Point mutations in the DNA- and cNMP-binding domains of the homologue of the cAMP receptor protein (CRP) in Mycobacterium bovis BCG: implications for the inactivation of a global regulator and strain attenuation

Claire L. Spreadbury1, Mark J. Pallen1, Tim Overton2, Marcel A. Behr3, Serge Mostowy3, Stephen Spiro4,{dagger}, Stephen J. W. Busby2 and Jeffrey A. Cole2

1 Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham B15 2TT, UK
2 School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
3 Division of Infectious Diseases and Medical Microbiology, McGill University Health Centre, Montreal, Canada H3G 1A4
4 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK

Correspondence
Claire L. Spreadbury
c.l.spreadbury{at}zen.co.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genome of Mycobacterium tuberculosis H37Rv includes a homologue of the CRP/FNR (cAMP receptor protein/fumarate and nitrate reduction regulator) family of transcription regulators encoded by Rv3676. Sequencing of the orthologous gene from attenuated Mycobacterium bovis Bacille Calmette–Guérin (BCG) strains revealed point mutations that affect the putative DNA-binding and cNMP-binding domains of the encoded protein. These mutations are not present in the published sequences of the Rv3676 orthologues in M. bovis, M. tuberculosis or Mycobacterium leprae. An Escherichia coli lacZ reporter system was used to show that the M. tuberculosis Rv3676 protein binds to DNA sites for CRP, but this DNA binding was decreased or abolished with the Rv3676 protein counterparts from BCG strains. The DNA-binding ability of the M. tuberculosis Rv3676 protein was decreased by the introduction of base changes corresponding to the BCG point mutations. Conversely, the DNA binding of the BCG Rv3676 proteins from BCG strains was restored by removing the mutations. These data show that in this reporter system the point mutations present in the Rv3676 orthologue in BCG strains render its function defective (early strains) or abolished (late strains) and suggest that this protein might be naturally defective in M. bovis BCG strains. This raises the possibility that a contributing factor to the attenuation of BCG strains may be an inability of this global regulator to control the expression of genes required for in vivo survival and persistence.


Abbreviations: BCG, Bacille Calmette–Guérin; CRP, cAMP receptor protein/catabolite repression protein; FNR, fumarate and nitrate reduction regulator; HTH, helix–turn–helix; RD, region of difference

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences of the Rv3676 homologue in M. bovis BCG-Russia, -Tokyo, -Moreau, -Birkhaug, -Danish, -Glaxo and -Pasteur reported in this paper are AY461387–AY461393, respectively.

{dagger}Present address: School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The tuberculosis vaccine strain, Mycobacterium bovis Bacille Calmette–Guérin (BCG), has been given safely to over three billion people around the world (Bloom & Murray, 1992), yet the genetic basis for its attenuation is not fully understood. BCG was derived from a virulent isolate of the bovine tubercle bacillus M. bovis. Attenuation was achieved between 1908 and 1921 after 230 serial passages on glycerinated potato medium containing beef bile (Calmette, 1927). After only 15 passages a new morphotype was observed with altered colony morphology and decreased virulence for animals (Calmette & Guérin, 1911). By 1921, Calmette and Guérin had shown that this new strain was safe in a variety of animal species and that it provided protective immunity to challenge with virulent Mycobacterium tuberculosis (Guérin & Rosenthal, 1957). The attenuated daughter strains were distributed and serially propagated for vaccine production until BCG Pasteur was lyophilized in 1961. Clinical trials have shown that the efficacy of the BCG vaccine ranges from zero to 80 % benefit (Comstock, 1988). It is possible that phenotypic differences between vaccine strains might have contributed to the variable results and it has been speculated that prolonged culture in vitro might have led to overattenuated strains (Behr & Small, 1997).

