More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per μm of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The “dry” colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water.     

Visualization of Flagella during Bacterial Swarming

When cells of Escherichia coli are grown in broth and suspended at low density in a motility medium, they swim independently, exploring a homogeneous, isotropic environment. Cell trajectories and the way in which these trajectories are determined by flagellar dynamics are well understood. When cells are grown in a rich medium on agar instead, they elongate, produce more flagella, and swarm. They move in coordinated packs within a thin film of fluid, in intimate contact with one another and with two fixed surfaces, a surfactant monolayer above and an agar matrix below: they move in an inhomogeneous, anisotropic environment. Here we examine swarm-cell trajectories and ways in which these trajectories are determined by flagellar motion, visualizing the cell bodies by phase-contrast microscopy and the flagellar filaments by fluorescence microscopy. We distinguish four kinds of tracks, defining stalls, reversals, lateral movement, and forward movement. When cells are stalled at the edge of a colony, they extend their flagellar filaments outwards, moving fluid over the virgin agar; when cells reverse, changes in filament chirality play a crucial role; when cells move laterally, they are pushed sideways by adjacent cells; and when cells move forward, they are pushed by flagellar bundles in the same way as when they are swimming in bulk aqueous media. These maneuvers are described in this report.Swarming is a common yet specialized form of surface translocation exhibited by flagellated bacteria and is distinct from swimming (23). When cells are grown on a moist nutrient-rich surface, they differentiate from a vegetative to a swarm state: they elongate, make more flagella, secrete wetting agents, and move across the surface in coordinated packs. Here we focus on the mechanics of bacterial swarming, as exhibited by the model organism Escherichia coli. Others have worked on swarm-cell differentiation in a variety of organisms, including Proteus, Salmonella, Pseudomonas, Serratia, Bacillus, and Vibrio. For example, screens for genes required for swarming in E. coli or Salmonella have been made by Inoue et al. (25) and Wang et al. (40, 41). Vibrio is a special case, because a single polar flagellum enables cells to swim while multiple lateral flagella promote swarming (32). For general reviews, see the work of Allison and Hughes (1), Shapiro (37), Fraser and Hughes (17), and Fraser et al. (16). Also see the work of Eberl et al. (15), Sharma and Anand (38), Harshey (18), Daniels et al. (11), Kaiser (26), O''Toole (33), and Copeland and Weibel (10).Swarming was first observed with Proteus mirabilis by Hauser (22), who named the genus for a sea god able to change his own form. Proteus is distinctive because cells switch periodically from the vegetative to the swarming state, building terraced colonies (36, 42). This is not observed with E. coli under the conditions used here, where swarms expand at a constant rate propelled by cells swimming vigorously in a monolayer behind a smooth outer boundary.Swarming in E. coli was discovered by Harshey, who found that K-12 strains, which lack the lipopolysaccharide O antigen, swarmed on Eiken agar (from Japan) but not on Difco agar (from the United States), presumably because the former is more wettable (19, 20). Chemotaxis is not required: cells lacking the chemotaxis response regulator CheY swarm perfectly well, provided that mutations in the motor protein FliM enable transitions between clockwise (CW) and counterclockwise (CCW) rotational states (31). It was suggested that these reversals promote wetness by causing cells to shed lipopolysaccharide.How do cells in E. coli swarms move across an agar surface? What are their flagella doing? We sought to answer such questions by performing a global analysis of videotaped data (of phase-contrast images) collected from 5 regions of 2 swarms, plotting body lengths, speeds, propulsion angles, local track curvatures, and temporal and spatial correlations, and we found that cells reorient on a time scale of a few tenths of a second, primarily by colliding with one another (13). Our previous report did not describe analyses of individual tracks or visualization of flagella. This aspect of the work is presented here.Most of the time, cells are driven forwards by a flagellar bundle in the usual way. Flagellar filaments from different cells can intertwine and form common bundles, but this is rare. However, cells in swarms do something not ordinarily seen with swimming cells: they back up. They do this without changing the orientation of the cell body by moving back through the middle of the flagellar bundle. This involves changes in filament shape (in polymorphic form), from normal to curly and back to normal. Polymorphic forms were classified by Calladine (7), on the basis of earlier work by Asakura (3), in terms of the relative lengths of 11 protofilaments, longitudinal arrays of protein subunits that comprise the filament. All polymorphic forms are helical, with some being left-handed (e.g., the normal form) and some being right-handed (e.g., the semicoiled and curly forms, which have half the pitch of the normal filament and half the pitch and half the amplitude, respectively). Transformations from one shape to another can be caused in various ways, e.g., by changes in pH, salinity, or temperature (21, 27, 28) or by application of torque (24). The changes observed with swarm cells are driven by the latter mechanism, when motors switch from CCW to CW rotation. When swimming cells tumble, polymorphic transformations also occur, in the order normal to semicoiled to curly and back to normal (14, 39). But we rarely see the semicoiled form with cells in swarms, and when it appears, it is quite transient. We wonder whether polymorphic transformations evolved to enable cells to escape confined environments when the only way out is to back up, keeping the filaments close to the sides of the cell body.     

