The swarming motility of Pseudomonas aeruginosa is blocked by cranberry proanthocyanidins and other tannin-containing materials

Bacterial motility plays a key role in the colonization of surfaces by bacteria and the subsequent formation of resistant communities of bacteria called biofilms. Derivatives of cranberry fruit, predominantly condensed tannins called proanthocyanidins (PACs) have been reported to interfere with bacterial adhesion, but the effects of PACs and other tannins on bacterial motilities remain largely unknown. In this study, we investigated whether cranberry PAC (CPAC) and the hydrolyzable tannin in pomegranate (PG; punicalagin) affected the levels of motilities exhibited by the bacterium Pseudomonas aeruginosa. This bacterium utilizes flagellum-mediated swimming motility to approach a surface, attaches, and then further spreads via the surface-associated motilities designated swarming and twitching, mediated by multiple flagella and type IV pili, respectively. Under the conditions tested, both CPAC and PG completely blocked swarming motility but did not block swimming or twitching motilities. Other cranberry-containing materials and extracts of green tea (also rich in tannins) were also able to block or impair swarming motility. Moreover, swarming bacteria were repelled by filter paper discs impregnated with many tannin-containing materials. Growth experiments demonstrated that the majority of these compounds did not impair bacterial growth. When CPAC- or PG-containing medium was supplemented with surfactant (rhamnolipid), swarming motility was partially restored, suggesting that the effective tannins are in part acting by a rhamnolipid-related mechanism. Further support for this theory was provided by demonstrating that the agar surrounding tannin-induced nonswarming bacteria was considerably less hydrophilic than the agar area surrounding swarming bacteria. This is the first study to show that natural compounds containing tannins are able to block P. aeruginosa swarming motility and that swarming bacteria are repelled by such compounds.     

The surprisingly diverse ways that prokaryotes move

Prokaryotic cells move through liquids or over moist surfaces by swimming, swarming, gliding, twitching or floating. An impressive diversity of motility mechanisms has evolved in prokaryotes. Movement can involve surface appendages, such as flagella that spin, pili that pull and Mycoplasma 'legs' that walk. Internal structures, such as the cytoskeleton and gas vesicles, are involved in some types of motility, whereas the mechanisms of some other types of movement remain mysterious. Regardless of the type of motility machinery that is employed, most motile microorganisms use complex sensory systems to control their movements in response to stimuli, which allows them to migrate to optimal environments.     

Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here, we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMHs) and show that conserved glutamate residues in GldL TMH2 are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane, suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

Motile bacteria usually rely on external apparatus like flagella or pili, but gliding bacteria do not rely on obvious surface appendages for their movement. This study shows that bacteria in the phylum Bacteroidetes use proton-dependent motors to power protein secretion and gliding motility.     

Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili

We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.     

Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system

Although flagella are the best-understood means of locomotion in bacteria [1], other bacterial motility mechanisms must exist as many diverse groups of bacteria move without the aid of flagella [2-4]. One unusual structure that may contribute to motility is the type IV pilus [5,6]. Genetic evidence indicates that type IV pili are required for social gliding motility (S-motility) in Myxococcus, and twitching motility in Pseudomonas and Neisseria [6,7]. It is thought that type IV pili may retract or rotate to bring about cellular motility [6,8], but there is no direct evidence for the role of pili in cell movements. Here, using a tethering assay, we obtained evidence that the type IV pilus of Myxococcus xanthus functions as a motility apparatus. Pili were required for M. xanthus cells to adhere to solid surfaces and to generate cellular movement using S-motility. Tethered cells were released from the surface at intervals corresponding to the reversal frequency of wild-type cells when gliding on a solid surface. Mutants defective in the control of directional movements and cellular reversals (frz mutants) showed altered patterns of adherence that correlate reversal frequencies with tethering. The behavior of the tethered cells was consistent with a model in which the pili are extruded from one cell pole, adhere to a surface, and then retract, pulling the cell in the direction of the adhering pili. Cellular reversals would result from the sites of pili extrusion switching from one cell pole to another and are controlled by the frz chemosensory system.     

Inhibition of swarming motility of Pseudomonas aeruginosa by branched-chain fatty acids.

