Since its initial discovery as an allosteric factor regulating cellulose biosynthesis in Gluconacetobacter xylinus , the list of functional outputs regulated by c-di-GMP has grown. We have focused this article on one of these c-di-GMP-regulated processes, namely, biofilm formation in the organism Pseudomonas aeruginosa . The majority of diguanylate cyclases and phosphodiesterases encoded in the P. aeruginosa genome still remain uncharacterized; thus, there is still a great deal to be learned about the link between c-di-GMP and biofilm formation in this microbe. In particular, while a number of c-di-GMP metabolizing enzymes have been identified that participate in reversible and irreversible attachment and biofilm maturation, there is a still a significant knowledge gap regarding the c-di-GMP output systems in this organism. Even for the well-characterized Pel system, where c-di-GMP-mediated transcriptional regulation is now well documented, how binding of c-di-GMP by PelD stimulates Pel production is not understood in any detail. Similarly, c-di-GMP-mediated control of swimming, swarming and twitching also remains to be elucidated. Thus, despite terrific advances in our understanding of P. aeruginosa biofilm formation and the role of c-di-GMP in this process since the last version of this book (indeed there was no chapter on c-di-GMP!) there is still much to learn.

To colonize the cystic fibrosis lung, Pseudomonas aeruginosa establishes sessile communities referred to as biofilms. Although the signaling molecule c-di-GMP governs the transition from motile to sessile growth, the environmental signal(s) required to modulate biofilm formation remain unclear. Using relevant in vivo concentrations of the 19 amino acids previously identified in cystic fibrosis sputum, we demonstrated that arginine, ornithine, isoleucine, leucine, valine, phenylalanine and tyrosine robustly promoted biofilm formation in vitro. Among the seven biofilm-promoting amino acids, only arginine also completely repressed the ability of P. aeruginosa to swarm over semi-solid surfaces, suggesting that arginine may be an environmental cue favoring a sessile lifestyle. Mutating two documented diguanylate cyclases required for biofilm formation (SadC and RoeA) reduced biofilm formation and restored swarming motility on arginine-containing medium. Growth on arginine increased the intracellular levels of c-di-GMP, and this increase was dependent on the SadC and RoeA diguanylate cyclases. Strains mutated in sadC, roeA or both also showed a reduction in biofilm formation when grown with the other biofilm-promoting amino acids. Taken together, these results suggest that amino acids can modulate biofilm formation and swarming motility, at least in part, by controlling the intracellular levels of c-di-GMP.

bWe constructed a library of in-frame deletion mutants targeting each gene in Pseudomonas aeruginosa PA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.

Pseudomonas aeruginosa is a Gram-negative bacterium that has become an indispensable model organism in our quest to understand the A-to-Z of bacterial biofilms (1). It is genetically tractable, has a sequenced and annotated genome (http:// www.pseudomonas.com), and boasts a number of useful tools (2, 3) that facilitate both in vivo and in vitro studies. Although it is commonly found as an environmental isolate, it is also an opportunistic pathogen capable of colonizing plants and mammalian hosts, and is particularly significant for its efficient colonization of lungs of cystic fibrosis (CF) patients (4-6). The versatility of P. aeruginosa is in large part attributed to a battery of traits that provide it selective advantage(s) across diverse environments. Like many other bacteria, P. aeruginosa is capable of transitioning between motile and sessile/biofilm lifestyles, which is believed to contribute to this bacterium's versatility. Bacteria were once perceived to be simple, single-celled organisms; however, it is quite clear that microbes can participate in a broad range of complex multicellular behaviors, including quorum sensing, the formation of complex spore-forming aggregates by Myxococcus and Bacillus, swarming motility, and the formation of bacterial biofilms (7-13). Biofilms are defined as a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to inert or living surfaces (14). The ability to form a biofilm is a common trait of a diverse array of microbes, including lower order eukaryotes. We now understand that in bacteria the intracellular second messenger molecule-3',5'-cyclic diguanylic acid, or c-di-GMP (Fig. 1)-appears to control many facets of group behavior, including biofilm formation in P. aeruginosa. c-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC), and is degraded by phosphodiesterases (PDE). The genome of P. aeruginosa PAO1 encodes 41 cdi-GMP proteins predicted to participate in c-di-GMP metabolism, while P. aeruginosa PA14 has 40 such proteins (15, 16). Interestingly, most of these proteins are linked to various sensory input domains on their N-terminus, including PAS, GAF, and REC domains (17, 18), presumably transducing environmental stimuli to cellular response(s). In fact, cdi-GMP has been implicated in numerous cellular functions including regulation of cell cycle, differentiation, biofilm formation and dispersion, motility, and virulence (19-28), adding credence to this prediction. With regards to biofilm formation in particular, the current general model associates high intracellular levels of c-di-GMP with biofilm formation or a sessile lifestyle, and low c-di-GMP levels are associated with a motile or planktonic

Swimming motility is a flagellum-dependent form of movement observed in the Gram-negative bacterium Pseudomonas aeruginosa. Swimming motility is defined as the movement in liquid or low-viscosity conditions (up to 0.3 % agar concentration). Unlike swarming motility, swimming motility requires a functional flagellum, but neither quorum sensing (QS) systems nor biosurfactants. While swimming motility can also be observed via microscopy, here we describe a reproducible plate-based method.