Bacteria that express lateral flagella enable dissection of the multifunctional roles of flagella in pathogenesis

Flagella are much more than organelles of locomotion and have multiple roles that contribute to pathogenesis. Bacteria, such as Vibrio parahaemolyticus and Aeromonas spp., that possess two distinct flagellar systems (a polar flagellum for swimming in liquid and lateral flagella for swarming over surfaces) are relatively uncommon and provide ideal models for the independent investigation of the contributions of these different types of motility and other flagellar functions to virulence and how they are controlled. Studies with the above organisms have already increased our understanding of how bacteria sense and colonize surfaces forming biofilms that enable them to survive and persist in hostile environments. These insights are helping to identify possible new targets for novel antimicrobials that will both prevent or disrupt these processes and enhance the effectiveness of existing antibiotics. Aeromonas lateral flagella, in addition to mediating swarming motility, appear to be adhesins in their own right, contribute to microcolony formation and efficient biofilm formation on surfaces, and possibly facilitate host cell invasion. It is, therefore, likely that the ability to express lateral flagella is a significant virulence determinant for the Aeromonas strains able to cause persistent and dysenteric infections in the gastrointestinal tract, but further work is needed to establish this.     

Lateral flagella are required for increased cell adherence,invasion and biofilm formation by Aeromonas spp

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.     

Spirochete periplasmic flagella and motility

Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.     

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.     

Lateral Flagella and Swarming Motility in Aeromonas Species

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.     

Chlamydomonas flagella

Chlamydomonas reinhardtii Dangard has been widely adopted as a model system for studies of eukaryotic cilia and flagella. Here I review recent progress in understanding flagellar ultrastructure, the mechanisms that generate and regulate flagellar beating and gliding motility, the flagellar assembly process, basal body structure and function, and adhesion-based signaling, all advanced by work with this single-celled organism.     

Layer-by-layer assembly of bioengineered flagella protein nanotubes

Flagella nanotubes present on the surface of E. coli bacteria were bioengineered to display arginine-lysine and glutamic acid-aspartic acid peptide loops. These protein bionanotubes were demonstrated to self-assemble, layer-by-layer, by atomic force microscopy (AFM) on gold-coated mica and quartz surfaces. Flagella with arginine-lysine loops were assembled in a bottom-up manner on a gold-coated mica surface by employing the molecular complementarity of the biotin-streptavidin interaction. Self-assembled monolayers of alkylamines on the gold surface were derivatized with biotin, followed by binding of streptavidin to the biotinylated surface. The amine groups of the flagella peptide loops were chemically attached to biotin through a polyethyleneoxide spacer and paired with streptavidin on the gold surface. This process could be repeated to generate multiple layers of flagella. Flagella with glutamic acid-aspartic acid peptide loops were self-assembled on quartz surfaces by electrostatic attraction to protonated amine groups. The quartz surface was silanized to obtain amine groups, which were used to assemble the first layer of glutamic acid-aspartic acid peptide loop flagella nanotubes. This layer was covered with polyethyleneimine through electrostatic attraction and employed to assemble a second layer of flagella. The self-assembled glutamic acid-aspartic acid flagella were also used to demonstrate the biomineralization of CaCO 3. The layer-by-layer self-assembly employing electrostatic attraction yielded a more uniform layer of flagella than the one obtained with the molecular complementarity of the biotin-streptavidin pair.     

Novel mechanisms power bacterial gliding motility

For many bacteria, motility is essential for survival, growth, virulence, biofilm formation and intra/interspecies interactions. Since natural environments differ, bacteria have evolved remarkable motility systems to adapt, including swimming in aqueous media, and swarming, twitching and gliding on solid and semi‐solid surfaces. Although tremendous advances have been achieved in understanding swimming and swarming motilities powered by flagella, and twitching motility powered by Type IV pili, little is known about gliding motility. Bacterial gliders are a heterogeneous group containing diverse bacteria that utilize surface motilities that do not depend on traditional flagella or pili, but are powered by mechanisms that are less well understood. Recently, advances in our understanding of the molecular machineries for several gliding bacteria revealed the roles of modified ion channels, secretion systems and unique machinery for surface movements. These novel mechanisms provide rich source materials for studying the function and evolution of complex microbial nanomachines. In this review, we summarize recent findings made on the gliding mechanisms of the myxobacteria, flavobacteria and mycoplasmas.     

Flagellin phase‐dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation

The flagellum is a sophisticated nanomachine and an important virulence factor of many pathogenic bacteria. Flagellar motility enables directed movements towards host cells in a chemotactic process, and near‐surface swimming on cell surfaces is crucial for selection of permissive entry sites. The long external flagellar filament is made of tens of thousands subunits of a single protein, flagellin, and many Salmonella serovars alternate expression of antigenically distinct flagellin proteins, FliC and FljB. However, the role of the different flagellin variants during gut colonisation and host cell invasion remains elusive. Here, we demonstrate that flagella made of different flagellin variants display structural differences and affect Salmonella's swimming behaviour on host cell surfaces. We observed a distinct advantage of bacteria expressing FliC‐flagella to identify target sites on host cell surfaces and to invade epithelial cells. FliC‐expressing bacteria outcompeted FljB‐expressing bacteria for intestinal tissue colonisation in the gastroenteritis and typhoid murine infection models. Intracellular survival and responses of the host immune system were not altered. We conclude that structural properties of flagella modulate the swimming behaviour on host cell surfaces, which facilitates the search for invasion sites and might constitute a general mechanism for productive host cell invasion of flagellated bacteria.     

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.     

Reversible inhibition of flagella formation after specific inhibition of spermidine synthesis by dicyclohexylamine in Pseudomonas aeruginosa

Dicyclohexylamine, which is an inhibitor of bacterial and mammalian spermidine synthase, greatly inhibited the synthesis of spermidine in Pseudomonas aeruginosa. The depletion of spermidine caused by dicyclohexylamine was accompanied by an inhibition of growth of bacteria. This inhibition was reversed by addition of 50 M spermidine (but not putrescine or spermine) to growth medium. When its growth was inhibited Ps. aeruginosa also lost its motility. Electron microscopy showed a loss of flagella in spermidine-deficient bacteria: after 24h 70% 85% of bacteria grown in the presence of dicyclohexylamine did not have flagella, whereas bacteria grown in the presence of dicyclohexylamine and spermidine had flagella. The loss of flagella was reversible, since after the inhibition of spermidine synthesis for 24 h, addition of 50 M spermidine (but not putrescine or spermine) to the growth medium was able to restore the bacterial motility almost completely after a further 12 h ghrowth period.     

Campylobacter flagella: not just for motility

Campylobacter jejuni and Campylobacter coli are among the major causes of diarrheal disease worldwide. The motility imparted by the polar flagella of these pathogens is required for colonization of the mucus lining of the gastrointestinal tract. However, recent studies have revealed a more complex role for flagella in Campylobacter pathogenesis that includes the ability to secrete non-flagellar proteins that modulate virulence and the co-regulation of secreted and non-secreted virulence factors with the flagella regulon. Campylobacter flagellins are heavily glycosylated and changes in glycan composition affect autoagglutination and microcolony formation on intestinal epithelial cells; these traits are associated with disease in an animal model. Here, these recent advances in our understanding of the multifaceted role of flagella in Campylobacter virulence are summarized.     

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafTlU genes resulted in non-motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.     

Transformation of straight flagella and recovery of motility in a mutant Escherichia coli.

The non-motile strain W3623 ha-177 of Escherichia coli (Kondoh &; Ozeki, 1976) is known to produce straight flagella as a result of a mutation in the structural gene for the flagellin. Under physiological conditions, however, flagella of this mutant undergo straight-to-helical transformation with small changes of pH. Evidence for this came from dark-field light microscope observations of reconstituted flagella. At pH values lower than 6.6 in the presence of 0.1 m-NaCl, the flagella were straight. When, however, the pH was raised above 7.3, they were transformed into left-handed helices with a pitch of 2.05 μm. The transformation was rapid and reversible. In the pH range between 6.6 and 7.3, straight and transformed flagella co-existed but no stable forms other than the two were found.Bacterial motility also depended on the pH of the medium: at pH values above 7.0, bacteria swam by means of the transformed flagella. Therefore, helically transformed flagella of the mutant strain were similar in morphology and function to normal-type flagella of the parent strain. The significance of this similarity is discussed on the basis of general considerations of polymorphism in bacterial flagella.     

FimZ Is a Molecular Link between Sticking and Swimming in Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium produces two types of filamentous appendages on its surface. Fimbriae mediate adherence to tissues and cells via receptor-specific interactions, and flagella are the organelles of motility. These appendages play a role in colonization and dissemination, respectively, from infected surfaces and may be important components of bacterial survival. Increased expression of FimZ in serovar Typhimurium resulted in bacteria which were hyperfimbriated but were nonmotile in soft agar. This lack of motility was associated with down regulation of the flhDC master flagellar operon. Therefore, FimZ represents a molecular connection between flagella and fimbrial formation in serovar Typhimurium, indicating that the synthesis of flagella and fimbriae are oppositely controlled.     

Role of divalent cations in the subunit associations of complex flagella from Rhizobium meliloti.

Rhizobium meliloti, a symbiotic, nitrogen-fixing soil bacterium with complex flagella, as well as other members of the family Rhizobiaceae, rapidly lost motility when suspended in buffers lacking divalent cations but retained good motility in buffers containing calcium, magnesium, barium, or strontium. Loss of motility was associated with loss of flagella from the cells. Analysis of flagella by sedimentation, gel electrophoresis, and electron microscopy revealed that removal of divalent cations from the complex flagella of R. meliloti resulted in extensive dissociation of the flagellar filaments into low-molecular-weight subunits. Accordingly, divalent cations such as calcium and magnesium that are normally present at high concentrations in the soil solution may be crucial to the assembly and rigidity of complex flagella.     

Biofilm formation by Pseudomonas aeruginosa wild type,flagella and type IV pili mutants

Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.