Relative to M. bovis, all strains of BCG have a chromosomal ‘region of difference’ (RD1) that was deleted at an early point in its attenuation (Brosch et al., 1998; Mahairas et al., 1996). Selected BCG strains have also subsequently lost other chromosomal regions (Brosch et al., 1998; Behr et al., 1999). Deletion of RD1 from M. tuberculosis attenuates it (Lewis et al., 2003), suggesting that loss of RD1 from M. bovis BCG contributed to its attenuation. Pym et al. (2002) have shown that a BCG : : RD1 knock-in exhibits colonial morphology more akin to pathogenic M. tuberculosis and M. bovis. In addition, the BCG : : RD1 knock-in grew more vigorously in a murine model of infection and induced greater pathological changes. Intriguingly, Pym et al. (2002) found that complementing BCG-Pasteur or BCG-Russia (a 1924 strain) with RD1 did not restore virulence to the levels shown by M. tuberculosis or M. bovis, suggesting that other attenuating mutations have occurred during the initial isolation of BCG.

Candidates for attenuation of virulence might include the deletion or the diminished function of a global regulator. For example, in Escherichia coli, CRP (cAMP receptor protein) and FNR (fumarate and nitrate reduction regulator) act as global transcription factors regulating genes in response to glucose starvation and anaerobic conditions, respectively (reviewed by Kolb et al., 1993; Spiro & Guest, 1990). CRP and FNR belong to the same family of transcription factors, and related proteins are widely distributed among bacteria where they play a variety of regulatory roles. M. tuberculosis H37Rv contains a CRP/FNR homologue, encoded by the ORF Rv3676 (Cole et al., 1998), with an N-terminal cNMP-binding domain and a C-terminal helix–turn–helix (HTH) DNA-binding domain. While comparing the proteomes of M. tuberculosis and M. bovis BCG, Mattow et al. (2001) noted a difference in the two-dimensional electrophoretic mobility of Rv3676 in M. tuberculosis and M. bovis BCG. Intrigued by this finding, we sequenced the Rv3676 gene from various strains of M. bovis BCG. We report that the M. bovis BCG Rv3676 gene has accumulated point mutations and we have investigated the effects of the resulting amino acid substitutions on the binding of the Rv3676 product to DNA.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycobacterial strains and culture.
Mycobacterial strains used are listed in Table 1. BCG-Glaxo, BCG-Pasteur and M. tuberculosis H37Rv were grown in 100 ml Middlebrook 7H9 medium supplemented with either 10 % oleic acid-albumin-glucose-catalase for the BCG cultures, or 10 % albumin-glucose-catalase for M. tuberculosis (supplements from Beckton-Dickinson) and 0·02 % Tween 80. Genomic DNA was extracted as described by van Soolingen et al. (1991). The identities of the BCG-Russia, -Tokyo, -Moreau, -Birkhaug and -Danish strains have been described previously (Behr & Small, 1999), but are included in Table 1 for ease of reference. They were grown and DNA was extracted as described by Behr et al. (2000).


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Table 1. Bacterial strains, plasmids and promoters used in this work

 
Rv3676 DNA amplification, sequencing and cloning.
Oligonucleotide primers are listed in Table 2. Primers Rv3676/For and Rv3676/Rev, derived from the M. tuberculosis H37Rv genome sequence (Cole et al., 1998), were used to amplify the gene encoding the M. bovis BCG Rv3676 by PCR and Expand High Fidelity Taq polymerase (Roche Systems). The 675 bp PCR product was cloned into the pCR2·1-TOPO vector (Invitrogen Life Sciences) and plasmid DNA was confirmed by PCR to contain the correct insert. The DNA insert was sequenced using the M13 Forward and Reverse primers supplied with the PCR cloning kit using the automated Plasmid to Profile service/3700 DNA Analyser (Applied Biosystems) at the Functional Genomics Laboratory, University of Birmingham. All mutations found were independently confirmed by direct sequencing of genomic DNA for these BCG strains at McGill University, Montreal, Canada.


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Table 2. Rv3676 oligonucleotide primers used this work

 
To express the Rv3676 protein, primers were designed based on the M. tuberculosis H37Rv sequence (Cole et al., 1998) to amplify the Rv3676 coding region plus 100 bp downstream using genomic DNA from M. tuberculosis H37Rv, BCG-Birkhaug (1927) and BCG-Danish (1931). The forward primer Rv3676/NcoI contained an NcoI site at the translation start codon. The reverse primer Rv3676/BglII contained a BglII site. The 775 bp amplicons were purified from an agarose gel and cloned between the NcoI and BamHI sites of the vector pGCFNR3 (see Table 1) so that the mycobacterial proteins would be expressed under the control of the E. coli fnr promoter. The resulting plasmids were designated pMtbRv3676, pBirkhaugRv3676 or pDanishRv3676 for M. tuberculosis, BCG-Birkhaug and BCG-Danish, respectively (Table 1).

Mutagenesis of Rv3676.
The QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used according to the manufacturer's instructions for site-directed mutagenesis of the Rv3676 gene carried in the pRv3676 plasmids. The mutagenic primers MtbHTH/For and MtbHTH/Rev (Table 2) were used to introduce the DNA-binding point mutation in helix 1 of the HTH motif in the M. tuberculosis Rv3676 gene carried in pMtbRv3676, and similarly the primers MtbcNMP/For and MtbcNMP/Rev were used to introduce the cNMP-binding point mutation. The DNA-binding mutation was removed from the BCG-Birkhaug Rv3676 gene (carried in pBirkhaugRv3676) using the primers BCGHTH/For and BCGHTH/Rev. The resulting plasmids were sequenced to confirm that the point mutations had been introduced and that no other bases had been changed in the gene sequence.

Promoter-activity assays.
{beta}-Galactosidase assays were used to measure promoter activity in E. coli {Delta}lac CRP and FNR mutants containing different CRP/FNR-dependent promoter : : lacZ fusions, in the presence of the M. tuberculosis and M. bovis BCG protein encoded by Rv3676 compared to E. coli CRP or FNR. E. coli strains, plasmids and test promoters are detailed in Table 1. E. coli M182{Delta}crp or JRG1728{Delta}fnr cells were transformed with the lac expression vector pRW50 carrying the test promoter and then co-transformed with one of the pRv3676 plasmids or with E. coli CRP (pDCRP) or FNR (pFNR) as appropriate. Negative controls were E. coli M182{Delta}crp cells co-transformed with pDU9 (no CRP) or JRG1728{Delta}fnr cells transformed with pRW50 vector alone (no FNR). Single colonies of the co-transformants were grown overnight in 2 ml Luria–Bertani (LB) broth supplemented with the appropriate antibiotics. For the CRP studies, 30 µl of an overnight culture were inoculated into sterile 100 ml conical flasks (duplicate cultures) containing 8 ml LB broth supplemented with tetracycline and ampicillin. The cultures were grown aerobically in a 37 °C shaking incubator until the OD650 had reached 0·5 or above (typically 4 h). For the FNR experiments, the bacteria were grown (in duplicate) both aerobically to an OD650 of between 0·19 and 0·23 (typically 2 h) and anaerobically by inoculating 400 µl culture into a 15 ml capped sterile glass test tube containing 10 ml LB supplemented with 0·4 % glucose and appropriate antibiotics. The cultures were grown at 37 °C without aeration until the OD650 was between 0·5 and 0·8 (typically 5 h). Anaerobic cultures were used to determine repression of expression from the FFgal{Delta}4 promoter. Between two and five independent clones were assayed in duplicate for {beta}-galactosidase activity as described by Jayaraman et al. (1989).

Homology searches, sequence retrieval and alignment.
Homologues of the protein encoded by Rv3676 were aligned using CLUSTALW/SEAVIEW (Galtier et al., 1996) following PSI-BLAST (Altschul et al., 1997) searches on the NCBI (http://www.ncbi.nlm.nih.gov) and the ViruloGenome (http://www.vge.ac.uk) websites.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Point mutations in the M. bovis BCG Rv3676 gene
M. tuberculosis Rv3676 is annotated as encoding a putative transcriptional regulator belonging to the CRP/FNR family, sharing 33 % identity over 188 amino acid residues with E. coli CRP (Cole et al., 1998). Upstream of Rv3676 is a gene that encodes a possible membrane protein (Rv3675) and downstream is a gene (Rv3677c) that encodes a possible hydrolase (Cole et al., 1998). Rv3676 does not appear to be part of an operon. Orthologues of Rv3676 are present in M. bovis, M. leprae, M. avium, M. marinum and M. smegmatis.

