Cyclic di‐GMP Signaling in Paracoccus denitrificans

Abstract

Cyclic di‐GMP (c‐di‐GMP) signaling has been discovered to regulate many functions such as biofilm formation, motility, and virulence in gram negative bacteria. Intracellular concentrations of this secondary messenger are controlled by activities of diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes, which synthesize and degrade it, respectively. In general, elevated levels of c‐di‐GMP are associated with increased biofilm formation. However, a hyperbiofilm phenotype in a DGC mutant of Paracoccus denitrificans suggests that c‐di‐GMP downregulates biofilm formation in this organism. Further, a gene for the heme‐based sensor protein H‐NOX, which has been implicated in biofilm formation in other organisms, is found adjacent to one of these DGC genes. Consistent with previous observation, we show that the Δhnox mutant of P. denitrificans is deficient in biofilm formation. We further use mass spectrometry to determine levels of intracellular c‐di‐GMP and proteomics to demonstrate altered expression and phosphorylation for several proteins, indicated signaling pathways perturbed by the mutation. In order to understand the mechanism of H‐NOX action, it and its cognate DGC were expressed and purified from E. coli. P. denitrificans H‐NOX lacks the proximal His residue found in other H‐NOX homologues to coordinate the heme cofactor. Indeed, this H‐NOX purifies with no heme bound, making it uncertain how it could act as a sensor of environmental conditions. We have performed activity assays for the DGC in the presence and absence of H‐NOX to test its function as a DGC inhibitor and are currently working to determine structures of these proteins by X‐ray crystallography or NMR. Taken together, this work advances our knowledge of the diverse mechanisms by which c‐di‐GMP regulates biofilm formation in bacteria.