Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria

Abstract

The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission.

Keywords: Pseudomonas aeruginosa (P. aeruginosa); antibiotic resistance; biofilm; cyclic di-GMP (c-di-GMP); signaling.

FIGURE 1.

Molecular basis of c-di-GMP signaling in P. aeruginosa. A, c-di-GMP is synthesized by diguanylate cyclases (green) that carry GGDEF domains and degraded by phosphodiesterases (red) that carry either EAL or HD-GYP domains. EAL phosphodiesterases linearize c-di-GMP into pGpG, which is successively hydrolyzed into 2 GMP molecules primarily by the oligoribonuclease Orn (orange) (34, 35). HD-GYP-phosphodiesterases are proposed to perform both steps of the c-di-GMP degradation process (31). Feedback inhibition mechanisms are illustrated by gray lines. In the cell, c-di-GMP regulates cellular processes at different levels (transcriptional, post-transcriptional, and post-translational). The diversity of c-di-GMP-binding receptors and effectors (blue) is the key of the c-di-GMP pleiotropic mechanisms. B, spatial localization signals and partner domain occurrence for GGDEF, EAL, and HD-GYP proteins of P. aeruginosa. Table based on the work of Seshasayee et al. (17) *: The sets of proteins corresponding to each of the category are not mutually exclusive. Organization of classes is in agreement as described previously (17). TM helices, transmembrane helices. C, pie chart illustrating numerical proportion of GGDEF, EAL, and HD-GYP proteins in P. aeruginosa.

FIGURE 2.

Coordinated action of c-di-GMP signaling pathways and two-component system cascades in the control of P. aeruginosa biofilm development. In the laboratory, biofilm formation is shown to be a cyclic process that initiates with attachment to the surface of planktonic bacteria (first reversible and then irreversible). A bacteria microcolony is subsequently formed, which evolves into a mature mushroom-shaped macrocolony until the biofilm-associated cells disperse to resume again a planktonic lifestyle. Planktonic, biofilm, and dispersed cells possess distinct physiological stages (green, black, and red outline, respectively) (1, 7). The upper panel illustrates DGC (green), PDE (red), and c-di-GMP receptors/effectors (blue) and the developmental stage in which they are proposed to act. Specific references to each DGC/PDE/effector are available in supplemental Tables S1 and S2. The lower panel illustrates biofilm stage-specific two-component regulatory systems (45). The gradient of the gray panels in the background of the figure indicates increasing intracellular c-di-GMP levels (also indicated with *, **, ***, and ****).

FIGURE 3.

The Gac/Rsm cascade in P. aeruginosa is genetically linked to c-di-GMP through SadC. The GacS/GacA two-component system is promoting the expression of two small regulatory RNAs, RsmY and RsmZ, which sequester the translational repressor RsmA. Titration of RsmA induces the production of sessile and biofilm determinants, whereas free RsmA leads to a planktonic and more virulent lifestyle (45, 99). Several additional regulators modulate the Gac/Rsm system, such as the two hybrid sensors RetS and LadS, as well as the histidine phosphotransfer protein HptB and other pathways. The elevated concentration of c-di-GMP in a hyperbiofilm-forming retS mutant was the first hint of the link between the Gac/Rsm and the c-di-GMP pathways (100). Later on, the molecular details of the link were elucidated: SadC, a DGC whose production is repressed by RsmA, is a central player for the Gac/Rsm regulation of biofilm formation (46). It appears therefore evident that the c-di-GMP signaling network and the Gsc/Rsm cascade are not independent to each other and that they are both instrumental for a proper development of the biofilm.