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

Abstract

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-Gué rin (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.

Characterization of Mycobacterium tuberculosis Rv3676 (CRP Mt ), a Cyclic AMP Receptor Protein-Like DNA Binding Protein

“…Only Rv0867c (rpfA) and Rv1552 ( frdA) are predicted by both their study and the present study to be members of the putative CRP Mt regulon (65). The regulation of rpfA by CRP Mt has also recently been experimentally confirmed (54).…”

Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins

“…A point mutation in the CRP-FNR-like regulator (Mb3700) alters global gene expression and attenuates the strain. This mutation has been traced back to post-1924 and affects the HTH domain of the regulatory protein (Spreadbury et al, 2005). This mutation is likely to be present in many of the daughter derivative strains that were passaged independently by different laboratories prior to the development of a seed-stock in the 1960s.…”

Regulation of the expression of whiB1 in Mycobacterium tuberculosis: role of cAMP receptor protein

“…Each of the Wbl family of proteins contains four invariant cysteine residues, which are believed to be involved in binding a [4Fe-4S] cluster (Jakimowicz et al, 2005). The functional importance of this cluster is emphasized by the observation that none of the four whiD alleles carrying mutations at these cysteine residues was able to complement the whiD mutant phenotype in S. coelicolor (Jakimowicz et al, 2005).It has recently been reported that the M. tuberculosis gene Rv3676 encoding a CRP-FNR family protein (hereafter referred to as CRP M ) is defective in the vaccine strain Mycobacterium bovis BCG (Spreadbury et al, 2005). Moreover, an M. tuberculosis mutant lacking an intact Rv3676 gene is attenuated for virulence and shows reduced expression of several mycobacterial genes including resuscitation promoting factor, rpfA (Rickman et al, 2005).…”

“…It has recently been reported that the M. tuberculosis gene Rv3676 encoding a CRP-FNR family protein (hereafter referred to as CRP M ) is defective in the vaccine strain Mycobacterium bovis BCG (Spreadbury et al, 2005). Moreover, an M. tuberculosis mutant lacking an intact Rv3676 gene is attenuated for virulence and shows reduced expression of several mycobacterial genes including resuscitation promoting factor, rpfA (Rickman et al, 2005).…”