Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa.
Ontology highlight
ABSTRACT:
SUBMITTER: Toutain CM
PROVIDER: S-EPMC543560 | biostudies-literature | 2005 Jan
REPOSITORIES: biostudies-literature
Publications
Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa.
Toutain Christine M CM Zegans Michael E ME O'Toole George A GA
Journal of bacteriology 20050101 2
Pseudomonas aeruginosa is a ubiquitous bacterium capable of twitching, swimming, and swarming motility. In this study, we present evidence that P. aeruginosa has two flagellar stators, conserved in all pseudomonads as well as some other gram-negative bacteria. Either stator is sufficient for swimming, but both are necessary for swarming motility under most of the conditions tested, suggesting that these two stators may have different roles in these two types of motility. ...[more]
Similar Datasets
Project description:Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol.IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.
| S-EPMC6707923 | biostudies-literature
Project description:Flagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in Pseudomonas aeruginosa and other microbes is cyclic diguanylate (c-di-GMP). High levels of this second messenger repress motility and stimulate biofilm formation. c-di-GMP levels regulate motility in P. aeruginosa in part by influencing the localization of its two flagellar stator sets, MotAB and MotCD. Here, we show that while c-di-GMP can influence stator localization, stators can in turn impact c-di-GMP levels. We demonstrate that the swarming motility-driving stator MotC physically interacts with the transmembrane region of the diguanylate cyclase SadC. Furthermore, we demonstrate that this interaction is capable of stimulating SadC activity. We propose a model by which the MotCD stator set interacts with SadC to stimulate c-di-GMP production under conditions not permissive to motility. This regulation implies a positive-feedback loop in which c-di-GMP signaling events cause MotCD stators to disengage from the motor; then disengaged stators stimulate c-di-GMP production to reinforce a biofilm mode of growth. Our studies help to define the bidirectional interactions between c-di-GMP and the flagellar machinery.IMPORTANCE The ability of bacterial cells to control motility during early steps in biofilm formation is critical for the transition to a nonmotile, biofilm lifestyle. Recent studies have clearly demonstrated the ability of c-di-GMP to control motility via a number of mechanisms, including through controlling transcription of motility-related genes and modulating motor function. Here, we provide evidence that motor components can in turn impact c-di-GMP levels. We propose that communication between motor components and the c-di-GMP synthesis machinery allows the cell to have a robust and sensitive switching mechanism to control motility during early events in biofilm formation.
| S-EPMC6707927 | biostudies-literature
Project description:The opportunistic human pathogen Pseudomonas aeruginosa uses two surface organelles, flagella and pili, for motility and adhesion in biotic and abiotic environments. Polar flagellar placement and number are influenced by FlhF, which is a signal recognition particle (SRP)-type GTPase. The FlhF proteins of Bacillus subtilis and Campylobacter jejuni were recently shown to have GTPase activity. However, the phenotypes associated with flhF deletion and/or mutation differ between these organisms and P. aeruginosa, making it difficult to generalize a role for FlhF in pseudomonads. In this study, we confirmed that FlhF of P. aeruginosa binds and hydrolyzes GTP. We mutated FlhF residues that we predicted would alter nucleotide binding and hydrolysis and determined the effects of these mutations on FlhF enzymatic activity, protein dimerization, and bacterial motility. Both hydrolytically active and inactive FlhF point mutants restored polar flagellar assembly, as seen for wild-type FlhF. However, differential effects on flagellar function were observed in single-cell assays of swimming motility and flagellar rotation. These findings indicate that FlhF function is influenced by its nucleotide binding and hydrolytic activities and demonstrate that FlhF affects P. aeruginosa flagellar function as well as assembly.
| S-EPMC3571332 | biostudies-literature
Project description:We report the transcriptome of Pseudomonas aeruginosa PA14 under swarming conditions as compared to a swimming control
2019-12-23 | GSE121504 | GEO
Project description:Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.
| S-EPMC97303 | biostudies-literature
Project description:Each Pseudomonas aeruginosa cell localizes two types of motility structures, a single flagellum and one or two clusters of type IV pili, to the cell poles. Previous studies suggested that these motility structures arrive at the pole through distinct mechanisms. Here we performed a swimming motility screen to identify polar flagellum localization factors and discovered three genes homologous to the TonB/ExbB/ExbD complex that have defects in both flagella-mediated swimming and pilus-mediated twitching motility. We found that deletion of tonB3, PA2983 or PA2982 led to non-polar localization of the flagellum and FlhF, which was thought to sit at the top of the flagellar localization hierarchy. Surprisingly, these mutants also exhibited pronounced changes in pilus formation or localization, indicating that these proteins may co-ordinate both the pilus and flagellum motility systems. Thus, we have renamed PA2983 and PA2982, pocA and pocB, respectively, for polar organelle co-ordinator to reflect this function. Our results suggest that TonB3, PocA and PocB may form a membrane-associated complex, which we term the Poc complex. These proteins do not exhibit polar localization themselves, but are required for increased expression of pilus genes upon surface association, indicating that they regulate motility structures through either localization or transcriptional mechanisms.
