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

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 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. Present address: School of Biology, Georgia Institute of Technology, Atlanta, GA