CHAPTER
1
Recent Advances in Understanding Quorum Sensing and Regulation of Virulence in Plant-Pathogenic Bacteria J. M A X WELL D OW BIOMERIT Research Centre, School of Microbiology, University College Cork, Ireland
VIT TORIO VENTURI International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
stability—can also affect the local density of signal molecules. The term “QS” is used to describe cell–cell signaling in a range of different contexts (Platt and Fuqua, 2010), and we will use it here in this broad sense. QS has been extensively studied in a range of gramnegative bacterial plant pathogens, including Agrobacterium tumefaciens (Smith & Townsend) Conn; Burkholderia glumae (Kurita & Tabei) Urakami et al.; Erwinia carotovora Jones (Bergey et al.); Pantoea stewartii (Smith) Mergaert et al.; Pseudomonas syringae van Hall; Ralstonia solanacearum (Smith) Yabuuchi et al.; Xylella fastidiosa Wells et al.; and Xanthomonas spp. (for review, see Von Bodman et al., 2003). Gram-positive pathogens, by contrast, have received scant attention. As noted earlier, these organisms synthesize a range of signals with different chemical structures (Fig. 1.1). The most common signal molecules found in gramnegative bacteria are the N-acylhomoserine lactones (N-AHLs). This class of molecules is found in all of the plant pathogens just identified, with the exception of Xylella fastidiosa and Xanthomonas spp., which
Many bacteria use cell–cell communication mediated by diffusible signal molecules to monitor aspects of their environment and modulate their behavior accordingly. Cell–cell signaling allows a colony or group of organisms to act in a coordinated fashion to regulate diverse processes such as the formation of biofilms, the synthesis of antibiotics, and the production of virulence factors in pathogenic bacteria. The signal molecules synthesized by bacteria belong to a wide range of chemical classes, and multiple systems using different types of signals often occur within a single organism. A diverse range of mechanisms for signal perception and transduction have been described. Bacteria can utilize cell–cell signaling systems to monitor their population density—a process referred to as “quorum sensing” (QS). The elevated levels of signal concentration that result from a high local population lead to activation of specific QS-regulated functions. Other aspects of the environment—such as confinement to particular niches in which diffusion may be limited and exposure to conditions that affect signal production or 3
4
PART I / VIRULENCE MECHANISMS
FIG. 1.1. Quorum‐sensing signal molecules from plant‐pathogenic bacteria have diverse structures.
utilize cis-unsaturated fatty acids as signals. R. solana cearum has an additional signaling system mediated by 3-hydroxy palmitic acid methyl ester. This chapter will discuss developments in understanding signaling systems and virulence regulation in the xanthomonads (as exemplified by Xylella and Xanthomonas spp.) and the plant-pathogenic pseudomonads. This review will highlight the considerable diversity of signaling mechanisms in plant pathogens. In addition, this chapter will discuss interference with QS as a strategy for the control of plant disease and conclude by considering outstanding research questions. ●●Quorum
Sensing in PlantPathogenic Pseudomonas spp.
Studies of the mechanisms and roles of cell–cell communication in several Pseudomonas spp. have demonstrated that the most common signal molecules
are N-AHLs. These signals were first described in the marine bioluminescent bacterium Vibrio fischeri (Beijerinck) Lehmann & Neumann, in which QS regulates light production (Ruby, 1996). The archetypal N-AHL QS system is rather simple (Fig. 1.2), comprising two proteins from the LuxI and the LuxR families (Fuqua et al., 2001; Whitehead et al., 2001). LuxI-family proteins are the cytoplasmic enzymes responsible for N-AHL synthesis. Several LuxI-family proteins have been studied at the biochemical and structural levels, and their precise mode of action has been revealed (Pappas et al., 2004). N-AHLs are synthesized from S-adenosyl methionine, which provides the homoserine lactone moiety, and acyl-acyl-carrier protein, which provides the fatty acyl moiety. After synthesis, the signal moves freely across the bacterial membranes and accumulates both intraand extracellularly in proportion to cell number. As indicated earlier, confinement of bacteria to niches in which diffusion of the signal may be limited can also influence the local concentration of signal molecules.