The Rv3676 orthologue from seven M. bovis BCG isolates with defined histories (Behr & Small, 1999) was sequenced. All BCG strains have a point mutation affecting the first helix of the DNA-binding HTH motif: a G-to-A base substitution at position 532 resulting in the amino acid change E178K (Fig. 1). BCG-Tokyo (1925) alone has a second mutation affecting this domain (V184A). Three strains obtained from the Pasteur Institute in 1931 and later, BCG-Danish (1931), BCG-Glaxo (1953) and BCG-Pasteur (1961), also have a point mutation affecting the cNMP-binding domain: a T-to-C base substitution at position 140 resulting in the amino acid change L47P. A conservative mutation, a T-to-C substitution at position 205, distinguished M. bovis AF2122/97 and all BCG strains from M. tuberculosis H37Rv.



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Fig. 1. Multiple alignments of HTH DNA-binding domains (a) and cNMP-binding domains (b) from Rv3676 and its orthologues. Numbers indicate the position of the first residue in the alignment. Periods show residues identical to the BCG Pasteur sequence. BCG mutations relative to M. tuberculosis are highlighted in bold type. Helices in the HTH motif are indicated by underlining, and turns by ‘T’. M. bovis BCG strains are designated by the first letters of the strain names: R, Russia; M, Moreau; B, Birkhaug; D, Danish; G, Glaxo; P, Pasteur; T, Tokyo. Sequences for BCG strains were determined experimentally. The overall percentage identity of each entire amino acid sequence to the M. tuberculosis Rv3676 complete amino acid sequence is included below. Protein sequences can be retrieved from Entrez using the following accession numbers: E70790 (M. tuberculosis), CAD95886(M. bovis Rv3676 orthologue, 100 % identity), CAC31818(M. leprae Rv3676 orthologue, 96 % identity), AAK58838(Corynebacterium glutamicum GlxR, 79 % identity), CAE48807(Corynebacterium diphtheriae putative transcriptional regulator, 78 % identity), CAB45551(Streptomyces coelicolor putative transcriptional regulator, 54 % identity) and AAA23601(E. coli CRP, 32 % identity). Sequences from incomplete genome sequencing projects (M. smegmatis, 97 % identity; M. avium, 96 % identity; M. marinum, 95 % identity) can be obtained through BLAST searches with Rv3676 on the ViruloGenome (http://www.vge.ac.uk) site or the TIGR site (http://www.tigr.org).

 
Comparison of the M. bovis BCG protein sequences with the predicted protein sequences of Rv3676 orthologues derived from mycobacterial genome sequences revealed that the coding mutations in the BCG strains, relative to M. tuberculosis H37Rv, are absent from other mycobacteria, including M. bovis AF2122/97 (Garnier et al., 2003), M. tuberculosis CDC1551, M. leprae (Cole et al., 2001), M. avium, M. marinum and M. smegmatis. We also sequenced the Rv3676 gene from several M. bovis strains with the BCG genotype (both as defined by genomic deletions and as defined by the same spoligotype) and the Leu-47 and Glu-178 mutations that we describe are not present. Furthermore, Leu-47 and Glu-178 are conserved in the Rv3676 orthologues from corynebacteria, Streptomyces and many other Gram-positive bacteria, and Leu-47 is even conserved in CRP from E. coli (Fig. 1).

Promoter-activity assays
An E. coli lacZ reporter system was used to monitor the function of the M. tuberculosis Rv3676 protein and the corresponding products from BCG strains. FNR and CRP (cAMP-CRP complex) bind to 22 bp sequences at target promoters and activate transcription by contacting RNA polymerase. To investigate whether Rv3676 could activate transcription initiation at a CRP- or FNR-dependent promoter, plasmid pMtbRv3676 was transformed into E. coli M182{Delta}crp containing a CC(–41·5) : : lac fusion carried by plasmid pRW50. CC(–41·5) is an artificial promoter, derived from the E. coli melR promoter, carrying a consensus site for CRP. This site is centred at position –41·5 such that bound CRP is optimally placed to activate transcription in E. coli (many naturally occurring CRP-dependent promoters carry a DNA site for CRP at –41·5 bp upstream of the transcript start site). Plasmid pMtbRv3676 was transformed into E. coli JRG1728{Delta}fnr containing an FF(–41·5) : : lac fusion carried by pRW50. FF(–41·5) is an artificial promoter, derived from the E. coli melR promoter, carrying a consensus DNA site for FNR. This site is centred at position –41·5 such that bound FNR is optimally placed to activate transcription in E. coli (many naturally occurring FNR-dependent promoters carry a DNA site for FNR at position –41·5). The M. tuberculosis Rv3676 protein encoded by pMtbRv3676 was unable to activate either the CRP-dependent promoter CC(–41·5) or the FNR-dependent promoter FF(–41·5) (data not shown), possibly because the mycobacterial protein was unable to interact productively with the E. coli RNA polymerase.

