Genomic mapping of cAMP receptor protein (CRP Mt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor.
Bottom Line: CRP(Mt) binding overlapped a palindromic consensus sequence.Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRP(Mt) during exponential growth, and in response to nutrient starvation.The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation.
Affiliation: Division of Mycobacterial Research, MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.Show MeSH
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Mentions: We were able to map 98% of the sequences uniquely to the H37Rv genome (allowing for up to two mismatches per read) and achieved a near-complete representation of the entire genome (98% of the genome was mapped). The remaining 2% of the genome includes PE/PPE genes, which contain highly repetitive sequences that are poorly resolved by short-read sequencing. To visualize the genome coverage, the number of reads mapping to each position on the M. tuberculosis chromosome was calculated and the traces visualized in the Artemis genome browser. Peaks were called using the CisGenome software (33) to identify enriched regions in the CRPMt-IP compared to the mock-IP (performed in the absence of antibody) and input (sheared genomic DNA). To validate the results, the data were also analysed using the BayesPeak package in R/Bioconductor, and peaks called in only one of the two methods were discarded. As seen in Figure 1A, there was no significant enrichment of any regions of the M. tuberculosis chromosome in the mock-IP (or the input; data not shown) indicating negligible non-specific binding to the resin or the antibody. In the CRPMt-IP, however, 191 peaks were found, denoting CRPMt-binding sites (here abbreviated to CBSs) on the chromosome (Figure 1A). The average length of the CBSs was 276 bp, and in total this represents 0.7% of the entire M. tuberculosis genome. No differences were observed in CRPMt-DNA binding between cells grown in Dubos medium and cells grown in Middlebrook 7H9 medium (data not shown). No ChIP-seq signals were detected for the crp-deletion strain.
Affiliation: Division of Mycobacterial Research, MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.