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Infect. Immun. doi:10.1128/IAI.01410-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Single nucleotide polymorphisms causing structural changes in the CRP transcriptional regulator of the TB vaccine strain Mycobacterium bovis BCG alter global gene expression without attenuating growth

Division of Mycobacterial Research, Division of Mathematical Biology, and Division of Molecular Neuroendocrinology, MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom School of Biosciences, and Division of Infection and Immunity, The Medical School, University of Birmingham, Birmingham, B15 2TT, United Kingdom

^* To whom correspondence should be addressed. Email: rbuxton{at}nimr.mrc.ac.uk.

	Abstract

Single nucleotide polymorphisms (SNPs) are present in the global transcriptional regulator, CRP (cAMP receptor protein), of the attenuated vaccine strain, M. bovis, BCG. We have found that these SNPs resulted in small but significant changes in expression of a number of genes in M. tuberculosis when a deletion of Rv3676 (CRP) was complemented by the BCG allele, compared to complementation by the M. tuberculosis allele. We can explain these changes in gene expression by modelling the structure of the mycobacterial protein on the known structure of CRP from E. coli. Thus the SNP change in the DNA-binding domain, Lys178, is predicted to form a hydrogen bond with the phosphate backbone of the DNA as does the equivalent residue in E. coli, whereas Glu178 in M. tuberculosis/M. bovis is not, thus explaining the reported stronger binding of CRP_BCG to CRP-binding sites in mycobacterial DNA. In contrast, the SNP change in the nucleotide binding domain (Leu47Pro) is predicted to result in the loss of one hydrogen bond which is accommodated by the structure, and would not therefore be expected to cause any change in function relating to cAMP binding. The BCG allele fully complemented the growth defect caused by deletion of Rv3676 in M. tuberculosis, both in vitro and in macrophage and mouse infections, suggesting that these SNPs do not play any role in the attenuation of BCG. However, they may have allowed BCG to grow better under the in vitro selective conditions used in its derivation from wild type M. bovis.

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