Diguanylate cyclase NicD‐based signalling mechanism of nutrient‐induced dispersion by Pseudomonas aeruginosa

Dispersion enables the transition from the biofilm to the planktonic growth state in response to various cues. While several Pseudomonas aeruginosa proteins, including BdlA and the c‐di‐GMP phosphodiesterases DipA, RbdA, and NbdA, have been shown to be required for dispersion to occur, little is known about dispersion cue sensing and the signalling translating these cues into the modulation c‐di‐GMP levels to enable dispersion. Using glutamate‐induced dispersion as a model, we report that dispersion‐inducing nutrient cues are sensed via an outside‐in signalling mechanism by the diguanylate cyclase NicD belonging to a family of seven transmembrane (7TM) receptors. NicD directly interacts with BdlA and the phosphodiesterase DipA, with NicD, BdlA, and DipA being part of the same pathway required for dispersion. Glutamate sensing by NicD results in NicD dephosphorylation and increased cyclase activity. Active NicD contributes to the non‐processive proteolysis and activation of BdlA via phosphorylation and temporarily elevated c‐di‐GMP levels. BdlA, in turn, activates DipA, resulting in the overall reduction of c‐di‐GMP levels. Our results provide a basis for understanding the signalling mechanism based on NicD to induce biofilm dispersion that may be applicable to various biofilm‐forming species and may have implications for the control of biofilm‐related infections.     

The phosphodiesterase DipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion

Although little is known regarding the mechanism of biofilm dispersion, it is becoming clear that this process coincides with alteration of cyclic di-GMP (c-di-GMP) levels. Here, we demonstrate that dispersion by Pseudomonas aeruginosa in response to sudden changes in nutrient concentrations resulted in increased phosphodiesterase activity and reduction of c-di-GMP levels compared to biofilm and planktonic cells. By screening mutants inactivated in genes encoding EAL domains for nutrient-induced dispersion, we identified in addition to the previously reported ΔrbdA mutant a second mutant, the ΔdipA strain (PA5017 [dispersion-induced phosphodiesterase A]), to be dispersion deficient in response to glutamate, nitric oxide, ammonium chloride, and mercury chloride. Using biochemical and in vivo studies, we show that DipA associates with the membrane and exhibits phosphodiesterase activity but no detectable diguanylate cyclase activity. Consistent with these data, a ΔdipA mutant exhibited reduced swarming motility, increased initial attachment, and polysaccharide production but only somewhat increased biofilm formation and c-di-GMP levels. DipA harbors an N-terminal GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain and two EAL motifs within or near the C-terminal EAL domain. Mutational analyses of the two EAL motifs of DipA suggest that both are important for the observed phosphodiesterase activity and dispersion, while the GAF domain modulated DipA function both in vivo and in vitro without being required for phosphodiesterase activity. Dispersion was found to require protein synthesis and resulted in increased dipA expression and reduction of c-di-GMP levels. We propose a role of DipA in enabling dispersion in P. aeruginosa biofilms.     

Delineating PAS‐HAMP interaction surfaces and signalling‐associated changes in the aerotaxis receptor Aer

The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer‐PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N‐terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signalling domains in the kinase‐off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS‐HAMP surfaces overlapped with a cluster of PAS kinase‐on lesions and with cysteine substitutions that crosslinked the PAS β ‐scaffold to the HAMP AS‐2 helix. A refined Aer PAS‐HAMP interaction model is presented. Compared to the kinase‐off state, the kinase‐on state increased the accessibility of HAMP residues (apparently relaxing PAS‐HAMP interactions), but decreased the accessibility of proximal signalling domain residues. These data are consistent with an alternating static‐dynamic model in which oxidized Aer‐PAS interacts directly with HAMP AS‐2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signalling domain, resulting in a kinase‐off output. When PAS‐FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signalling domain convey a kinase‐on output.     

The Diguanylate Cyclase GcbA Facilitates Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA

Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion.     

Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix

HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathic and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain.     

PAS domain residues involved in signal transduction by the Aer redox sensor of Escherichia coli

PAS domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation. Although PAS domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL. We investigated the signalling mechanism in the PAS domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Forty-two residues in Aer were substituted using cysteine-replacement mutagenesis. Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen. Four of them also led to the loss of the non-covalently bound FAD cofactor. Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal-on bias. One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa. Residues critical for signalling were mapped onto a three-dimensional model of the Aer PAS domain, and an FAD-binding site and 'active site' for signal transduction are proposed.     

Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer

The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 alpha-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.     

Minimal requirements for oxygen sensing by the aerotaxis receptor Aer

The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared.     

Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa

It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis. In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified. Aerotaxis assays of a number of deletion-insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis. Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis. A double mutant deficient in tlpC and tlpG was negative for aerotaxis. TlpC has 45% amino acid identity with the Escherichia coli aerotactic transducer Aer. The TlpG protein has a predicted C-terminal segment with 89% identity to the highly conserved domain of the E. coli serine chemoreceptor Tsr. A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG. A PAS motif was found in the N-terminal domains of TlpC and TlpG. On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2.     

Aer on the inside looking out: paradigm for a PAS-HAMP role in sensing oxygen, redox and energy

Aer, the Escherichia coli aerotaxis (oxygen-sensing) receptor, is representative of a small class of receptors that face the cytoplasm in bacteria. Instead of sensing oxygen directly, Aer detects redox changes in the electron transport system or cytoplasm when the bacteria enter or leave a hypoxic microniche. As a result, Aer sensing also enables bacteria to avoid environments where carbon deficiency, unfavourable reduction potential or other insults would limit energy production. An FAD-binding PAS domain is the sensor for Aer and a HAMP domain interacts with the PAS domain to form an input-output module for signal transduction. By analogy to the first solution structure of an isolated HAMP domain from Archaeoglobus, Aer HAMP is proposed to fold into a four-helix bundle that rotates between a signal-on and signal-off conformation. Aer is the first protein in which a PAS-HAMP input-output module has been investigated. The structure and signal transduction mechanism of Aer is providing important insights into signalling by PAS and HAMP domains.     

PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding

The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold.     

Role of the F1 region in the Escherichia coli aerotaxis receptor Aer

In Escherichia coli, the aerotaxis receptor Aer is an atypical receptor because it senses intracellular redox potential. The Aer sensor is a cytoplasmic, N-terminal PAS domain that is tethered to the membrane by a 47-residue F1 linker. Here we investigated the function, topology, and orientation of F1 by employing random mutagenesis, cysteine scanning, and disulfide cross-linking. No native residue was obligatory for function, most deleterious substitutions had radically different side chain properties, and all F1 mutants but one were functionally rescued by the chemoreceptor Tar. Cross-linking studies were consistent with the predicted α-helical structure in the N-terminal F1 region and demonstrated trigonal interactions among the F1 linkers from three Aer monomers, presumably within trimer-of-dimer units, as well as binary interactions between subunits. Using heterodimer analyses, we also demonstrated the importance of arginine residues near the membrane interface, which may properly anchor the Aer protein in the membrane. By incorporating these data into a homology model of Aer, we developed a model for the orientation of the Aer F1 and PAS regions in an Aer lattice that is compatible with the known dimensions of the chemoreceptor lattice. We propose that the F1 region facilitates the orientation of PAS and HAMP domains during folding and thereby promotes the stability of the PAS and HAMP domains in Aer.     

Conserved residues in the HAMP domain define a new family of proposed bipartite energy taxis receptors

HAMP domains, found in many bacterial signal transduction proteins, generally transmit an intramolecular signal between an extracellular sensory domain and an intracellular signaling domain. Studies of HAMP domains in proteins where both the input and output signals occur intracellularly are limited to those of the Aer energy taxis receptor of Escherichia coli, which has both a HAMP domain and a sensory PAS domain. Campylobacter jejuni has an energy taxis system consisting of the domains of Aer divided between two proteins, CetA (HAMP domain containing) and CetB (PAS domain containing). In this study, we found that the CetA HAMP domain differs significantly from that of Aer in the predicted secondary structure. Using similarity searches, we identified 55 pairs of HAMP/PAS proteins encoded by adjacent genes in a diverse group of microorganisms. We propose that these HAMP/PAS pairs form a new family of bipartite energy taxis receptors. Within these proteins, we identified nine residues in the HAMP domain and proximal signaling domain that are highly conserved, at least three of which are required for CetA function. Additionally, we demonstrated that CetA contributes to the invasion of human epithelial cells by C. jejuni, while CetB does not. This finding supports the hypothesis that members of HAMP/PAS pairs possess the capacity to act independently of each other in cellular traits other than energy taxis.     

