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Single Nucleotide Polymorphisms That Cause Structural Changes in the Cyclic AMP Receptor Protein Transcriptional Regulator of the Tuberculosis Vaccine Strain Mycobacterium bovis BCG Alter Global Gene Expression without Attenuating Growth

Debbie M. Hunt,^1, José W. Saldanha,^2, John F. Brennan,¹ Pearline Benjamin,¹ Molly Strom,³ Jeffrey A. Cole,⁴ Claire L. Spreadbury,^5, and Roger S. Buxton^1*

Division of Mycobacterial Research,¹ Division of Mathematical Biology,² Division of Molecular Neuroendocrinology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom,³ School of Biosciences and Division of Infection and Immunity,⁴ The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom⁵

Received 19 October 2007/ Returned for modification 20 November 2007/ Accepted 26 February 2008

Single nucleotide polymorphisms (SNPs) are present in the global transcriptional regulator cyclic AMP (cAMP) receptor protein (CRP) of the attenuated vaccine strain Mycobacterium bovis, bacillus Calmette-Guérin (BCG). We have found that these SNPs resulted in small but significant changes in the expression of a number of genes in M. tuberculosis when a deletion of the 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 modeling the structure of the mycobacterial protein on the known structure of CRP from Escherichia 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 does not, thus explaining the stronger binding reported for CRP of 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 the deletion of the Rv3676 protein 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 the M. bovis wild type.

Published ahead of print on 10 March 2008.

Editor: R. P. Morrison

The first two authors contributed equally to this work.

Present address: Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom.