Paralogous cAMP Receptor Proteins in Mycobacterium smegmatis Show Biochemical and Functional Divergence

ABSTRACT: The cyclic AMP receptor protein (CRP) family of transcription factors consists of global regulators of bacterial gene expression. Here, we identify two paralogous CRPs in the genome of Mycobacterium smegmatis that have 78 % identical sequences and characterize them biochemically and function-ally. The two proteins (MSMEG_0539 and MSMEG_6189) show differences in cAMP binding affinity, trypsin sensitivity, and binding to a CRP site that we have identified upstream of the msmeg_3781 gene. MSMEG_6189 binds to the CRP site readily in the absence of cAMP, while MSMEG_0539 binds in the presence of cAMP, albeit weakly. msmeg_6189 appears to be an essential gene, while the Δmsmeg_0539 strain was readily obtained. Using promoter−reporter constructs, we show that msmeg_3781 is regulated by CRP binding, and its transcription is repressed by MSMEG_6189. Our results are the first to characterize two paralogous and functional CRPs in a single bacterial genome. This gene duplication event has subsequently led to the evolution of two proteins whose biochemical differences translate to differential gene regulation, thus catering to the specific needs of the organism. Cyclic AMP, via the cAMP receptor protein (CRP), playsan important role in the regulation of gene expression in diverse bacteria.1 CRP is a transcription factor that belongs to the CRP/FNR family, which includes members that are global regulators associated with functions that are highly diverse in different bacterial species.2 The biological processes under the regulation of CRP include adaptation to starvation, extreme temperature, low oxygen energy metabolism, cell division, toxin production, quorum sensing, and cellular motility.3−7 Mycobacteria, including Mycobacterium tuberculosis, which is the causative agent of tuberculosis, contain extraordinarily high levels of cAMP8,9 produced by a large number of adenylyl cyclases encoded in their genomes.10 The genome of M. tuberculosis H37Rv contains two CRP-like genes, encoded b