Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) modulates the transition between planktonic and biofilm life styles. In response to c-di-GMP, the enhancer binding protein FleQ from Pseudomonas aeruginosa derepresses the expression of Pel exopolysaccharide genes required for biofilm formation when a second protein, FleN is present. A model is that binding of c-di-GMP to FleQ induces its dissociation from the pelA promoter allowing RNA polymerase to access this site. To test this, we analyzed pelA DNA footprinting patterns with various combinations of FleQ, FleN and c-di-GMP, coupled to in vivo promoter activities. FleQ binds to two sites called box 1 and 2. FleN binds to FleQ bound at these sites causing the intervening DNA to bend. Binding of c-di-GMP to FleQ relieves the DNA distortion but FleQ remains bound to the two sites. Analysis of wild type and mutated versions of pelA-lacZ transcriptional fusions suggests that FleQ represses gene expression from box 2 and activates gene expression in response to c-di-GMP from box 1. The role of c-di-GMP is thus to convert FleQ from a repressor to an activator. The mechanism of action of FleQ is distinct from that of other bacterial transcription factors that both activate and repress gene expression from a single promoter.

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ 54 -interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.enhancer binding protein | flagella | structure | gene expression B acterial adaptations to diverse environments, including human hosts, involve collaborative group behaviors, such as quorum sensing, swarming, and biofilm formation (1-5). In general, quorum-sensing during host tissue colonization is associated with virulence gene expression and acute-phase infections, whereas biofilm formation facilitates the development of chronic infections, evasion of host immune response, and increased tolerance to treatments (6). It is now well appreciated that these social behaviors result from highly regulated signal transduction processes, which in many bacteria are choreographed by the nucleotide-based second messenger c-di-GMP (7-9). Synthesized by GGDEF domain-containing diguanylate cyclases and hydrolyzed by EAL or HD-GYP domain-containing phosphodiesterases, c-di-GMP is sensed by a variety of protein-and RNA-based effectors to exert control at transcriptional, translational, and posttranslational levels (10, 11).In Pseudomonas aeruginosa, an opportunistic pathogen that causes severe chronic infections in cystic fibrosis patients, burn victims, and other immunocompromised individuals, the transcription factor FleQ acts as a master regulator of flagellar...

The transcription factor FleQ is a bacterial AAA+ ATPase enhancerbinding protein that is the master activator of flagella gene expression in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Homologs of FleQ are present in all Pseudomonas species and in many polarly flagellated gamma proteobacteria. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls the transition between planktonic and biofilm modes of growth in bacteria in response to diverse environmental signals. C-di-GMP binds to FleQ to dampen its activity, causing down-regulation of flagella gene expression. This action is potentiated in the simultaneous presence of another protein, FleN. We explored the effect of c-di-GMP and FleN on the ATPase activity of FleQ and found that a relatively low concentration of c-di-GMP competitively inhibited FleQ ATPase activity, suggesting that c-di-GMP competes with ATP for binding to the Walker A motif of FleQ. Confirming this, a FleQ Walker A motif mutant failed to bind c-di-GMP. FleN, whose gene is regulated by FleQ, also inhibited FleQ ATPase activity, and FleQ ATPase activity was much more inhibited by c-di-GMP in the presence of FleN than in its absence. These results indicate that FleN and c-di-GMP cooperate to inhibit FleQ activity and, by extension, flagella synthesis in P. aeruginosa. The Walker A motif of FleQ is perfectly conserved, opening up the possibility that other AAA+ ATPases may respond to c-di-GMP.an ubiquitous second messenger in bacteria, where it affects the transition between a motile planktonic lifestyle and an adhesive biofilm lifestyle by binding to various effector proteins to modulate enzymatic activity, protein-protein interactions, transcription, and translation (1-6). One of these effector proteins is FleQ (PA1097), an enhancer-binding protein (EBP) transcription factor from the opportunistic pathogen Pseudomonas aeruginosa.FleQ contains an N-terminal FleQ domain, a central AAA+ ATPase domain, and a C-terminal helix-turn-helix DNA-binding domain. It activates expression of biofilm-related genes, including genes for Pel and Psl exopolysaccharide (EPS) in response to binding of c-di-GMP at an unknown site (7,8). EBPs typically act in conjunction with σ 54 -RNA polymerase; however, FleQ regulates EPS gene expression independent of σ 54 , most likely with σ 70 (8, 9).In addition to regulating genes for biofilm formation, FleQ has a second, better-known role as a σ 54 -dependent master regulator of P. aeruginosa flagella gene expression (9, 10). FleQ activates expression of the two-component regulatory genes fleSR, as well as genes for the assembly of the flagella export apparatus and for the initiation of flagella basal body assembly. FleSR controls genes to complete the basal body-hook structure (9, 11).The regulation of both flagella and biofilm genes is also under the control of a second protein, FleN. Mutations in fleN (PA1454) lead to an up-regulation of flagella gene expression and a smalldown-regulation of biofilm genes (7, 12). The expression ...

