB1 in Corynebacterium glutamicum, WhcE, may be involved in the regulation of genes sensing redox changes at the different stages of cell growth.
Based on a recent report (Rickman et al., 2005), we have chosen to focus on CRP-dependent regulation of the expression of M. tuberculosis whiB1. In E. coli, the CRP-dependent promoters are grouped into three classes. Class I has a single CRP-binding site, centred at one of the positions –61.5, –71.5, –81.5 or –91.5; class II possesses a CRP-recognition site centred at –41.5, which thus overlaps with the –35 sequence; and class III requires two or more CRP-binding sites in the promoter for full activation (Busby & Ebright, 1999; Ebright, 1993; Ushida & Aiba, 1990). Based on the position of the CRP-recognition sequence –58.5 nt upstream of the TSP, the M. tuberculosis whiB1 promoter region most resembles the class I CRP-dependent promoters of E. coli.
Our study indicates that CRPM binds to a CRP recognition site upstream of the M. tuberculosis whiB1 promoter in a cAMP-dependent manner in vitro, and that db-cAMP administered exogenously strongly induces M. tuberculosis whiB1 transcription in vivo. The higher activity of the wild-type whiB1 promoter in comparison to its mutated derivative, lacking a functional CRP-recognition sequence, indicates a requirement for CRPM binding to drive full-level expression of whiB1 in M. tuberculosis. However, despite the presence of a functional CRP recognition site, we observed constitutive expression of the wild-type promoter through the growth cycle. This may reflect relatively constant intracellular cAMP levels during mycobacterial growth in the glucose-rich OADC-supplemented 7H9 medium. We also observed constitutive expression from the mutant whiB1 promoter lacking the CRP recognition sequence at levels approximately three- to fourfold lower than those with an intact CRP recognition site, and we postulate that this represents a basal expression level from the 
70-like promoter consensus sequences at the –35 (TTGACA) and –10 positions (AACGAT) in the core whiB1 promoter.
An earlier study by Rickman et al. (2005) found that whiB1 transcription was reduced in an M. tuberculosis mutant lacking an intact Rv3676 gene, which encodes CRPM. However, the CRP-recognition consensus sequence in the M. tuberculosis whiB1 5'UTR lacks perfect twofold symmetry and diverges from the canonical E. coli consensus sequence at several positions. Indeed, a recent report which identified 73 CRP binding motifs in M. tuberculosis using a Gibbs sampling computational search model did not detect the whiB1 promoter region as a significant match (Bai et al., 2005). Despite the lack of perfect twofold symmetry and several differences from the E. coli consensus sequence, our observations, together with those of Rickman et al. (2005), indicate that certain divergent CRPM-recognition sites such as that of whiB1 are likely to retain biologically significant activity in M. tuberculosis.
cAMP is an important signalling molecule regulating various cellular functions, including virulence factors from a diverse range of pathogens (Alspaugh et al., 2002; Caler et al., 2000; D'Souza & Heitman, 2001; Gross et al., 2003; Lee et al., 2003; Li et al., 2002). There is little information about the role of cAMP in mycobacteria, despite its presence in both fast- and slow-growing, as well as pathogenic and nonpathogenic, species (Padh & Venkitasubramanian, 1977). The genome sequence of M. tuberculosis revealed 15 ORFs that contain a cyclase homology domain (Cole et al., 1998; McCue et al., 2000), which implies that cAMP-mediated signal transduction may be a central and versatile tool that this pathogen may employ to combat multiple environmental challenges. Indeed, in one novel role for cAMP in mycobacterial pathogenesis, Mycobacterium microti, a member of the M. tuberculosis complex, has been reported to secrete cAMP during infection of macrophages. It is postulated that secreted cAMP may prevent phagolysosome formation and thereby protect bacilli from host-mediated destruction (Lowrie et al., 1975, 1979). Recently, it has been observed that exogenous cAMP induced the expression of several biologically significant genes in M. bovis BCG, thus implicating cAMP-mediated regulation of gene expression as an important mechanism in the M. tuberculosis complex (Gazdik & McDonough, 2005).
We conclude that whiB1 is the first gene of the wbl family shown to be regulated by cAMP in M. tuberculosis. Future studies of the effects of cAMP on whiB1 expression and subsequent downstream effects mediated by whiB1 will better define the role of cAMP signalling and Wbl-mediated gene regulation in pathogenic mycobacteria.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 15 February 2006;
revised 17 May 2006;
accepted 8 June 2006.
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