JBC Papers in Press. Published on April 21, 2016 as Manuscript R115.711507 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.R115.711507 Biofilm & c-di-GMP signaling MINIREVIEW Biofilms and c-di-GMP Signaling: Lessons from Pseudomonas aeruginosa and other Bacteria Martina Valentini and Alain Filloux From the MRC Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom Running title: Biofilm & c-di-GMP signaling Downloaded from To whom correspondence should be addressed: M. Valentini, E-mail: [email protected] and A. Filloux, E-mail: [email protected], MRC Centre for Molecular Microbiology and Infection, http://www.jbc.org/ Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom Bacteria can live as planktonic cells exploring ABSTRACT aqueous environments or as a sessile biofilm at Imperial College London on May 17, 2016 The cyclic-di-GMP (c-di-GMP) second messenger community. The switch from planktonic to sessile represents a signaling system that regulates many occurs when, under propitious conditions, bacterial behaviors and is of key importance for individual cells encounter a surface and undergo a driving the lifestyle switch between motile loner series of dramatic physiological, metabolic and cells and biofilm formers. This review provides an phenotypic changes. Among these changes are the up-to-date compendium of c-di-GMP pathways slowdown of metabolic activities and the connected to biofilm formation, biofilm- production of an extracellular matrix, a complex associated motilities and other functionalities in mixture of exopolysaccharides, proteins and the ubiquitous and opportunistic human pathogen nucleic acids (1). In the case of pathogens, the two Pseudomonas aeruginosa. This bacterium is bacterial lifestyles also differ in terms of virulence frequently adopted as model organism to study factor production and infection strategies. While bacterial biofilm formation. Importantly, its planktonic cells cause fulminant acute infections, versatility and adaptation capabilities are linked the formation of a biofilm correlates with deep- with a broad range of complex regulatory rooted chronic infections and resistance to both networks, including a large set of genes involved phagocytosis and antimicrobial agents (2). in c-di-GMP biosynthesis, degradation and Cyclic di-GMP (c-di-GMP) is recognized as an transmission. intracellular signaling molecule coordinating the “lifestyle transition” from motility to sessility and vice versa (i.e. dispersion) (3). The correlation 1 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Biofilm & c-di-GMP signaling between high c-di-GMP concentration in the cell diguanylate cyclases (DGCs) and is degraded into and biofilm formation or between low c-di-GMP pGpG and/or GMP by phosphodiesterases (PDEs) levels and motility has been demonstrated in (Figure 1A). Using bioinformatics, biochemical several bacteria species, e.g. Escherichia coli, and structural approaches, the catalytic domains Pseudomonas aeruginosa, Salmonella enterica of DGCs and PDEs have been identified and serovar Typhimurium (4). P. aeruginosa biofilms characterized; the former carrying a GGDEF are estimated to contain on average 75-110 pmol active site motif, the latter carrying either EAL or c-di-GMP per mg total cell extract; while HD-GYP domains (9,10). These domains can planktonic cells contain less than 30 pmol.mg-1 stand alone in a protein or can be present in (5). This concept is widely accepted but does not association with receiver or transmission domains, include the multiplicity of c-di-GMP transmission suggesting a modulation of their enzymatic cascades operating during biofilm. Biofilm activity in response to external/internal signals, Downloaded from determinants modulated by c-di-GMP range from while several have multiple hydrophobic flagella rotation to type IV pili retraction, segments suggesting membrane localization exopolysaccharides production, surface adhesins (Figure 1B). This indicates a possible post- http://www.jbc.org/ expression, antimicrobial resistance and other translational regulation of DGCs and PDEs that stress responses, secondary metabolites may segregate their activity temporally or production and biofilm dispersion (3). How do we spatially. Moreover, GGDEF- and EAL- domains at Imperial College London on May 17, 2016 reconcile the global effect of the intracellular c-di- can be both present in the same protein. In these GMP concentration on stimulating the biofilm so called “hybrid” proteins either only one of the lifestyle with the discrete actions of c-di-GMP on two domains is catalytically active, the other biofilm formation? Biofilm formation is having acquired a regulatory function, or a third considered as a developmental process that regulatory domain is present, probably disjoining includes attachment to and movement on the the GGDEF and EAL domains activity (11,12). surface, formation of microcolonies, maturation Recently, examples of proteins with dual DGC and ultimately dispersal (1,6,7). It is proposed that and