Genomic binding for cAMP responsive transcription factors, CRP (Rv3676) and Cmr (Rv1675c) in TB complex mycobacteria

We identified the genome-wide binding sites of CRP/FNR family transcription factors CRP and Cmr in M. bovis BCG using ChIP-seq, and cross-referenced these binding sites to orthologous regions in M. tuberculosis. Selected binding sites for each transcription factor were further characterized for their importance to gene regulation. Overall design: Chromatin immunoprecipitation was performed in M. bovis BCG Pasteur strain using antibodies to CRP or Cmr. Genome-wide binding profiles for each transcription factor were compared with and without elevated levels of cAMP. CRP studies were performed with native CRP expression levels, while Cmr binding was analyzed in the presence of cmr overexpression using the tuf promoter. cAMP levels were elevated exogenously by addition of dibutyryl cAMP treatment, or endogenously by expression of the Rv1264 catalytic domain. Bacteria were analyzed in late log phase during growth in ambient air (21% O2) or hypoxia (1.3% O2) supplemented with 5% CO2. Please note that polyclonal antisera were generated in New Zealand white rabbits against CRP, as described in Bai et al. 2007. Infect Immun 75(11): 5509-5517, and against Cmr, as described in Ranganathan et al. 2016. Nucleic Acids Res 44(1):134-151.