Flagella and Motility in Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae has been considered nonmotile and nonflagellate. In this work, it is demonstrated that A. pleuropneumoniae produces flagella composed of a 65-kDa protein with an N-terminal amino acid sequence that shows 100% identity with those of Escherichia coli, Salmonella, and Shigella flagellins. The DNA sequence obtained through PCR of the fliC gene in A. pleuropneumoniae showed considerable identity (93%) in its 5' and 3' ends with the DNA sequences of corresponding genes in E. coli, Salmonella enterica, and Shigella spp. The motility of A. pleuropneumoniae was observed in tryptic soy or brain heart infusion soft agar media, and it is influenced by temperature. Flagella and motility may be involved in the survival and pathogenesis of A. pleuropneumoniae in pigs.     

A Colonization Factor (Production of Lateral Flagella) of Mesophilic Aeromonas spp. Is Inactive in Aeromonas salmonicida Strains

The nine laf (lateral flagellum) genes of mesophilic aeromonads are in the Aeromonas salmonicida genome. The laf genes are functional, except for lafA (flagellin gene), which was inactivated by transposase 8 (IS3 family). A pathogenic characteristic of mesophilic aeromonads (lateral flagella) is abolished in this specialized pathogen with a narrow host range.     

Laboratory Strains of Bacillus subtilis Do Not Exhibit Swarming Motility

We redemonstrate that SwrA is essential for swarming motility in Bacillus subtilis, and we reassert that laboratory strains of B. subtilis do not swarm. Additionally, we find that a number of other genes, previously reported to be required for swarming in laboratory strains, are dispensable for robust swarming motility in an undomesticated strain. We attribute discrepancies in the literature to a lack of reproducible standard experimental conditions, selection for spontaneous swarming suppressors, inadvertent genetic linkage to swarming mutations, and auxotrophy.Many species of bacteria are capable of flagellum-mediated swimming motility in liquid broth. Of those species, a subset is also capable of a related, but genetically separable, form of flagellum-mediated surface movement called swarming motility (17). Examples of swarming-proficient species include Proteus mirabilis, Vibrio parahaemolyticus, Serratia marcescens, Escherichia coli, Salmonella enterica, and Bacillus subtilis (1, 15, 16, 20, 28). In general, swarming requires a surfactant or wetting agent to reduce surface tension, an increase in flagellar number per cell, and other genetic features that are distinct from swimming (7, 14).There is confusion in the literature concerning the genetic requirements of the swarming phenotype of B. subtilis. It is generally accepted that the ancestral undomesticated strain B. subtilis 3610 exhibits robust swarming motility (18, 20, 33). Swarming motility of strain 3610 requires the production of a secreted surfactant, called surfactin (6, 20), to reduce surface tension and permit surface spreading, and it also requires the protein SwrA to activate flagellar biosynthesis gene expression and increase the number of flagella on the cell surface (5, 20). Some reports claim that domesticated derivatives of 3610, such as the commonly used laboratory strain 168, are also swarming proficient (10, 18, 19, 24). Strain 168, however, is defective in both surfactin production (9, 25) and SwrA (5, 21, 31), and thus, swarming 168 strains challenge the genetic definition of