The Mycobacterium bovis BCG Cyclic AMP Receptor-Like Protein Is a Functional DNA Binding Protein In Vitro and In Vivo, but Its Activity Differs from That of Its M. tuberculosis Ortholog, Rv3676

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Abstract
Mycobacterium tuberculosis Rv3676 encodes a cyclic AMP (cAMP) receptor-like protein (CRP Mt ) that has been implicated in global gene regulation and may play an important role during tuberculosis infection. The CRP Mt ortholog in Mycobacterium bovis BCG, CRP BCG , is dysfunctional in an Escherichia coli CRP competition assay and has been proposed as a potential source of M. bovis BCG's attenuation. We compared CRP BCG and CRP Mt in vitro and in vivo, in M. bovis BCG and M. tuberculosis , to evaluate CRP BCG 's potential function in a mycobacterial system. Both proteins formed dimers in mycobacterial lysates, bound to the same target DNA sequences, and were similarly affected by the presence of cAMP in DNA binding assays. However, CRP Mt and CRP BCG differed in their relative affinities for specific DNA target sequences and in their susceptibilities to protease digestion. Surprisingly, CRP BCG DNA binding activity was stronger than that of CRP Mt both in vitro and in vivo, as measured by electrophoretic mobility shift and chromatin immunoprecipitation assays. Nutrient starvation-associated regulation of several CRP Mt regulon members also differed between M. bovis BCG and M. tuberculosis . We conclude that CRP BCG is a functional cAMP-responsive DNA binding protein with an in vivo DNA binding profile in M. bovis BCG similar to that of CRP Mt in M. tuberculosis . However, biologically significant functional differences may exist between CRP BCG and CRP Mt with respect to gene regulation, and this issue warrants further study.