To monitor binding of the Rv3676 protein to potential target sites (which does not require interaction with the E. coli RNA polymerase) we exploited the CCgal{Delta}4 and FFgal{Delta}4 promoters, previously shown to be repressed by the binding of CRP or FNR, respectively (Bell et al., 1990). These synthetic promoters are derived from E. coli galP1 by cloning a consensus DNA site for CRP/FNR at position –37·5. Plasmid pMtbRv3676 was transformed into M182{Delta}crp containing a CCgal{Delta}4 : : lac fusion carried by pRW50 or into JRG1728{Delta}fnr containing an FFgal{Delta}4 : : lac fusion carried by pRW50. The M. tuberculosis Rv3676 product repressed expression of the CCgal{Delta}4 : : lac fusion (Fig. 2a) but not the FFgal{Delta}4 : : lac fusion (Fig. 2b). This shows that the Rv3676 product efficiently recognizes DNA sites for CRP but not DNA sites for FNR. In contrast the BCG-Danish Rv3676 product was unable to repress the CCgal{Delta}4 : : lac fusion (Fig. 2a). The phenotypes on MacConkey lactose/tetracycline/ampicillin agar of M182{Delta}crp cells carrying the CCgal{Delta}4 : : lac fusion and either E. coli pDCRP or pMtbRv3676 were the same – white colonies, indicating repression of lactose catabolism. Cells carrying the negative control (pDU9) or the pDanishRv3676 product formed red colonies, showing lack of repression in the absence of CRP. Thus the mutations in the M. bovis BCG protein are responsible for a phenotypic change. DNA sequencing of the Rv3676 genes in the pRv3676 series showed that no other mutations had been introduced by the PCR step prior to cloning that could have accounted for the differences in repression observed between the M. tuberculosis and the M. bovis BCG Rv3676 gene products.



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Fig. 2. Repression of promoter activity by the M. tuberculosis Rv3676 protein compared to the M. bovis BCG (Danish) counterpart in an E. coli CRP or FNR background. The figure illustrates {beta}-galactosidase activities in M182{Delta}crp cells containing a CCgal{Delta}4 : : lacZ fusion (carried by pRW50) in the presence of E. coli CRP (encoded by pDCRP), the M. tuberculosis Rv3676 protein (encoded by pMtbRv3676), the BCG-Danish Rv3676 protein (encoded by pDanishRv3676) or no E. coli CRP (pDU9) (a), or in JRG1728{Delta}fnr cells containing an FFgal{Delta}4 : : lacZ fusion (carried by pRW50) in the presence of E. coli FNR (encoded by pFNR), the M. tuberculosis Rv3676 protein (encoded by pMtbRv3676), the BCG-Danish Rv3676 protein (encoded by pDanishRv3676) or no E. coli FNR (b). The bars represent the mean of two independent assays and the error bars show one standard deviation either side of the mean.

 
The M. tuberculosis but not the M. bovis BCG Rv3676 protein competes with E. coli CRP for binding to a CRP-inducible promoter
To provide further evidence that the mycobacterial Rv3676 encodes a CRP homologue, plasmid pMtbRv3676 was transformed into the crp+ strain of E. coli, M182 (K12{Delta}lac crp+) containing a CC(–41·5) : : lac fusion carried by pRW50. The M. tuberculosis Rv3676 protein competed with the native E. coli CRP for binding to the promoter, inhibiting expression by 46 % compared to pDCRP (Fig. 3), indicating trans-dominance of the M. tuberculosis CRP homologue. As expected, the BCG-Danish counterpart (pDanishRv3676) showed no competition for binding to the promoter (Fig. 3).