| S-EPMC4666538 | biostudies-literature
Project description:The bacterial flagellar motor drives the rotation of flagellar filaments and enables many species of bacteria to swim. Torque is generated by interaction of stator units, anchored to the peptidoglycan cell wall, with the rotor. Recent experiments [Yuan J, Berg HC (2008) Proc Natl Acad Sci USA 105:1182-1185] show that at near-zero load the speed of the motor is independent of the number of stators. Here, we introduce a mathematical model of the motor dynamics that explains this behavior based on a general assumption that the stepping rate of a stator depends on the torque exerted by the stator on the rotor. We find that the motor dynamics can be characterized by two timescales: the moving-time interval for the mechanical rotation of the rotor and the waiting-time interval determined by the chemical transitions of the stators. We show that these two timescales depend differently on the load, and that their cross-over provides the microscopic explanation for the existence of two regimes in the torque-speed curves observed experimentally. We also analyze the speed fluctuation for a single motor by using our model. We show that the motion is smoothed by having more stator units. However, the mechanism for such fluctuation reduction is different depending on the load. We predict that the speed fluctuation is determined by the number of steps per revolution only at low load and is controlled by external noise for high load. Our model can be generalized to study other molecular motor systems with multiple power-generating units.
| S-EPMC2656151 | biostudies-literature
Project description:The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili (T4P). Single cells twitch by sequential T4P extension, attachment, and retraction. How single cells coordinate T4P to efficiently navigate surfaces remains unclear. We demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from T4P in a process called mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. Collisions between twitching cells stimulate reversals, but Chp mutants either always or never reverse. As a result, while wild-type cells colonize surfaces uniformly, collision-blind Chp mutants jam, demonstrating a function for mechanosensing in regulating group behavior. On surfaces, Chp senses T4P attachment at one pole, thereby sensing a spatially resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization, inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local excitation-global inhibition in higher-order organisms, identifying a conserved strategy to transduce spatially resolved signals.
| S-EPMC8325320 | biostudies-literature
Project description:BackgroundThe bacterium Pseudomonas aeruginosa is capable of three types of motilities: swimming, twitching and swarming. The latter is characterized by a fast and coordinated group movement over a semi-solid surface resulting from intercellular interactions and morphological differentiation. A striking feature of swarming motility is the complex fractal-like patterns displayed by migrating bacteria while they move away from their inoculation point. This type of group behaviour is still poorly understood and its characterization provides important information on bacterial structured communities such as biofilms. Using GeneChip® Affymetrix microarrays, we obtained the transcriptomic profiles of both bacterial populations located at the tip of migrating tendrils and swarm center of swarming colonies and compared these profiles to that of a bacterial control population grown on the same media but solidified to not allow swarming motility.ResultsMicroarray raw data were corrected for background noise with the RMA algorithm and quantile normalized. Differentially expressed genes between the three conditions were selected using a threshold of 1.5 log2-fold, which gave a total of 378 selected genes (6.3% of the predicted open reading frames of strain PA14). Major shifts in gene expression patterns are observed in each growth conditions, highlighting the presence of distinct bacterial subpopulations within a swarming colony (tendril tips vs. swarm center). Unexpectedly, microarrays expression data reveal that a minority of genes are up-regulated in tendril tip populations. Among them, we found energy metabolism, ribosomal protein and transport of small molecules related genes. On the other hand, many well-known virulence factors genes were globally repressed in tendril tip cells. Swarm center cells are distinct and appear to be under oxidative and copper stress responses.ConclusionsResults reported in this study show that, as opposed to swarm center cells, tendril tip populations of a swarming colony displays general down-regulation of genes associated with virulence and up-regulation of genes involved in energy metabolism. These results allow us to propose a model where tendril tip cells function as «scouts» whose main purpose is to rapidly spread on uncolonized surfaces while swarm center population are in a state allowing a permanent settlement of the colonized area (biofilm-like).
| S-EPMC3091734 | biostudies-literature
Project description:The single polar flagellum of Pseudomonas aeruginosa plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the Pseudomonas genome database, an open reading frame (ORF), flanked by flagellar genes flhF and fliA, that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in P. aeruginosa PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in P. aeruginosa. The ORF was named fleN. An intact copy of fleN on a plasmid complemented the mutant by restoring motility and monoflagellate status. The beta-galactosidase activities of eight flagellar operon or gene promoters in the wild-type and fleN mutant strains revealed a direct correlation between six promoters that were upregulated in the fleN mutant (fliLMNOPQ, flgBCDE, fliEFG, fliDS orf126, fleSR, and fliC) and positive regulation by FleQ, an NtrC-like transcriptional regulator for flagellar genes. Based on these results, we propose a model where FleN influences FleQ activity (directly or indirectly) in regulating flagellar number in P. aeruginosa.
| S-EPMC94283 | biostudies-literature