CHAPTER 1 / RECENT ADVANCES IN UNDERSTANDING QUORUM SENSING AND REGULATION OF VIRULENCE
FIG. 1.2. Archetypal N-acylhomoserine lactone (N-AHL)
quorum-sensing (QS) system of gram-negative bacteria. The archetypal system comprises a LuxI-family protein that is responsible for N-AHL synthesis and a LuxR-family protein that binds to the promoters of QS-regulated genes to influence transcription. After synthesis, the N-AHL signal moves freely across the bacterial membranes and accumulates both intra- and extracellularly as the population size increases. Above a critical threshold concentration, N-AHLs bind directly to the LuxR-family protein. These complexes can then bind at specific promoter DNA sequences called lux-boxes, which are located in the promoter regions of target QS-regulated genes, affecting their expression. One target can be the luxI gene, so that recognition of the N-AHL can lead to autoinduction of N-AHL synthesis.
Above a critical threshold concentration or cell density, N-AHLs interact directly with the LuxR-family protein, which in most cases results in the formation of homodimers. These complexes can then bind at specific promoter DNA sequences called “lux-boxes,” which are located in the promoter regions of target QS-regulated genes, affecting their expression. More than 70 gram-negative bacteria have been reported to produce N-AHLs that differ mainly in acyl chain length (4–18 carbons, most commonly) and in the oxidation state of position C3 (which is either part of the methylene group or carries an oxo- or hydroxyl group). Individual LuxI proteins can synthesize several N-AHLs with closely related structures, indicating some latitude in substrate specificity. Some organisms have several LuxI-family proteins that direct the synthesis of multiple N-AHL signal molecules, often with diverse acyl moieties. Each LuxI protein has an associated LuxR protein, and the different LuxI/R systems usually interact extensively and are organized hierarchically.
5
Numerous biochemical and structural studies have elucidated the mechanism of action of the LuxRfamily proteins. These proteins comprise two domains: an amino-terminal region in N-AHL binding and a C-terminal domain in DNA binding (Pappas et al., 2004). Favored binding of a particular N-AHL by its cognate LuxR-family protein ensures a good degree of specificity in signal transduction. Many traits in plantassociated and plant-pathogenic bacteria are regulated by N-AHL signaling. For example, the TraI/R of A. tumefaciens controls Ti plasmid conjugation, the CarI/R of Pectobacterium carotovorum (Jones) Waldee regulates exoenzyme and antibiotic production, the EsaI/R of Pantoea stewartii regulates exopolysaccharide (EPS) biosynthesis, and the PhzI/R and CsaI/R systems of Pseudomonas aureofaciens Kluyver regulate phenazine and wheat colonization (Loh et al., 2002; Pierson et al., 1998; Von Bodman et al., 2003; White and Winans, 2007) (see Chapter 11). Pseudomonads are ubiquitous gram-negative bacteria that can live in several environmental niches and undergo transition to become important and dangerous human pathogens. Pseudomonads are studied for their role as plant pathogens (e.g., Pseudomonas syringae) and human-opportunistic pathogens (e.g., infections by P. aeruginosa (Schroeter) Migula). They are also studied for their ability to colonize plant-related niches, such as the rhizosphere (e.g., P. fluorescens Migula and P. chloro raphis (Guignard & Sauvageau) Bergey et al.), where they can act as beneficial bacteria by antagonizing deleterious microorganisms and producing traits that directly influence plant disease resistance and growth. P. aeruginosa, which can also be pathogenic to plants, is by far the most studied pseudomonad, and consequentially, its QS response is well understood. Two N-AHL QS systems—the Las and Rhl systems—are present in P. aeruginosa. In the Las system, the lasI gene product directs the synthesis of N-(3-oxododecanoyl)L-homoserine lactone (3-oxo-C12-HSL), which interacts with LasR and activates target promoters. In the Rhl system, rhlI directs the synthesis of N-butanoylL-homoserine lactone (C4 -HSL), which interacts with the cognate regulator RhlR and then activates its gene promoters. The Las and Rhl systems are connected and regulate the production of multiple virulence factors, including elastase, alkaline protease, exotoxin A, rhamnolipids, pyocyanin, lectins, superoxide dismutases, and biofilm formation (Smith and Iglewski, 2003). The effects of the two AHL QS systems have been exhaustively tested in various mouse models of P. aeruginosa infection and also using alternative infection models of Caenorhabditis elegans (Maupas) Dougherty,
6
PART I / VIRULENCE MECHANISMS
Arabidopsis thaliana (L.) Heynh., and Dictyostelium discoideum Raper (Juhas et al., 2005; Smith and Iglewski, 2003). Since the role of QS in P. aeruginosa virulence is studied in relation to opportunistic human infections and not as a plant pathogen, it will not be reviewed here. Several years ago, two studies systematically determined that many of the 18 validly described species of plant-pathogenic Pseudomonas (Höfte and De Vos, 2006) produced N-AHLs (Cha et al., 1998; Elasri et al., 2001). Despite these reports, surprisingly few species have been studied in detail for the role of QS in plant virulence. N -AHL SIGNALING IN PSEUDOMONAS SYRINGAE