The transcription factor FleQ from Pseudomonas aeruginosa derepresses expression of genes involved in biofilm formation when intracellular levels of the second messenger cyclic diguanosine monophosphate (c-di-GMP) are high. FleQ also activates transcription of flagellar genes, and the expression of these genes is highest at low intracellular c-di-GMP. FleQ thus plays a central role in mediating the transition between planktonic and biofilm lifestyles of P. aeruginosa. Previous work showed that FleQ controls expression of the pel operon for Pel exopolysaccharide biosynthesis by converting from a repressor to an activator upon binding c-di-GMP. To explore the activity of FleQ further, we carried out DNase I footprinting at three additional biofilm gene promoters, those of psl, cdrAB, and PA2440. The expression of cdrAB, encoding a cell surface adhesin, was sufficiently responsive to FleQ to allow us to carry out in vivo promoter assays. The results showed that, similarly to our observations with the pel operon, FleQ switches from a repressor to an activator of cdrAB gene expression in response to c-di-GMP. From the footprinting data, we identified a FleQ DNA binding consensus sequence. A search for this conserved sequence in bacterial genome sequences led to the identification of FleQ binding sites in the promoters of the siaABCD operon, important for cell aggregation, and the bdlA gene, important for biofilm dispersal, in P. aeruginosa. We also identified FleQ binding sites upstream of lapA-like adhesin genes in other Pseudomonas species. IMPORTANCEThe transcription factor FleQ is widely distributed in Pseudomonas species. In all species examined, it is a master regulator of flagellar gene expression. It also regulates diverse genes involved in biofilm formation in P. aeruginosa when intracellular levels of the second messenger c-di-GMP are high. Unlike flagellar genes, biofilm-associated genes are not always easy to recognize in genome sequences. Here, we identified a consensus DNA binding sequence for FleQ. This allowed us to survey Pseudomonas strains and find new genes that are likely regulated by FleQ and possibly involved in biofilm formation. Cyclic diguanosine monophosphate (c-di-GMP) is an intracellular second messenger that is produced by bacteria and has diverse effects on bacterial physiology. c-di-GMP binds to riboswitches and effector proteins to modulate transcription, translation, and protein activities (1, 2). In the opportunistic pathogen Pseudomonas aeruginosa, the transcription factor FleQ binds c-di-GMP to derepress expression of genes for biofilm components (3, 4). Biofilms are surface-attached communities of bacteria embedded in a matrix made of exopolysaccharides, proteins, and DNA. Biofilm infections are particularly difficult to treat because bacteria are protected by the biofilm matrix, making them resistant to antimicrobial treatment compared to planktonic cells (5). Genes controlled by FleQ include pel genes, coding for Pel exopolysaccharide; psl genes, coding for Psl exopolys...

SummaryShewanella oneidensis uses a wide range of terminal electron acceptors for respiration. In this study, we show that the chemotactic response of S. oneidensis to anaerobic electron acceptors requires functional electron transport systems. Deletion of the genes encoding dimethyl sulphoxide and trimethylamine N-oxide reductases, or inactivation of these molybdoenzymes as well as nitrate reductase by addition of tungstate, abolished electron acceptor taxis. Moreover, addition of nigericin prevented taxis towards trimethylamine N-oxide, dimethyl sulphoxide, nitrite, nitrate and fumarate, showing that this process depends on the DpH component of the proton motive force. These data, together with those concerning response to metals (Bencharit and Ward, 2005), support the idea that, in S. oneidensis, taxis towards electron acceptors is governed by an energy taxis mechanism. Surprisingly, energy taxis in S. oneidensis is not mediated by the PAS-containing chemoreceptors but rather by a chemoreceptor (SO2240) containing a Cache domain. Four other chemoreceptors also play a minor role in this process. These results indicate that energy taxis can be mediated by new types of chemoreceptors.