The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris.

Abstract Cyclic-di-GMP [bis-(3′-5′)-cyclic diguanosine monophosphate] controls a wide range of functions in eubacteria, yet little is known about the underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris , expression of a subset of virulence genes is regulated by c-di-GMP and also by the CAP (catabolite activation protein)-like protein Xc CLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and functional insights into the interplay between Xc CLP and c-di-GMP in regulation of gene expression. Xc CLP bound target promoter DNA with submicromolar affinity in the absence of any ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to Xc CLP with micromolar affinity. The crystal structure of Xc CLP showed that the protein adopted an intrinsically active conformation for DNA binding. Alteration of residues of Xc CLP implicated in c-di-GMP binding through modeling studies caused a substantial reduction in binding affinity for the nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by c-di-GMP. Together, these findings reveal the structural mechanism behind a novel class of c-di-GMP effector proteins in the CRP/FNR superfamily and indicate that Xc CLP regulates bacterial virulence gene expression in a manner negatively controlled by the c-di-GMP concentrations.