Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction

Abstract

In many bacterial pathogens, the second messenger c-di-GMP stimulates the production of an exopolysaccharide (EPS) matrix to shield bacteria from assaults of the immune system. How c-di-GMP induces EPS biogenesis is largely unknown. Here, we show that c-di-GMP allosterically activates the synthesis of poly-b-1,6-N-acetylglucosamine (poly-GlcNAc), a major extracellular matrix component of Escherichia coli biofilms. C-di-GMP binds directly to both PgaC and PgaD, the two inner membrane components of the poly-GlcNAc synthesis machinery to stimulate their glycosyltransferase activity. We demonstrate that the PgaCD machinery is a novel type c-di-GMP receptor, where ligand binding to two proteins stabilizes their interaction and promotes enzyme activity. This is the first example of a c-di-GMP-mediated process that relies on protein-protein interaction. At low c-di-GMP concentrations, PgaD fails to interact with PgaC and is rapidly degraded. Thus, when cells experience a c-di-GMP trough, PgaD turnover facilitates the irreversible inactivation of the Pga machinery, thereby temporarily uncoupling it from c-di-GMP signalling. These data uncover a mechanism of c-di-GMP-mediated EPS control and provide a frame for c-di-GMP signalling specificity in pathogenic bacteria.

Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3

“…Recent studies also have demonstrated homo-and heteromeric complexes between cell wall-synthesizing enzymes, i.e. chitin, cellulose, and poly-␤-1,6-N-acetylglucosamine synthases (29,31,32). These enzymes resemble HASs in that they are also thought to form a pore in plasma membrane for extrusion of the growing polysaccharide chain.…”

“…For example, the extrusion pore of the poly-␤-1,6-N-acetylglucosamine synthesis machinery of Yersinia pestis (28) and Escherichia coli (29) is suggested to require the formation of a heterodimer with altogether 6 transmembrane domains. On the other hand, channels such as lactose permease contain 12 transmembrane ␣-helices (30), whereas the two protein subunits of bacterial cellulose synthase contain altogether 9 transmembrane domains and couple polymerization with membrane penetration (31).…”

Evolutionary convergence in experimental Pseudomonas populations

“…This constitutes a plausible system for transcriptional control, thus it appears that PGA is subject to both transcriptional control and post-translational control. In E. coli, c-di-GMP functions primarily as an allosteric activator that binds directly to PgaC and PgaD to increase glycosyltransferase activity (Steiner et al, 2013). The presence of a homologous two-component system (PA4032 and PA4036) in P. aeruginosa, which lacks the pgaABCD genes, suggests that the PFLU0005-PFLU0007 system might not exclusively regulate PGA expression or that its regulatory role is indirect.…”

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane

“…Finally, PgaD, a small membrane protein with two predicted transmembrane helices, assists the glycosyltransferase in polymerizing PNAG and is involved in the regulation of PNAG synthesis. The two inner membrane proteins PgaC and PgaD form a complex that functions as a novel type of c-di-GMP receptor wherein ligand binding to the two proteins stabilizes their interaction and allosterically activates PNAG synthesis (25). The expression of the E. coli pgaABCD operon is tightly regulated.…”