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
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ABSTRACT |
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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 AY461387AY461393, respectively.
Present address: School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA.
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INTRODUCTION |
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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 helixturnhelix (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.
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METHODS |
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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.
-Galactosidase assays were used to measure promoter activity in E. coli
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
crp or JRG1728
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
crp cells co-transformed with pDU9 (no CRP) or JRG1728
fnr cells transformed with pRW50 vector alone (no FNR). Single colonies of the co-transformants were grown overnight in 2 ml LuriaBertani (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
4 promoter. Between two and five independent clones were assayed in duplicate for
-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.
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RESULTS |
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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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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 M182crp 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
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 CCgal4 and FFgal
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
crp containing a CCgal
4 : : lac fusion carried by pRW50 or into JRG1728
fnr containing an FFgal
4 : : lac fusion carried by pRW50. The M. tuberculosis Rv3676 product repressed expression of the CCgal
4 : : lac fusion (Fig. 2a
) but not the FFgal
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
4 : : lac fusion (Fig. 2a
). The phenotypes on MacConkey lactose/tetracycline/ampicillin agar of M182
crp cells carrying the CCgal
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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DISCUSSION |
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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
-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 FFgal4 : : 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 104 (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.
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ACKNOWLEDGEMENTS |
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Received 29 June 2004;
revised 15 October 2004;
accepted 20 October 2004.
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