Pseudomonas aeruginosa is capable of moving by swimming, swarming, and twitching motilities. In this study, we investigated the effects of fatty acids on Pseudomonas aeruginosa PAO1 motilities. A branched-chain fatty acid (BCFA)--12-methyltetradecanoic acid (anteiso-C15:0)--has slightly repressed flagella-driven swimming motility and completely inhibited a more complex type of surface motility, i.e. swarming, at a concentration of 10 microg mL(-1). In contrast, anteiso-C15:0 exhibited no effect on pili-mediated twitching motility. Other BCFAs and unsaturated fatty acids tested in this study showed similar inhibitory effects on swarming motility, although the level of inhibition differed between these fatty acids. These fatty acids caused no significant growth inhibition in liquid cultures. Straight-chain saturated fatty acids such as palmitic acid were less effective in swarming inhibition. The wetness of the PAO1 colony was significantly reduced by the addition of anteiso-C15:0; however, the production of rhamnolipids as a surface-active agent was not affected by the fatty acid. In addition to motility repression, anteiso-C15:0 caused 31% repression of biofilm formation by PAO1, suggesting that BCFA could affect the multiple cellular activities of Pseudomonas aeruginosa.     

Mutations that impair swarming motility in Serratia marcescens 274 include but are not limited to those affecting chemotaxis or flagellar function.

Serratia marcescens exists in two cell forms and displays two kinds of motility depending on the type of growth surface encountered (L. Alberti and R. M. Harshey, J. Bacteriol. 172:4322-4328, 1990). In liquid medium, the bacteria are short rods with few flagella and show classical swimming behavior. Upon growth on a solid surface (0.7 to 0.85% agar), they differentiate into elongated, multinucleate, copiously flagellated forms that swarm over the agar surface. The flagella of swimmer and swarmer cells are composed of the same flagellin protein. We show in this study that disruption of hag, the gene encoding flagellin, abolishes both swimming and swarming motility. We have used transposon mini-Mu lac kan to isolate mutants of S. marcescens defective in both kinds of motility. Of the 155 mutants obtained, all Fla- mutants (lacking flagella) and Mot- mutants (paralyzed flagella) were defective for both swimming and swarming, as expected. All Che- mutants (chemotaxis defective) were also defective for swarming, suggesting that an intact chemotaxis system is essential for swarming. About one-third of the mutants were specifically affected only in swarming. Of this class, a large majority showed active "swarming motility" when viewed through the microscope (analogous to the active "swimming motility" of Che- mutants) but failed to show significant movement away from the site of initial inoculation on a macroscopic scale. These results suggest that bacteria swarming on a solid surface require many genes in addition to those required for chemotaxis and flagellar function, which extend the swarming movement outward. We also show in this study that nonflagellate S. marcescens is capable of spreading rapidly on low-agar media.     

Myxococcus xanthus dif genes are required for biogenesis of cell surface fibrils essential for social gliding motility

Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.     

Gliding motility in bacteria: insights from studies of Myxococcus xanthus.

Gliding motility is observed in a large variety of phylogenetically unrelated bacteria. Gliding provides a means for microbes to travel in environments with a low water content, such as might be found in biofilms, microbial mats, and soil. Gliding is defined as the movement of a cell on a surface in the direction of the long axis of the cell. Because this definition is operational and not mechanistic, the underlying molecular motor(s) may be quite different in diverse microbes. In fact, studies on the gliding bacterium Myxococcus xanthus suggest that two independent gliding machineries, encoded by two multigene systems, operate in this microorganism. One machinery, which allows individual cells to glide on a surface, independent of whether the cells are moving alone or in groups, requires the function of the genes of the A-motility system. More than 37 A-motility genes are known to be required for this form of movement. Depending on an additional phenotype, these genes are divided into two subclasses, the agl and cgl genes. Videomicroscopic studies on gliding movement, as well as ultrastructural observations of two myxobacteria, suggest that the A-system motor may consist of multiple single motor elements that are arrayed along the entire cell body. Each motor element is proposed to be localized to the periplasmic space and to be anchored to the peptidoglycan layer. The force to glide which may be generated here is coupled to adhesion sites that move freely in the outer membrane. These adhesion sites provide a specific contact with the substratum. Based on single-cell observations, similar models have been proposed to operate in the unrelated gliding bacteria Flavobacterium johnsoniae (formerly Cytophaga johnsonae), Cytophaga strain U67, and Flexibacter polymorphus (a filamentous glider). Although this model has not been verified experimentally, M. xanthus seems to be the ideal organism with which to test it, given the genetic tools available. The second gliding motor in M. xanthus controls cell movement in groups (S-motility system). It is dependent on functional type IV pili and is operative only when cells are in close proximity to each other. Type IV pili are known to be involved in another mode of bacterial surface translocation, called twitching motility. S-motility may well represent a variation of twitching motility in M. xanthus. However, twitching differs from gliding since it involves cell movements that are jerky and abrupt and that lack the organization and smoothness observed in gliding. Components of this motor are encoded by genes of the S-system, which appear to be homologs of genes involved in the biosynthesis, assembly, and function of type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae. How type IV pili generate force in S-motility is currently unknown, but it is to be expected that ongoing physiological, genetic, and biochemical studies in M. xanthus, in conjunction with studies on twitching in P. aeruginosa and N. gonorrhoeae, will provide important insights into this microbial motor. The two motility systems of M. xanthus are affected to different degrees by the MglA protein, which shows similarity to a small GTPase. Bacterial chemotaxis-like sensory transduction systems control gliding motility in M. xanthus. The frz genes appear to regulate gliding movement of individual cells and movement by the S-motility system, suggesting that the two motors found in this bacterium can be regulated to result in coordinated multicellular movements. In contrast, the dif genes affect only S-system-dependent swarming.     