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Fig. 3. Competition between E. coli CRP and the M. tuberculosis CRP homologue for binding to a CRP-inducible promoter in a crp+ strain of E. coli. The figure illustrates {beta}-galactosidase activities in M182 cells containing a CC(–41·5) : : lacZ fusion (carried by pRW50) in the presence of no E. coli CRP (pDU9), E. coli CRP (encoded by pDCRP), the M. tuberculosis Rv3676 protein (encoded by pMtbRv3676) or the BCG-Danish Rv3676 protein (encoded by pDanishRv3676). The bars represent the mean of three independent assays and the error bars show one standard deviation either side of the mean.

 
Effect of introducing the DNA- and cNMP-binding mutations into the M. tuberculosis CRP homologue
To dissect out the effects of each point mutation on the function of the regulator encoded by Rv3676, site-directed mutagenesis was used to introduce point mutations into the M. tuberculosis Rv3676 gene, or to remove point mutations from the corresponding gene in two strains of M. bovis BCG (BCG-Birkhaug and BCG-Danish). Introduction of the DNA-binding mutation in the first helix of the HTH motif into the M. tuberculosis Rv3676 gene decreased the binding of the substituted protein to almost the same level as shown for the protein from wild-type BCG-Birkhaug (Fig. 4). In contrast, when the DNA-binding mutation was removed from the BCG-Birkhaug protein (Fig. 4; pBirkhaug–HTH) the DNA-binding ability was restored to the same level as the wild-type M. tuberculosis Rv3676 protein (pMtbRv3676). These data show that the DNA-binding mutation in the M. bovis BCG Rv3676 protein decreased its ability to bind to DNA in CRP targets.



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Fig. 4. Effects of introducing or removing point mutations in the DNA-binding or cNMP-binding domains in the mycobacterial CRP homologues on the repression of the CCgal{Delta}4 promoter. The figure illustrates {beta}-galactosidase activities in M1821{Delta}crp cells containing a CCgal{Delta}4 : : lacZ fusion (carried by pRW50) in the presence of no E. coli CRP (pDU9), E. coli CRP (encoded by pDCRP), wild-type M. tuberculosis, BCG-Birkhaug or BCG-Danish CRP homologues encoded by pMtbRv3676, pBirkhaugRv3676 or pDanishRv3676, respectively, or mutagenized M. tuberculosis, BCG-Birkhaug or BCG-Danish CRP homologues encoded by pMtb+HTH, pBirkhaug–HTH, pMtb+cNMP, pMtb+both or pDanish–HTH, respectively. The bars represent the mean of three to five independent assays and the error bars show one standard deviation either side of the mean. Abbreviations: Mtb, M. tuberculosis; +HTH, where the DNA-binding mutation has been introduced by site-directed mutagenesis; –HTH, where the DNA-binding mutation has been removed; +cNMP, where the cNMP-binding mutation has been introduced; +both, where both mutations have been introduced.

 
Introducing the cNMP-binding mutation into the M. tuberculosis Rv3676 gene also decreased the ability of the protein product to repress the CCgal{Delta}4 : : lac fusion (Fig. 4; pMtb+cNMP) to a greater extent than that shown by introducing the DNA-binding mutation, suggesting that the presence of the cNMP-binding mutation had a greater detrimental effect. The standard deviation from the mean was high in this series, but the observed trend was verified by introducing both mutations into the M. tuberculosis Rv3676 gene. The inability of the protein (pMtb+both) to repress expression was almost equivalent to having no CRP (pDU9), showing that when the protein contains both mutations, its DNA-binding ability is abolished as is the case for the wild-type BCG-Danish protein (pDanishRv3676). Removing the DNA-binding mutation from the BCG-Danish gene (pDanish–HTH) to leave the protein with only the cNMP-binding domain mutation decreased the DNA-binding ability of the protein to a level similar to the M. tuberculosis protein containing the introduced cNMP-binding mutation (pMtb+cNMP). This suggests that the cNMP-binding mutation alone affects the ability of the Rv3676 protein to bind CRP sites. Its effect appears to be more pronounced than when the protein contains only the DNA-binding mutation. Taken together, these findings suggest that strains of M. bovis BCG obtained from the Pasteur Institute in 1927 and earlier have a CRP homologue with decreased DNA-binding ability, while in later strains (1931 onwards) the DNA-binding function of this potential global regulator appears to be abolished.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We describe the presence of point mutations in the key functional domains of a gene (Rv3676) encoding a CRP homologue in vaccine strains of M. bovis BCG. This is significant because CRP belongs to the CRP/FNR family of global transcription regulators that control the expression of many genes involved in the metabolism of carbon, nitrogen and sulphur, aerobic and anaerobic respiration, denitrification, nitrogen fixation, aromatic acid degradation, bacterial luminescence, haemolysin synthesis and virulence in response to different stimuli (Green et al., 2001). This family of transcription regulators are closely related by their primary amino acid sequence, notably a DNA-binding domain based on the HTH structural motif and a characteristic {beta}-roll that is involved in nucleotide binding in CRP. CRP and FNR share 22 % identity over 207 equivalent positions and share a common structure, but FNR differs from CRP in that it has an N-terminal extension containing a cluster of four cysteine residues required to form a 4Fe–4S cluster which senses the oxygen level in the cell.