Plant-pathogenic strains of P. syringae are known for their diverse and host-specific interactions with different plant species. Specific strains are classified based on their ability to infect various plant species, and more than 50 pathovars have been identified (PPI, n.d.). These strains utilize various factors during colonization of their plant hosts and production of symptoms, such as exopolysaccharides (EPSs), elicitors of hypersensitive response, avirulence gene products, plant growth hormones, and phytotoxins. Many of these factors, including phenotypes such as movement, are regulated in a growth-phase-dependent manner, as shown by studies of several P. syringae pathovars. Shaw et al. (1997) first reported the production of N-AHLs by P. syringae—specifically, production of N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6 HSL) by P. syringae pv. tabaci (Wolf & Foster) Young et al. This work was extended by Dumenyo et al. (1998), who showed the production of these signals by various P. syringae pathovars. Initial experiments showed that the inactivation of ahlI (a luxI homolog in P. syringae pv. syringae (van Hall) Janse strains B3A and 301D) did not alter the pathovars’ ability to elicit the hypersensitive response or to produce phytotoxins (Dumenyo et al., 1998). However, subsequent work revealed the role of N-AHL QS in epiphytic survival, EPS synthesis, and virulence of P. syringae (Quiñones et al., 2004, 2005). P. syringae pv. syringae is the causal agent of brown spot disease of bean and incites frost injury to plants (Hirano and Upper, 2000). This bacterium lives as an epiphyte on the surfaces of leaves and can reach a high population density (107 cells per gram) before initiating disease (Monier and Lindow, 2003). Leaf-epiphytic colonization is therefore considered the first step in infection, and bacterial colonization usually takes place
along the veins and near trichomes. P. syringae pv. syringae actually forms aggregates in leaf surfaces, which are quite similar to bacterial biofilms; key characteristics include a localized high spatial density of cells embedded in a mucoid matrix. This aggregative behavior results in creating a protected environment that promotes the ability of P. syringae pv. syringae to survive desiccation stress when compared to solitary cells (Monier and Lindow, 2003). Using P. syringae pv. syringae strain B728a, researchers demonstrated that this aggregation is necessary to colonize the leaf and start the infection process (Monier and Lindow, 2003). This finding suggests that the epiphytic fitness of the bacterium is cell-density dependent and that the expression of certain traits within these bacterial communities contributes to plant colonization. As demonstrated for P. syringae pv. tabaci and pv. maculicola (McCulloch) Young et al., strain B728a contains the AhlI/AhlR QS system, which produces and responds to 3-oxo-C6 -HSL (Quiñones et al., 2004). Importantly, ahlI and ahlR mutants were shown to be impaired in their ability to colonize and survive epiphytically on bean leaves under stressful dry conditions, indicating that N-AHL QS is pivotal for aggregate formation by P. syringae pv. syringae on leaf surfaces. The expression of ahlI in strain B728a is positively regulated by a TetR-family regulator AefR (i.e., AHL and epiphytic fitness regulator) and by the GacS/GacA twocomponent regulatory system. GacS and GacA have a global regulatory influence on expression of a large number of genes, although AefR and GacS/GacA activate the AhlI/R system via independent pathways. The precise cascades and modes of action involving GacA/ GacS, AhlI/R, and AefR are unclear, but findings suggest that N-AHL QS in P. syringae pv. syringae is connected with other global regulatory systems (Quiñones et al., 2004). This interplay probably ensures the proper timing of cell-density-dependent gene expression with respect to the growth phase and physiological state of the cell. Further studies of P. syringae pv. syringae strain B728a have revealed that the AhlI/R system regulates several important virulence-related phenotypes (Quiñones et al., 2005). Strains with mutations in ahlI or ahlR displayed much more swarming motility, especially during the early phases of QS, and consequently were able to invade more sites in the leaf tissue in the initial phases of colonization. Wild-type P. syringae pv. syringae caused sunken, water-soaked lesions when injected into bean pods, indicating tissue necrosis and fluid release. The ahlI or ahlR mutants inoculated into bean pods
CHAPTER 1 / RECENT ADVANCES IN UNDERSTANDING QUORUM SENSING AND REGULATION OF VIRULENCE