swarming motility. Our lab has never observed swarming in laboratory strains, and here we investigated swarming motility in a reportedly swarming-proficient 168 strain.We obtained a reportedly swarming-proficient 168 strain (13) (generous gift of Simone Séror, Orsay University, Paris-Sud, France) (Table ​(Table1)1) and compared its swarming phenotype to that of 3610 under our standard conditions (20). Swarm plates were prepared one day prior to use with 25 ml of LB medium (10 g Bacto tryptone, 5 g Bacto yeast extract, 5 g NaCl per liter) fortified with 0.7% Bacto agar. To minimize water on the agar surface and thus minimize the potentially confounding influence of swimming motility, plates were dried 20 min prior to inoculation and 10 min postinoculation open-faced in a laminar flow hood. For qualitative swarm assays, plates were centrally inoculated with cells from a freshly grown overnight colony using a sterile stick. For quantitative swarm expansion assays, 1 ml of cells grown to mid-exponential phase (optical density at 600 nm [OD₆₀₀], 0.5) was resuspended in PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄ per liter, pH 7.0) containing 0.5% India ink (Higgins) to an OD₆₀₀ of 10 and centrally spotted (10 μl). Swarm expansion was measured at 0.5-h intervals along a transect on the plate. Plates were incubated at 37°C in 20 to 30% humidity. Whereas strain 3610 was swarming proficient, strain 168 (Orsay) was swarming deficient (Fig. ​(Fig.1A).1A). Thus, strain 168 (Orsay) appeared to behave similarly to all other laboratory strains we have tested previously (20, 21).Open in a separate windowFIG. 1.Swarming motility on LB and B media. In qualitative plate images, colonized agar appears white and uncolonized agar appears black on LB and B media, as indicated. Swarming cells colonize a larger surface area than nonswarming cells. All strains are derivatives of strain 3610 unless otherwise indicated. Bar, 2 cm. (A) Quantitative swarm expansion assays on solid medium and growth in liquid medium of the indicated strains on LB medium (closed symbols) and on B medium (open symbols). To indicate variability in a particular experiment, we have reproduced the quantitative swarm expansion assay of strain 3610 on LB and B media with error bars in Fig. S5 in the supplemental material. (B) Quantitative swarm expansion assays on LB (closed symbols) and B (open symbols) media. The following strains were used: DS3337 (sfp), DS2415 (swrA), DS5106 (168 swrA⁺), DS5758 (168 sfp⁺), and DS5759 (168 swrA⁺ sfp⁺). In all assays, B medium was made according to reference 2 except for strain DS5759, for which B medium was supplemented with 780 μM threonine to compensate for thrC auxotrophy. (C) Swarm plates of the indicated strains on LB medium made with equal parts peptone instead of tryptone. (D) Quantitative swarm expansion assays of the indicated 3610-derived mutant strains on LB medium (closed symbols) and on B medium (open symbols). The following strains were used: DS72 (yvzB), DS2268 (epr), DS3903 (phrC), DS4978 (rapC), DS4979 (oppD), DS2509 (swrB), and DS3649 (degU). All points are averages for three replicates.

TABLE 1.

Strains

Quorum Sensing Controls Swarming Motility of Burkholderia glumae through Regulation of Rhamnolipids

Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of rhamnolipids in B. glumae and that rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous rhamnolipids. Impaired rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour.     

