Biofilms and c-di-GMP Signaling: Lessons from Pseudomonas aeruginosa and other Bacteria

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 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. exploring aqueous a sessile biofilm community. The switch from planktonic to sessile occurs when, cells and series physiological, metabolic and phenotypic changes. are the slowdown of an extracellular matrix, a complex mixture of exopolysaccharides, proteins and nucleic the case of the two bacterial in terms of virulence

. It is proposed that cells use c-di-GMP as a checkpoint to proceed through the distinct stages of biofilm development until they fully commit to the biofilm lifestyle, although they may still be offered the choice to revert the decision at any time (3,8).

The c-di-GMP metabolism
The levels of c-di-GMP in the cell are the GGDEF and EAL domains activity (11,12).
Recently, examples of proteins with dual DGC and PDE activities have been described, shedding some light on this "biochemical conundrum" (13)(14)(15)

PDEs: EAL or HD-GYP domain proteins
The EAL domain hydrolyzes c-di-GMP into linear pGpG ( Figure 1). HD-GYP domain-containing proteins belong to the HD superfamily of metal-dependent phosphohydrolases (11). This enzyme hydrolyzes c-di-GMP in a two-step reaction, producing as final product two molecules of GMP ( Figure 1).  (Table S1) YfiB is an outer membrane lipoprotein and an antagonist of YfiR (53). Finally, exposure to subinhibitory concentration of antibiotic triggers SCVs formation (54,55) and in the case of kanamycin, this effect is linked to c-di-GMP via the PvrR PDE (55).

Molecular mechanisms of c-di-GMP regulation
The regulation of cellular functions by c-di-   (11,56).
In P. aeruginosa, PelD is a c-di-GMP receptor whose expression and binding to c-di-GMP are required for Pel polysaccharide production (57).
PelD is an inner membrane protein with a GAF domain and a degenerated GGDEF domain with a conserved I-site (Table S2)

Cross-talk between second messengers
While c-di-GMP is the second messenger associated with biofilm and chronic infection, cyclic AMP (cAMP) has been shown as being a hallmark for P. aeruginosa virulence (i.e. acute infection) (86). The dichotomy between these two

c-di-GMP regulation of antimicrobial resistance
Cells in a biofilm can be up to 1000 times less susceptible to antimicrobial agents than planktonic cells (92).   of the category are not mutually exclusive. Organization of classes is in agreement as described previously (17). 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 a cyclic process that initiates with attachment to the surface of planktonic bacteria (first reversible than irreversible). A bacteria microcolony is subsequently formed, which evolve 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). Upper panel illustrated 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 Table   S1 and S2. Lower panel illustrates biofilm stage-specific two-component regulatory systems (45). Gradient of the grey panels in the background of the figure indicates increasing intracellular c-di-GMP levels (also indicated with *, **, ***, ****). RsmZ, which sequester the translational repressor RsmA. Titration of RsmA induces the production of sessile and biofilm determinants, whilst 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 others pathways. The elevated concentration of cdi-GMP in a hyperbiofilm forming retS mutant was the first hint of the link between the Gac/Rsm and the cdi-GMP pathways (100). The link has been later on elucidated in molecular details: SadC, a DGC which 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.