Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.
Bacteria can switch from a single-cell, free-floating behavioral mode to a community life-form via colonization of surfaces and the secretion of an extracellular matrix. This process, called biofilm formation, has been attributed to a majority of chronic infections, including the lungs, as occurs in patients with cystic fibrosis. Recently, a small intracellular signaling molecule, the nucleotide cyclic dimeric guanosine monophosphate (c-di-GMP), and enzymes for its production and degradation have been discovered that relay environmental cues to changes in secretion, cell adhesion and ultimately, biofilm formation and virulence. We have studied the molecular mechanism and mode of regulation of WspR, an enzyme from Pseudomonas and related pathogenic bacteria responsible for the generation of c-di-GMP and biofilm formation. On the basis of its crystal structure and functional assays, we elucidated a sophisticated regulatory mechanism in WspR that is controlled by feedback inhibition mediated by c-di-GMP. We hypothesize that WspR is primed for the (re)activation by enzymatic degradation of the inhibitory nucleotide. In addition, we identified mutations at the inhibitory site of WspR in a subset of bacteria that are frequently found in cystic fibrosis patients, suggesting that altered c-di-GMP signaling, mediated by modified WspR, may contribute to the pathogenicity of these strains. Furthermore, we present a structural comparison with GAF domains, which are widely used conserved regulatory signaling domains, suggesting a similar mechanism of regulation.
We present a model for the regulation of a conserved diguanyate cyclase fromPseudomonas that is responsible for cyclic di-GMP production and biofilm formation, providing insight into the molecular mechani7sm controlling cell signaling and virulence.