Putrescine Importer PlaP Contributes to Swarming Motility and Urothelial Cell Invasion in Proteus mirabilis

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437–446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.     

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24, 26, 34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34, 38, 54).Type IV pili are typically composed of major pilins, the primary structural components of the pilus, and several minor pilin-like proteins that play important roles in pilus assembly or function (15, 17, 46). Pilin precursor proteins are transported across the cytoplasmic membrane via the Sec translocation pathway (7, 20). Most Sec substrates contain either a class I or a class II signal peptide that is cleaved at a recognition site that lies subsequent to the hydrophobic portion of the signal peptide (18, 43). However, the precursors of type IV pilins contain class III signal peptides, which are processed at recognition sites that precede the hydrophobic domain by a prepilin-specific peptidase (SPase III) (38, 43, 45). Similarly, archaeal flagellin precursors contain a class III signal peptide that is processed by a prepilin-specific peptidase homolog (FlaK/PibD) (3, 8, 10, 11). Moreover, flagellar assembly involves homologs of components involved in the biosynthesis of bacterial type IV pili, including FlaI, an ATPase homologous to PilB, and FlaJ, a multispanning membrane protein that may provide a platform for flagellar assembly, similar to the proposed role for PilC in pilus assembly (38, 44, 53, 54). These genes, as well as a number of others that encode proteins often required for either flagellar assembly or function (flaCDEFG and flaH), are frequently coregulated with the flg genes (11, 26, 44, 54).Interestingly, most sequenced archaeal genomes also contain diverse sets of genes that encode type IV pilin-like proteins with little or no homology to archaeal flagellins (3, 39, 52). While often coregulated with pilB and pilC homologs, these genes are never found in clusters containing the motility-specific flaCDEFG and flaH homologs; however, the proteins they encode do contain class III signal peptides (52). Several of these proteins have been shown to be processed by an SPase III (4, 52). Moreover, in Sulfolobus solfataricus and Methanococcus maripaludis, some of these archaeal type IV pilin-like proteins were confirmed to form surface filaments that are distinct from the flagella (21, 22, 56). These findings strongly suggest that the genes encode subunits of pilus-like surface structures that are involved in functions other than swimming motility.In bacteria, type IV pili are multifunctional filamentous protein complexes that, in addition to facilitating twitching motility, mediate adherence to abiotic surfaces and make close intercellular associations possible (15, 17, 46). For instance, mating between Escherichia coli in liquid medium has been shown to require type IV pili (often referred to as thin sex pili), which bring cells into close proximity (29, 30, 57). Recent work has shown that the S. solfataricus pilus, Ups, is required not only for efficient adhesion to surfaces of these crenarchaeal cells but also for UV-induced aggregation (21, 22, 58). Frols et al. postulate that autoaggregation is required for DNA exchange under these highly mutagenic conditions (22). Halobacterium salinarum has also been shown to form Ca²⁺-induced aggregates (27, 28). Furthermore, conjugation has been observed in H. volcanii, which likely requires that cells be held in close proximity for a sustained period to allow time for the cells to construct the cytoplasmic bridges that facilitate DNA transfer between them (35).To determine the roles played by haloarchaeal flagella and other putative type IV pilus-like structures in swimming and surface motility, surface adhesion, autoaggregation, and conjugation, we constructed and characterized two mutant strains of H. volcanii, one lacking the genes that encode the flagellins and the other lacking pibD. Our analyses indicate that although this archaeon was previously thought to be nonmotile (14, 36), wild-type (wt) H. volcanii can swim in a flagellum-dependent manner. Consistent with the involvement of PibD in processing flagellins, the peptidase mutant is nonmotile. Unlike nonhalophilic archaea, however, the flagellum mutant can adhere to glass as effectively as the wild type. Conversely, the ΔpibD strain fails to adhere to glass surfaces, strongly suggesting that in H. volcanii surface adhesion involves nonflagellar, type IV pilus-like structures.