Bacterial lateral flagella: an inducible flagella system

Flagella are complex surface organelles that allow bacteria to move towards favourable environments and that contribute to the virulence of pathogenic bacteria through adhesion and biofilm formation on host surfaces. There are a few bacteria that possess functional dual flagella systems, such as Vibrio parahaemolyticus, some mesophilic Aeromonas spp., Rhodospirillum centenum and Azospirillum brasilense. These bacteria are able to express both a constitutive polar flagellum required for swimming motility and a separate lateral flagella system that is induced in viscous media or on surfaces and is essential for swarming motility. As flagella synthesis and motility have a high metabolic cost for the bacterium, the expression of the inducible lateral flagella system is highly regulated by a number of environmental factors and regulators.     

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.     

Self-produced extracellular stimuli modulate the Pseudomonas aeruginosa swarming motility behaviour

Pseudomonas aeruginosa presents three types of motilities: swimming, twitching and swarming. The latter is characterized by rapid and coordinated group movement over a semisolid surface resulting from morphological differentiation and intercellular interactions. A striking feature of P. aeruginosa swarming motility is the formation of migrating tendrils producing colonies with complex fractal-like patterns. Previous studies have shown that normal swarming motility is intimately related to the production of extracellular surface-active molecules: rhamnolipids (RLs), composed of monorhamnolipids (mono-RLs) and dirhamnolipids (di-RLs), and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). Here, we report that (i) di-RLs attract active swarming cells while HAAs behave as strong repellents, (ii) di-RLs promote and HAAs inhibit tendril formation and migration, (iii) di-RLs and HAAs display different diffusion kinetics on a surface as di-RLs spread faster than HAAs in agar, (iv) di-RLs and HAAs have no effect on swimming cells, suggesting that swarming cells are different from swimming cells not only in morphology but also at the regulatory level and (v) mono-RLs act as wetting agents. We propose a model explaining how HAAs and di-RLs together modulate the behaviour of swarming migrating cells by acting as self-produced negative and positive chemotactic-like stimuli.     

Motility and chemotaxis in Agrobacterium tumefaciens surface attachment and biofilm formation

Bacterial motility mechanisms, including swimming, swarming, and twitching, are known to have important roles in biofilm formation, including colonization and the subsequent expansion into mature structured surface communities. Directed motility requires chemotaxis functions that are conserved among many bacterial species. The biofilm-forming plant pathogen Agrobacterium tumefaciens drives swimming motility by utilizing a small group of flagella localized to a single pole or the subpolar region of the cell. There is no evidence for twitching or swarming motility in A. tumefaciens. Site-specific deletion mutations that resulted in either aflagellate, flagellated but nonmotile, or flagellated but nonchemotactic A. tumefaciens derivatives were examined for biofilm formation under static and flowing conditions. Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms. In contrast, a nonmotile mutant with unpowered flagella was clearly debilitated for biofilm formation relative to the wild type. A nontumbling chemotaxis mutant was only weakly affected with regard to biofilm formation under nonflowing conditions but was notably compromised in flow, generating less adherent biomass than the wild type, with a more dispersed cellular arrangement. Extragenic suppressor mutants of the chemotaxis-impaired, straight-swimming phenotype were readily isolated from motility agar plates. These mutants regained tumbling at a frequency similar to that of the wild type. Despite this phenotype, biofilm formation by the suppressor mutants in static cultures was significantly deficient. Under flowing conditions, a representative suppressor mutant manifested a phenotype similar to yet distinct from that of its nonchemotactic parent.     

A spider web strategy of type IV pili‐mediated migration to build a fibre‐like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms

Bacterial motilities participate in biofilm development. However, it is unknown how/if bacterial motility affects formation of the biofilm matrix. Psl polysaccharide is a key biofilm matrix component of Pseudomonas aeruginosa. Here we report that type IV pili (T4P)‐mediated bacterial migration leads to the formation of a fibre‐like Psl matrix. Deletion of T4P in wild type and flagella‐deficient strains results in loss of the Psl‐fibres and reduction of biofilm biomass in flow cell biofilms as well as pellicles at air‐liquid interface. Bacteria lacking T4P‐driven twitching motility including those that still express surface T4P are unable to form the Psl‐fibres. Formation of a Psl‐fibre matrix is critical for efficient biofilm formation, yet does not require flagella and polysaccharide Pel or alginate. The Psl‐fibres are likely formed by Psl released from bacteria during T4P‐mediated migration, a strategy similar to spider web formation. Starvation can couple Psl release and T4P‐driven twitching motility. Furthermore, a radial‐pattern Psl‐fibre matrix is present in the middle of biofilms, a nutrient‐deprived region. These imply a plausible model for how bacteria respond to nutrient‐limited local environment to build a polysaccharide‐fibre matrix by T4P‐dependent bacterial migration strategy. This strategy may have general significance for bacterial survival in natural and clinical settings.     

