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A polar flagella operon (flg) of Aeromonas hydrophila contains genes required for lateral flagella expression.

Aeromonas spp. are pathogens of both humans and poikilothermic animals, causing a variety of diseases. Certain strains are able to produce two distinct types of flagella; polar flagella for swimming in liquid and lateral flagella for swarming over surfaces. Although, both types of flagella have been associated as colonisation factors, little is known about their organisation and expression. Here we characterised a complete flagellar locus of Aeromonas hydrophila (flg) containing 16 genes, this was analogous to region 1 of the Vibrio parahaemolyticus polar flagellum, with the difference that no flagellin genes were found on A. hydrophila while V. parahaemolyticus showed three flagellin genes. The flg region was present in all Aeromonas strain tested. Defined insertion mutants in flgL, were unable to swim, had a drastic reduction in swarming, lateral flagella, HEp-2 cell adhesion and biofilm formation. Mutations in flgN caused a drastic reduction in lateral flagella, inability to swarm, but these strains were still able to swim. Whereas the cheV mutants still produced both types of flagella and were able to swim and swarm. These results suggest that FlgN is required for lateral flagella formation and swarming motility, but not for polar flagellum-mediated swimming.

Aeromonas hydrophila↗

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.

Biofilms↗

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.

Aeromonas↗

Analysis of the movement of Chlamydomonas flagella:" the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella.

The mutation uni-1 gives rise to uniflagellate Chlamydomonas cells which rotate around a fixed point in the microscope field, so that the flagellar bending pattern can be photographed easily. This has allowed us to make a detailed analysis of the wild-type flagellar bending pattern and the bending patterns of flagella on several mutant strains. Cells containing uni-1, and recombinants of uni-1 with the suppressor mutations, suppf-1 and suppf-3, show the typical asymmetric bending pattern associated with forward swimming in Chlamydomonas, although suppf-1 flagella have about one-half the normal beta frequency, apparently as the result of defective function of the outer dynein arms. The pf-17 mutation has been shown to produce nonmotile flagella in which radial spoke heads and five characteristic axonemal polypeptides are missing. Recombinants containing pf-17 and either suppf-2 or suppf-3 have motile flagella, but still lack radial-spoke heads and the associated polypeptides. The flagellar bending pattern of these recombinants lacking radial-spoke heads is a nearly symmetric, large amplitude pattern which is quite unlike the wild-type pattern. However, the presence of an intact radial-spoke system is not required to convert active sliding into bending and is not required for bend initiation and bend propagation, since all of these processes are active in suppfpf-17 recombinants. The function of the radial-spoke system appears to be to convert the symmetric bending pattern displayed by these recombinants into the asymmetric bending pattern required for efficient swimming, by inhibiting the development of reverse bends during the recovery phase of the bending cycle.

Chlamydomonas↗

Bending patterns of Chlamydomonas flagella: II. Calcium effects on reactivated Chlamydomonas flagella.

Ca2+ has profound effects on the movement of cilia and eukaryotic flagella, including those of Chlamydomonas. Two clear changes seen in Chlamydomonas flagella with changes in Ca2+ are beat frequency and symmetry. Photographic and computer assisted analysis of flagellar bending patterns on a uniflagellate mutant of Chlamydomonas have been used to examine details of the effects of Ca2+ on the movement of ATP-reactivated, demembranated flagella. In addition to the forward mode bending pattern seen at low Ca2+ concentrations (10(-9)M), which has a frequency of about 50 Hz and the reverse mode bending pattern seen at high Ca2+ concentrations (10(-4)M) with a frequency around 70 Hz, we carefully examined bending patterns in the intermediate Ca2+ concentration range of 1-6.5 X 10(-6)M. In this intermediate range, the bending patterns have significantly reduced asymmetry and slightly increased frequency, compared to the motility observed at low Ca2+ concentrations. These observations indicate that changes in these two parameters of motion do not occur in parallel and suggest that the effects of Ca2+ may be a multicomponent process. Physiologically, these changes in the beat pattern at intermediate Ca2+ may signal either 1) the beginning stages of transition to the symmetrical, high-frequency beating seen at high Ca2+, or 2) a more normal forward mode motility for the trans flagellum as suggested by Kamiya and Witman [1984]. No large amplitude bending patterns associated with transitions between forward and reverse mode beating in intact cells were seen at the intermediate Ca2+ concentrations.

