A c-di-GMP Effector System Controls Cell Adhesion by Inside-Out Signaling and Surface Protein Cleavage

Abstract

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit—from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…Another example involves the GGDEF-EAL c-di-GMP receptor LapD from Pseudomonas fluorescens (166,167). While neither the GGDEF nor EAL domain of this receptor is enzymatically active, c-di-GMP binds to the degenerate EAL domain of LapD with a high affinity (68,166). The Bacillus subtilis EAL-YkuI_C protein YkuI (BSU14090) is another candidate receptor belonging to this class.…”

“…However, the membrane fusion protein domain present in the periplasmic fragment of Alg44 suggests that it is involved in protein-protein interactions within the alginate translocation complex. Deletion of a short C-terminal sequence abolished alginate polymerization (157), suggesting that c-di-GMP binding to the cytoplasmic PilZ domain of Alg44 can be transferred via an inside-out signaling mechanism (67,68) to the periplasmic domain to activate alginate polymerization.…”

“…Elegant studies by the O'Toole group have uncovered the molecular mechanisms regulating LapA proteolysis in a c-di-GMP-dependent manner. Specifically, at low cdi-GMP levels, LapA is proteolytically processed by the periplasmic protease LapG (68). The proteolytic activity of LapG is regulated by the transmembrane protein LapD, which binds c-di-GMP via its degenerate EAL domain (166).…”

The role of cell-surface interactions in bacterial initial adhesion and consequent biofilm formation on nanofiltration/reverse osmosis membranes

“…Likewise, the presence of external elements present in the environment, such as salts, was also shown to influence bioadhesion and biofilm formation. Recent studies demonstrated that the presence of inorganic phosphate played a key role in the biofilm formation of Pseudomonas fluorescens and depending of the level found in the environment, determined the adhesive action of LapA, an adhesin localized outside the bacterial cell membrane [44,63].…”

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix

“…As functional and dynamic communities, biofilms are able to modify the matrix components and remodel their structures and functions as an adaptive strategy to encounter the fluctuation of various environment conditions as well as stresses. Protease and nuclease have been recently reported to be secreted by biofilm cells, which leads to disruption of protein adhesions and eDNA of biofilm matrix, resulting in remodeling of the biofilm architectures [10][11][12]. However, little is known about the biofilm self-generated factors that target the essential and universal matrix component, exopolysaccharide.…”