BdlA, a chemotaxis regulator essential for biofilm dispersion in Pseudomonas aeruginosa

Multiple environmental cues have been shown to trigger biofilm detachment, the transition from surface-attached, highly organized communities known as biofilms to the motile lifestyle. The goal of this study was to identify a gene product involved in sensing environmental cues that trigger biofilm dispersion in Pseudomonas aeruginosa. To do so, we focused on novel putative chemotaxis transducer proteins that could potentially be involved in environmental sensing. We identified a locus encoding such a protein that played a role in detachment, as indicated by the observation that an isogenic mutant biofilm could not disperse in response to a variety of environmental cues. The locus was termed bdlA for biofilm dispersion locus. The BdlA protein harbors an MCP (methyl-accepting chemotaxis protein) domain and two PAS (Per-Arnt-Sint) domains that have been shown to be essential for responding to environmental signals in other proteins. The dispersion-deficient phenotype of the bdlA mutant was confirmed by treatment with the biocide H(2)O(2) and by microscopic observations. The dispersion response was independent of motility. bdlA mutant biofilms were found to have increased adherent properties and increased intracellular levels of cyclic di-GMP (c-di-GMP). Our findings suggest that BdlA may be a link between sensing environmental cues, c-di-GMP levels, and detachment. Based on our findings, a possible involvement of BdlA in a signaling cascade resulting in biofilm dispersion is discussed.     

Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer

Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.     

Biofilm dispersal: deciding when it is better to travel

Bacteria live predominantly in biofilms, and the internal signal cyclic diguanylate (c‐di‐GMP) is a universal signal that governs the formation and the dispersal of these communities. Pseudomonas aeruginosa is one of the most important reference systems for studying bacterial biofilms and contains numerous diguanylate cyclases (DGCs) for synthesizing c‐di‐GMP and phosphodiesterases (PDEs) for degrading c‐di‐GMP. However, few studies have discerned how cells in biofilms respond to their environment to regulate c‐di‐GMP concentrations through this sophisticated network of enzymes. Basu Roy and Sauer (2014) provide insights on how cells disperse in response to an increase in nutrient levels. Their results show that the inner membrane protein NicD is a DGC that controls dispersal by sensing nutrient levels: when glutamate concentrations are increased, NicD is dephosphorylated, which increases c‐di‐GMP levels and leads to phosphorylation and processing of dispersal regulator BdlA. Processing of BdlA leads to activation of PDE DipA, which results in a net reduction of c‐di‐GMP and biofilm dispersal. These results suggest biofilm dispersal relies on surprisingly dynamic c‐di‐GMP concentrations as a result of a sophisticated interaction between DGCs and PDEs.     

Function of the N-terminal cap of the PAS domain in signaling by the aerotaxis receptor Aer

Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23 degrees C was attenuated at 10 degrees C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.     

An Archaeal Aerotaxis Transducer Combines Subunit I Core Structures of Eukaryotic Cytochrome c Oxidase and Eubacterial Methyl-Accepting Chemotaxis Proteins

Signal transduction in the archaeon Halobacterium salinarum is mediated by three distinct subfamilies of transducer proteins. Here we report the complete htrVIII gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome c oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the htrVIII gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.     

The Pseudomonas aeruginosa Diguanylate Cyclase GcbA,a Homolog of P. fluorescens GcbA,Promotes Initial Attachment to Surfaces,but Not Biofilm Formation,via Regulation of Motility

Cyclic di-GMP is a conserved signaling molecule regulating the transitions between motile and sessile modes of growth in a variety of bacterial species. Recent evidence suggests that Pseudomonas species harbor separate intracellular pools of c-di-GMP to control different phenotypic outputs associated with motility, attachment, and biofilm formation, with multiple diguanylate cyclases (DGCs) playing distinct roles in these processes, yet little is known about the potential conservation of functional DGCs across Pseudomonas species. In the present study, we demonstrate that the P. aeruginosa homolog of the P. fluorescens DGC GcbA involved in promoting biofilm formation via regulation of swimming motility likewise synthesizes c-di-GMP to regulate surface attachment via modulation of motility, however, without affecting subsequent biofilm formation. P. aeruginosa GcbA was found to regulate flagellum-driven motility by suppressing flagellar reversal rates in a manner independent of viscosity, surface hardness, and polysaccharide production. P. fluorescens GcbA was found to be functional in P. aeruginosa and was capable of restoring phenotypes associated with inactivation of gcbA in P. aeruginosa to wild-type levels. Motility and attachment of a gcbA mutant strain could be restored to wild-type levels via overexpression of the small regulatory RNA RsmZ. Furthermore, epistasis analysis revealed that while both contribute to the regulation of initial surface attachment and flagellum-driven motility, GcbA and the phosphodiesterase DipA act within different signaling networks to regulate these processes. Our findings expand the complexity of c-di-GMP signaling in the regulation of the motile-sessile switch by providing yet another potential link to the Gac/Rsm network and suggesting that distinct c-di-GMP-modulating signaling pathways can regulate a single phenotypic output.