caused larger water-soaked lesions than those caused by the parent wild-type strain, however, indicating that the QS system of P. syringae pv. syringae plays an important role in early disease symptoms (Quiñones et al., 2005). By contrast, ahlI and ahlR mutants did not cause the internal tissue maceration and dehydration seen with the wild type. AhlIR also positively regulated the production of the EPS alginate and oxidative stress tolerance in P. syringae pv. syringae. The production of EPS is known to be important for the growth, survival, and virulence of plant-pathogenic bacteria (Von Bodman et al., 2003) (see Chapter 4). In P. syringae pv. syringae, the production of the EPS alginate has been associated with increased epiphytic fitness and resistance to desiccation and toxic molecules (Keith and Bender, 1999) (see Chapter 4). The limited survival of ahlI or ahlR mutants on leaves in dry conditions may be attributable in part to the decreased production of alginate by the mutants (Quiñones et al., 2005). Studies of the interference of QS in P. syringae have provided interesting insights into the importance of timing in the expression of virulence-associated factors. These studies were performed using transgenic tobacco plants that produced the N-AHL signal (3-oxoC6 -HSL) of the AhlIR system (Fray et al., 1999). The tobacco pathogen P. syringae pv. tabaci, which has an orthologous AhlIR system to P. syringae pv. syringae, was used in these studies. Inoculation by infiltration of P. syringae pv. tabaci into transgenic tobacco plants produced necrotic lesions that spread less quickly and were less chlorotic than those produced in wild-type tobacco plants (Quiñones et al., 2005). One interpretation of these experiments is that N-AHL production by the transgenic plant induces early expression of virulence-associated factors. (That is, production occurs before the bacterial population has reached the quorum.) Recognition of these factors by the plant (either directly or indirectly) initiates defense responses that are more effective against the incipient disease, since a smaller bacterial population is present. In contrast, in the nontransgenic plant, the production of virulence factors by the pathogen occurs only when a larger bacterial population is present. In this case, the production of virulence factors increases, since more cells are producing them, and the rapid production by this larger population overwhelms the plant’s defenses. This scenario had been proposed earlier to explain the outcomes of similar experiments using the bacterium Pectobacterium carotovorum (Mäe et al., 2001), a plant pathogen for which QS regulates the expression of plant cell-wall-degrading enzymes.
7
The effect of exogenous N-AHLs on the disease process has also been demonstrated by assessing the ability of P. syringae pv. tabaci to induce lesions after being sprayinoculated onto the leaves of transgenic and untransformed plants. This assay tested in part the contribution of QS to the epiphytic fitness of the bacterial population. The results clearly indicated that bacteria growing epiphytically on transgenic tobacco leaves (which provide exogenous N-AHLs) induced significantly fewer lesions than bacteria growing on untransformed plants (Quiñones et al., 2005). A possible explanation for these results is that exposure to N-AHLs of small numbers of solitary bacteria results in the suppression of motility and thus an inefficient invasion of the leaf. Alternatively or additionally, the plant response to premature expression of virulence traits may control epiphytic invasion, as discussed earlier for bacteria infiltrated into leaves of transgenic plants expressing N-AHLs. Epiphytically, P. syringae pv. syringae grows in nature in the presence of many other bacterial species. It has been reported that indigenous epiphytic bacteria producing the same N-AHLs as P. syringae pv. syringae can suppress or interfere with the disease process (Dulla and Lindow, 2009). This report provides evidence of N-AHL-mediated cross-talk in the plant phyllosphere, which produces changes in behavior (mainly motility, which means less invasion) and consequently reduces the virulence of P. syringae pv. syringae. The ability of indigenous bacterial flora to influence pathogenesis through interspecies communication with pathogens may have applications in the control of disease. (This point will be discussed later.) As already mentioned, the AhlIR system is involved in regulatory interplay with another global regulatory system—the GacA/GacS two-component system— and with the AefR regulator (Dumenyo et al., 1998; Quiñones et al., 2004, 2005) (see Chapter 11). This interplay suggests that the production of N-AHLs and QS regulation both respond to environmental inputs, although the cues recognized by the GacS sensor are unknown. One cue unrelated to GacS is the availability of iron, which is assimilated into cells via siderophores. The ferric uptake regulator (Fur) was found to regulate the production of N-AHLs in P. syringae pv. tabaci (Cha et al., 2008). Mutants of P. syringae pv. tabaci that were unable to synthesize siderophores displayed a pleiotropic phenotype, including reduced virulence on tobacco and altered N-AHL production (Taguchi et al., 2010). Iron acquisition is therefore linked to QS in P. syringae, reflecting the connection of QS with other global regulatory systems.