PDE activities have been described, shedding cells use c-di-GMP as a checkpoint to proceed some light on this “biochemical conundrum” (13- through the distinct stages of biofilm development 15). In P. aeruginosa the GGDEF and the EAL until they fully commit to the biofilm lifestyle, domains of MucR are activated differently so that although they may still be offered the choice to in planktonic cells MucR functions as DGC and as revert the decision at any time (3,8). positive regulator of alginate biosynthesis; while in biofilms it functions as a PDE and is a positive The c-di-GMP metabolism regulator of biofilm dispersal induced by nitric The levels of c-di-GMP in the cell are oxide or glutamate (16). modified by the rate of its synthesis and Large-scale genome sequencing led to the degradation. The molecule is synthesized from discovery that GGDEF- and EAL-containing two molecules of GTP by enzymes called proteins are nearly ubiquitous in the bacterial 2 Biofilm & c-di-GMP signaling kingdom and that bacterial genomes contain aeruginosa wrinkly spreader phenotype that is multiple copies of genes encoding GGDEF, EAL, correlated with a thick biofilm due to an increased or HD-GYP domain-containing proteins (17). A production of exopolysaccharides (21). The census of all the GGDEF, EAL, HD-GYP control of WspR activity occurs by three different domains in bacterial genomes is available at routes that are proposed to occur sub-sequentially. http://www.ncbi.nlm.nih.gov/Complete_Genomes First, upon sensing growth on surface the Wsp /c-di-GMP.html (18). The abundance of DGCs signal transduction complex phosphorylates and PDEs in a genome may be correlated to the WspR and triggers c-di-GMP synthesis (21,22). In number of complex cellular functions linked with turn, the WspR phosphorylation triggers c-di-GMP signaling and to the diversity of subcellular WspR oligomerization and cluster possible signals coordinating these functions. The formation, which further increases the DGC P. aeruginosa genome encodes one of the highest activity (23). Finally, the feedback inhibition of Downloaded from numbers of DGCs and PDEs: eighteen GGDEF, WspR activity occurs by c-di-GMP binding at the five EAL, sixteen GGDEF/EAL and three HD- I-site (24). The mechanisms of WspR regulation GYP predicted proteins (Table S1). are supported by structural studies, which http://www.jbc.org/ revealed that, in solution, the protein can exist in DGCs: GGDEF domain proteins three stable forms: a globular dimer (active), a DGCs function as homodimers. The GGDEF tetramer (more active), and an elongated dimer at Imperial College London on May 17, 2016 catalytic site is placed at the dimer interface and is (less active due to c-di-GMP binding) (25,26). involved in the binding of two molecules of GTP and in their conversion into c-di-GMP, with Mg2+ PDEs: EAL or HD-GYP domain proteins as cofactor. Five amino acids upstream of the The EAL domain hydrolyzes c-di-GMP into GGDEF active site is the inhibitory (I-) site linear pGpG (Figure 1). Contrary to DGCs, the RxxD, where the feedback inhibition of the EAL activity of PDEs seems to be independent of cyclase activity occurs. Binding of c-di-GMP at protein oligomerization, while it is dependent on the I-site prevents the formation of enzymatically binding metal ions (requiring Mg2+ or Mn2+ and active DGC dimers (19). The first experimental inhibited by Ca2+ and Zn2+) (27). The glutamate demonstration of a DGC activity comes from the residue (E) in the EAL signature motif is work on PleD, a response regulator in essential, while a change of the alanine residue Caulobacter crescentus (20). Nowadays the PleD (A) into tyrosine or valine (ETL and EVL) still activity is well defined together with its receiver sustains the enzymatic activity. In P. aeruginosa, (REC) domain and the phosphorylation-induced the CheY-EAL-domain protein RocR was dimerization. In P. aeruginosa, the first identified as a response regulator in the RocSAR biochemical characterization of a DGC stems signaling system (28). This system is composed of from the work on WspR, which contains a REC- a membrane sensor RocS1 and two response- GGDEF domain organization (Table S1). The regulators, RocA1 and RocR. RocR activity is DGC was named after its regulatory role on the P. triggered by phosphorylation at the CheY domain 3 Biofilm & c-di-GMP signaling and the protein competes with RocA1 for the formation. Careful examination of dgc and pde phosphoryl transfer from the RocS1 sensor. mutants phenotypes, combined with epistasis Overall, the Roc system regulates biofilm analysis, pointed at specific features about the role formation and virulence genes expression (cup of for example SadC and RoeA (DGC) or BifA fimbriae gene clusters and Type III secretion (PDE) (Table S1). This resulted in a more global system genes) (28,29). understanding of their relative importance at HD-GYP domain-containing proteins belong different stages of the biofilm development to the HD superfamily of metal-dependent process (36,37).