Dokument

Summary:
Cyclic dimeric GMP (c-di-GMP) is a widespread second messenger regulating several processes including bacterial motility, biofilm formation, and virulence. The enzymes responsible for c-di-GMP production and degradation, diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively, are abundant and often present in multiple copies within bacterial genomes. DGCs possess a characteristic GGDEF domain, whereas PDEs have either an EAL or an HD-GYP domain. In the Escherichia coli K-12 strain MG1655, 29 proteins containing GGDEF and/or EAL domains have been identified. In E. coli, the diguanylate cyclase DgcZ (formerly YdeH) is the major DGC controlling the production of the exopolysaccharide poly-N-Acetylglucosamine (poly-GlcNAc, PGA), which is involved in biofilm formation. DgcZ contains a GGDEF domain, responsible for c-di-GMP production, and a sensory domain. First identified in the chemoreceptor TlpD of Helicobacter pylori, this domain was named chemoreceptor zinc-binding (CZB) domain after its capability to bind zinc. Researchers from the University of Basel’s Biozentrum solved the three-dimensional structure of the DgcZ protein and indeed found a zinc ion bound to the 3His/1Cys motif of the CZB domain. Additionally, zinc was shown to inhibit the activity of DgcZ in vitro with a subfemtomolar constant Ki. This study investigates the regulation and function of the diguanylate cyclase DgcZ in E. coli. To ascertain the role of zinc in the function of DgcZ activity in vivo, site-directed mutagenesis was employed to construct dgcZ alleles encoding protein variants with amino acid exchanges in the CZB domain involved in zinc coordination, thus reducing or abolishing binding. The activity of these DgcZ variants was derived by measuring the levels of PgaD, an enzyme involved in exopolysaccharide production, and of the exopolysaccharide poly-GlcNAc (PGA) produced, both proportional to DgcZ-derived c-di-GMP. Although single exchanges of zinc binding amino acids did not strongly affect the protein activity, a DgcZ variant carrying two such exchanges (H79L and H83L) displayed a significant increase of protein activity. The influence of zinc ions on DgcZ activity was further tested by applying increasing concentrations of ZnSO4 and measuring the ability of bacteria to form a PGA biofilm. Externally applied zinc inhibited PGA biofilm formation in a DgcZ- and c-di-GMP-dependent fashion. The evidence obtained in vivo therefore confirms the results from the in vitro experiments showing that the diguanylate cyclase DgcZ is allosterically regulated by zinc. The relevance of this regulatory mechanism is still unsettled, but potential explanations are that it might help the bacteria discriminate among different niches, characterized by high or low levels of zinc, or that it could “signal” the cell´s own physiological condition. Following these studies on allosteric regulation, the physiological role of DgcZ was examined, as its primary function and the conditions in which the protein is active are still not well defined. Analyses of DgcZ protein localization performed in this study revealed that a combination of carbon starvation and alkaline pH (8.7) induces localization at one bacterial cell pole. Polar localization occurred in non-dividing bacteria and disappeared after restoring nutrient-sufficient conditions. The role of this localization phenotype until now remains elusive. Further, Co-Immunoprecipitation analyses were performed and 11 proteins identified with a significant score. Among these, FrdB, a subunit of the fumarate reductase complex (FRD), interacted with DgcZ within a bacterial two-hybrid system. The FRD complex proved essential in the superoxide-stimulated increase of DgcZ-dependent biofilm, suggesting new roles of this complex and oxidative stress in DgcZ-mediated biofilm formation. Finally, the role of DgcZ in CpxAR-mediated surface adhesion was investigated, as it had already been established that dgcZ is transcriptionally regulated by this two-component system. The Cpx complex in turn had been shown to be responsible for surface sensing and to stimulate bacterial adhesion through an up to now unknown mechanism. This work reveals an involvement of DgcZ in Cpx-mediated surface adhesion, providing evidence for a physiological function of this diguanylate cyclase in connecting the processes of surface sensing and surface attachment.

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