8
PART I / VIRULENCE MECHANISMS
N -AHL SIGNALING IN OTHER PLANT-PATHOGENIC PSEUDOMONADS
Many of the validly described plant-pathogenic Pseudomonas spp. (Höfte and De Vos, 2006) produce N-AHLs, although in most cases, their roles have not been studied (Cha et al., 1998; Elasri et al., 2001). However, two plant-pathogenic Pseudomonas spp. have been studied in some detail, and QS has been reported to play an important role in the virulence of P. corrugata Roberts & Scarlett and P. fuscovaginae (ex Tanii et al.) Miyajima et al. Further examples of the pivotal role of N-AHL QS in plant-pathogenic Pseudomonas spp. will likely follow. P. corrugata is the causal agent of tomato pith necrosis (TPN) and has been isolated in all tomatogrowing areas of the world (Scarlett et al., 1978; Sutra et al., 1997). The genetic and molecular mechanisms of TPN are unknown, but the role of a toxin in the disease has been demonstrated (Chun and Leary, 1989). Strains of P. corrugata from various parts of the world have been shown to possess one N-AHL QS system, PcoI/R, which produces and responds to N-hexanoylL-homoserine lactone (C6 -HSL) (Licciardello et al., 2007). The PcoI/R system is involved in regulation of the hypersensitive response in nonhost tobacco, in swarming motility, and in disease, as evidenced by the reduced TPN phenotype displayed by PcoI/R mutants (Licciardello et al., 2007). The PcoI/R QS system is unique, because pcoI is cotranscribed with the regulatory gene rfiA (Licciardello et al., 2009). RfiA contains a DNA-binding domain that belongs to the LuxR family of regulators at the C-terminus, but it is devoid of the N-AHL autoinducerbinding domain and hence does not respond to N-AHLs. RfiA regulates expression of the operon pcoABC, which is located downstream of pcoI-rfiA and encodes the three components (PcoA, B, and C) of a tripartite resistance nodulation-cell-division (RND) transporter system (Licciardello et al., 2009). The PcoABC system is highly similar to the PseABC system of P. syringae pv. syringae, which is involved in transporting the anti microbial and phytotoxic compounds syringomycin and syringopeptin (Kang and Gross, 2005). P. corrugata produces three phytotoxic and antimicrobial compounds (i.e., corpetin A and B and cormycin), and researchers have suggested that the PcoABC system is involved in their transport (Licciardello et al., 2009). RfiA is also involved in TPN; an rfiA mutant was shown to display significantly reduced disease symptoms compared with the wild type. In P. corrugata, N-AHL QS clearly plays a pivotal role in disease and is part of a network that includes at least one other global regulator.