Flagellated ectosymbiotic bacteria propel a eucaryotic cell

A devescovinid flagellate from termites exhibits rapid gliding movements only when in close contact with other cells or with a substrate. Locomotion is powered not by the cell's own flagella nor by its remarkable rotary axostyle, but by the flagella of thousands of rod bacteria which live on its surface. That the ectosymbiotic bacteria actually propel the protozoan was shown by the following: (a) the bacteria, which lie in specialized pockets of the host membrane, bear typical procaryotic flagella on their exposed surface; (b) gliding continues when the devescovinid's own flagella and rotary axostyle are inactivated; (c) agents which inhibit bacterial flagellar motility, but not the protozoan's motile systems, stop gliding movements; (d) isolated vesicles derived from the surface of the devescovinid rotate at speeds dependent on the number of rod bacteria still attached; (e) individual rod bacteria can move independently over the surface of compressed cells; and (f) wave propagation by the flagellar bundles of the ectosymbiotic bacteria is visualized directly by video-enhanced polarization microscopy. Proximity to solid boundaries may be required to align the flagellar bundles of adjacent bacteria in the same direction, and/or to increase their propulsive efficiency (wall effect). This motility-linked symbiosis resembles the association of locomotory spirochetes with the Australian termite flagellate Mixotricha (Cleveland, L. R., and A. V. Grimstone, 1964, Proc. R. Soc. Lond. B Biol. Sci., 159:668-686), except that in our case propulsion is provided by bacterial flagella themselves. Since bacterial flagella rotate, an additional novelty of this system is that the surface bearing the procaryotic rotary motors is turned by the eucaryotic rotary motor within.     

Evidence for two flagellar stators and their role in the motility of Pseudomonas aeruginosa

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.     

Pseudomonas aeruginosa exhibits sliding motility in the absence of type IV pili and flagella

Pseudomonas aeruginosa exhibits swarming motility on 0.5 to 1% agar plates in the presence of specific carbon and nitrogen sources. We have found that PAO1 double mutants expressing neither flagella nor type IV pili (fliC pilA) display sliding motility under the same conditions. Sliding motility was inhibited when type IV pilus expression was restored; like swarming motility, it also decreased in the absence of rhamnolipid surfactant production. Transposon insertions in gacA and gacS increased sliding motility and restored tendril formation to spreading colonies, while transposon insertions in retS abolished motility. These changes in motility were not accompanied by detectable changes in rhamnolipid surfactant production or by the appearance of bacterial surface structures that might power sliding motility. We propose that P. aeruginosa requires flagella during swarming to overcome adhesive interactions mediated by type IV pili. The apparent dependence of sliding motility on environmental cues and regulatory pathways that also affect swarming motility suggests that both forms of motility are influenced by similar cohesive factors that restrict translocation, as well as by dispersive factors that facilitate spreading. Studies of sliding motility may be particularly well-suited for identifying factors other than pili and flagella that affect community behaviors of P. aeruginosa.     

Swimming and twitching motility are essential for attachment and virulence of Pantoea ananatis in onion seedlings

Pantoea ananatis is a widespread phytopathogen with a broad host range. Despite its ability to infect economically important crops, such as maize, rice and onion, relatively little is known about how this bacterium infects and colonizes host tissue or spreads within and between hosts. To study the role of motility in pathogenicity, we analysed both swimming and twitching motility in P. ananatis LMG 20103. Genetic recombineering was used to construct four mutants affected in motility. Two flagellar mutants were disrupted in the flgK and motA genes, required for flagellar assembly and flagellar rotation, respectively. Similarly, two twitching motility mutants were generated, impaired in the structure (pilA) and functioning (pilT) of the type IV pili. The role of swimming and twitching motility during the infection cycle of P. ananatis in onion seedlings was determined by comparing the mutant‐ and wild‐type strains using several in vitro and in planta assays. From the results obtained, it was evident that flagella aid P. ananatis in locating and attaching to onion leaf surfaces, as well as in pathogenicity, whereas twitching motility is instrumental in the spread of the bacteria on the surface once attachment has occurred. Both swimming and twitching motility contribute towards the ability of P. ananatis to cause disease in onions.