In E. coli, CRP is activated by the small intracellular signalling molecule, cAMP and forms an active cAMP.CRP complex. This complex is able to recognize and bind target DNA sites and regulate the transcription of specific genes in E. coli. In the absence of glucose, E. coli is able to synthesize enzymes to catabolize alternative sugars, requiring the function of the cya gene encoding adenylate cyclase and the crp gene encoding CRP. Both cAMP and CRP are required together to form a complex to regulate transcription, and in addition to its role in catabolite repression, this complex also participates in the regulation of a large number of E. coli genes at distant promoters (reviewed by Botsford & Harman, 1992), including those involved in the control of the catabolism of amino acids and nucleotides (Busby & Kolb, 1996; Busby & Ebright, 1999).

Phylogenetically, M. tuberculosis Rv3676 falls within the CRP group (Green et al., 2001). Orthologues of this protein exist in all other members of the Actinomycetes (for which genome sequence data are available), suggesting that this protein plays an important role in cellular physiology. Furthermore, its retention in M. leprae, which possesses the minimal gene set for a pathogenic mycobacterium (Cole et al., 2001), suggests that Rv3676 is important for pathogenicity.

The sequence of the HTH motif in the DNA-binding domain is virtually identical in all Actinomycetes and shows nearly 50 % identity (11/23 residues) with the equivalent region in the E. coli CRP, strongly suggesting conservation of structure and function. It is thus surprising that such a potentially important regulator should accumulate point mutations in both of its functional domains. The DNA-binding mutation present in the Rv3676 sequence from all BCG strains introduces a radical Glu-to-Lys change into the first helix of the DNA-binding domain. Using the lacZ reporter system we have shown that this point mutation in the M. bovis BCG Rv3676 protein decreases its ability to repress a CRP-repressible promoter and, therefore, potentially diminishes the function of this protein in its natural host. As this mutation is present in the earliest available strain, BCG-Russia (1924), this mutation might have compromised the function of this protein early in the BCG attenuation process.

Judging from its presence only in strains derived from the stock culture held at the Institute Pasteur after 1927, the mutation affecting the cNMP-binding domain appears to have arisen in the stock culture between 1927 and 1931, in common with a mutation in the mma3 gene, which results in impaired methoxymycolic acid production (Behr et al., 2000) and the deletion of the chromosomal region RD2 (Mahairas et al., 1996; Behr et al., 1999). The cNMP-binding mutation results in a radical Leu-to-Pro change which will disrupt a {beta}-sheet (Chou & Fasman, 1974, 1978). This may account for the electrophoretic mobility changes seen on 2-D gels with the Rv3676 protein from the late BCG strains, BCG-Chicago (1934) and BCG-Copenhagen (=BCG-Danish, 1931) (Mattow et al., 2001). Judging by our promoter-activity results, the cNMP-binding mutation appears to further decrease the DNA-binding ability of the protein as BCG-Danish is more compromised in this respect than the earlier strain, BCG-Birkhaug, to the point where its function appears to be abolished. Presumably the conformation of the protein is so disrupted that a cNMP can no longer bind and activate the regulator. It is possible that this mutation played a role in the further attenuation of BCG vaccine strains, lending support to the reports that in the laboratory, BCG strains could be distinguished in terms of virulent and attenuated forms until around 1929 (Frimodt-Moller, 1939) and that strains may have become overattenuated (Behr & Small, 1997).