P. fuscovaginae causes brown sheath rot on several cereals, including rice, maize, and wheat (Duveiller et al., 1989, 1990; Miyajima et al., 1983). As with P. corrugata, the mechanisms of virulence of P. fuscovaginae are largely unknown. Recent studies of QS in P. fuscovaginae have revealed the presence of unusually complex circuitry (Mattiuzzo et al., 2011). P. fuscovaginae possesses two N-AHL QS systems: (1) PfsI/R, which produces and responds to N-decanoyl-L-homoserine lactone (C10 -HSL) and N-dodecanoyl-L-homoserine lactone (C12-HSL) and (2) PfvI/R, which produces and responds to N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10 -HSL) and 3-oxo-C12-HSL. Different small repressor proteins act to stringently and negatively regulate the expression of these systems. The pfvI/R system is repressed by RsaL, which has also been reported to regulate QS systems in P. putida (Trevisan) Migula and P. aeruginosa (Bertani and Venturi, 2004; Mattiuzzo et al., 2011; Rampioni et al., 2007). The pfsI/R system is repressed by RsaM, a novel regulator in bacteria (Mattiuzzo et al., 2011). RsaL is a DNA-binding repressor that blocks transcription of the N-AHL synthase gene, whereas the mode of action of the RsaM repressor is unknown. The PfvI/R and PfsI/R systems are not organized in a hierarchical fashion (in contrast to LasIR and RhlIR in P. aeruginosa). Even so, the two systems can share the N-AHL response, because at a high N-AHL concentration, PfvI/R can respond to the N-AHLs produced by the PfsI/R system. The tight regulation of both systems by repressors indicates levels of control in addition to cell density, and QS in P. fuscovaginae most probably requires or integrates additional stimuli for its activation and/or derepression. Importantly, both QS systems play a role in virulence to rice and wheat (Mattiuzzo et al., 2011). ●●Cell–Cell
Signaling in Xanthomonads
rpf GENE CLUSTER AND CELL–CELL SIGNALING IN XANTHOMONAS CAMPESTRIS
Cell–cell signaling in the xanthomonads is mediated by molecules of the diffusible signal factor (DSF) family, which are cis-unsaturated fatty acids (Fig. 1.1) that were first described in Xanthomonas campestris pv. campestris (Dowson) Dye et al. (Barber et al., 1997; Ryan and Dow 2011; Wang et al., 2004) (see Chapter 6). Study of this pathovar established that synthesis and perception of the DSF signal requires products of genes within
CHAPTER 1 / RECENT ADVANCES IN UNDERSTANDING QUORUM SENSING AND REGULATION OF VIRULENCE
the rpf (i.e., regulation of pathogenicity factors) cluster (Barber et al., 1997; Slater et al., 2000). This cluster was originally named rpf because mutation of the genes leads to coordinated down-regulation of the synthesis of the extracellular enzymes (such as endoglucanase and protease) and the extracellular polysaccharide xanthan and reduced virulence to plants. The synthesis of DSF is totally dependent on RpfF, which is similar in amino acid sequence to enoyl CoA hydratase and partially dependent on RpfB, which is a long-chain fatty acyl CoA ligase (Barber et al., 1997). The rpfB and rpfF genes are adjacent within the X. cam pestris pv. campestris chromosome and transcribed as an operon, although rpfF also has its own promoter. The
9
sensing and transduction of the DSF signal depends on a two-component regulatory system encoded by the rpfGHC operon, which is adjacent to rpfF but convergently transcribed (Slater et al., 2000). RpfC is a complex sensor kinase comprising an N-terminal, membrane-associated sensory input domain; histidine kinase and histidine kinase acceptor (HisKA) domains; a CheY-like, two-component receiver (REC) domain; and a C-terminal, histidine phosphotransfer (HPT) domain (Fig. 1.3). The RpfG regulator comprises an REC domain and an HD-GYP domain, which is a phosphodiesterase involved in degradation of the second messenger, cyclic diguanosine monophosphate (di-GMP) (Ryan et al., 2006a, 2006b). RpfH is a novel
FIG. 1.3. Diffusible signal factor (DSF) signaling and regulation of
virulence in Xanthomonas campestris. Synthesis and perception of the DSF signal involves protein products of the rpf gene cluster. RpfF catalyzes the synthesis of DSF, and the RpfGC two-component system is involved in signal perception and transduction. The sensor kinase RpfC is associated with the cytoplasmic membrane. The regulator component RpfG has an HD-GYP domain, which is a phosphodiesterase that degrades the second messenger cyclic diguanosine monophosphate (di-GMP). Perception of the DSF cell–cell signal leads to phosphorylation of RpfG, thus activating it as a cyclic di-GMP phosphodiesterase and reducing the cellular levels of cyclic di-GMP. At high levels, cyclic di-GMP inhibits synthesis of extracellular enzymes through binding to the transcriptional activator Clp, preventing promoter binding. Degradation of cyclic di-GMP relieves this inhibition. Phosphorylation of RpfG also promotes its physical interaction with two GGDEF domain diguanylate cyclases (DGCs). This interaction, which serves to regulate motility, depends on the GYP motif. In contrast, effects of RpfG on extracellular enzyme synthesis do not require the GYP motif. The influence of the Rpf/DSF signaling system on biofilm formation is apparently independent of both Clp and the two DGCs, indicating additional complexity.