The function of the regulator encoded by Rv3676 is not yet known. Our experimental work points to Rv3676 playing a role equivalent to CRP. The fact that it did not repress expression of the FFgal{Delta}4 : : lac fusion and the absence of a cysteine-rich N terminus in the Rv3676 amino acid sequence suggests that it is not an FNR homologue. Using limiting dilution RT-PCR we found that Rv3676 mRNA is upregulated during stationary phase and glucose starvation. Microarray analysis of M. tuberculosis gene expression during adaptation to a nutrient starvation model of persistence has shown that Rv3676 is upregulated during the first 4 h (Betts et al., 2002). Taken together, these data suggest that Rv3676 responds to low levels of glucose and thus most probably acts in a way similar to CRP by potentially binding cAMP and mediating catabolite repression.

An in silico search of the M. tuberculosis H37Rv genome for potential CRP-binding sites (using the E. coli CRP-binding consensus sequence, 5'-TGTGA-N6-TCACA-3') revealed matches (80 % or greater) upstream of genes whose E. coli homologues are known to be regulated by CRP, including ansA, ilvG, galU (galE in E. coli) and acs. Potential sites were also found upstream of genes associated with regulation by FNR in E. coli, notably frdA, nirA, nirB, bioF2, ndh and sdhC. Other noteworthy matches included potential CRP-binding sites upstream of M. tuberculosis genes that have recently been characterized to play a role in in vivo persistence. For example, we found a potential CRP-binding site (TGTTA-N6-TCACA, one mismatch) 279 bp upstream of the glyoxylate bypass gene, icl (McKinney et al., 2000). The C. glutamicum isocitrate lyase gene (aceB) has recently been shown to be under the control of a CRP homologue, GlxR (see Fig. 1). GlxR forms dimers that bind to the aceB promoter in the presence of cAMP and repress the glyoxylate bypass genes (Kim et al., 2004). Other potential CRP-binding sites were found upstream of pcaA, which encodes a cyclopropane synthase required for in vivo persistence (Glickman et al., 2000) and genes (rpfA, rpfD and rpfE) thought to be involved in the resuscitation of dormancy (Mukamolova et al., 2002).

The transcriptional upregulation in response to stationary phase and glucose starvation together with the in silico findings point to the possibility that this gene might be required when the bacilli enter a developmental programme of gene expression required for in vivo growth and persistence. Given the paucity of mutations in M. tuberculosis complex isolates at about 10–4 (Fleischmann et al., 2002), the rate of mutation observed across this gene appears remarkably high. This suggests a selective pressure for the emergence of these mutants in BCG strains. As CRP could function as a global regulator, this could indicate that the inactivation of this regulator would have been beneficial in the unvaried conditions of life in vitro. While this might have led to in vitro benefits for the growth of BCG strains, the impact on in vivo survival would have been potentially detrimental with implications for the attenuation of BCG.

In summary, our findings suggest that M. bovis BCG might be a regulatory mutant and that the DNA-binding and cNMP-binding point mutations in this CRP homologue may have contributed to the attenuation of M. bovis BCG strains, possibly by rendering this transcription factor defective or unable to regulate many of the genes required for in vivo growth and persistence. This will be the focus of future work.


   ACKNOWLEDGEMENTS
 
This work was supported by a research grant from the Wellcome Trust (grant number GRO059718MA). We thank the BBSRC (grant number 6/JIF13209) for supporting the University of Birmingham Functional Genomics laboratory where the majority of the DNA sequencing was performed. We also thank Roy Chaudhuri for help with the multiple sequence alignments and Susan Turner for help with the figures. We gratefully acknowledge the Wellcome Trust Sanger Institute, Hinxton, UK, and The Institute for Genomic Research (TIGR, Rockville, MD, USA) for making complete genome sequence data publicly available.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 29 June 2004; revised 15 October 2004; accepted 20 October 2004.



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