Calcium↗

Synthesis of bacterial flagella. II. PBS1 transduction of flagella-specific markers in Bacillus subtilis.

The linkage relationship of mutants involved in the synthesis of flagella was determined by PBSl transduction. Mutants that affect the structure of flagellin (hag) and temperature-sensitive mutants (flaTS) that produce flagella when grown at 37 C but not when grown at 46 C were examined. All of the mutants were found to be linked to the hisA1 marker. The flaTS mutants fell into three clusters. Group A contained the majority of mutants which were loosely grouped around the hag locus. Group B mutants were segregated from the hag locus and appeared closely linked to the phage adsorption site gene (gtaA), and group C was only loosely linked to hisA1 and thus far contains only one mutant. A flagella locus (ifm) affecting both the degree of motility and level of flagellation was shown to map near group A. Mutants affecting motility (mot) were not linked to hisA1 by PBSl transduction. Several markers previously shown to link to hisA1 were ordered with respect to hisA1 and the flagellar genes.

Antigens↗

Synthesis of bacterial flagella: chromosomal synchrony and flagella synthesis.

Synchronous cultures of Bacillus subtilis 168 M were obtained from light-density spores germinated at 46 C and grown at 37 C. This procedure synchronizes both cell division and chromosome replication. The chromosome synchrony was demonstrated by using transformation to measure changes in marker frequency during the cell cycle. The synthesis of two enzymes and of bacterial flagellar protein was also followed. All of the proteins were found to be synthesized continuously with an abrupt doubling in the rate of synthesis at a specific time in the cell cycle. The time at which the doubling occurred for each enzyme corresponded to the time at which the structural gene for the enzyme was replicated. The doubling of the rate of flagella synthesis corresponded to the time of replication of the hisA1 gene. We conclude that the genetic locus for the factors involved in the rate-limiting steps in flagella synthesis are located on the genetic map near the hisA1 locus.

Bacillus subtilis↗

Archaeal flagella, bacterial flagella and type IV pili: a comparison of genes and posttranslational modifications.

The archaeal flagellum is a unique motility organelle. While superficially similar to the bacterial flagellum, several similarities have been reported between the archaeal flagellum and the bacterial type IV pilus system. These include the multiflagellin nature of the flagellar filament, N-terminal sequence similarities between archaeal flagellins and bacterial type IV pilins, as well as the presence of homologous proteins in the two systems. Recent advances in archaeal flagella research add to the growing list of similarities. First, the preflagellin peptidase that is responsible for processing the N-terminal signal peptide in preflagellins has been identified. The preflagellin peptidase is a membrane-bound enzyme topologically similar to its counterpart in the type IV pilus system (prepilin peptidase); the two enzymes are demonstrated to utilize the same catalytic mechanism. Second, it has been suggested that the archaeal flagellum and the bacterial type IV pilus share a similar mode of assembly. While bacterial flagellins and type IV pilins can be modified with O-linked glycans, N-linked glycans have recently been reported on archaeal flagellins. This mode of glycosylation, as well as the observation that the archaeal flagellum lacks a central channel, are both consistent with the proposed assembly model. On the other hand, the failure to identify other genes involved in archaeal flagellation by homology searches likely implies a novel aspect of the archaeal flagellar system. These interesting features remain to be deciphered through continued research. Such knowledge would be invaluable to motility and protein export studies in the Archaea.

Amino Acid Sequence↗

Parenteral application of a Pseudomonas aeruginosa flagella vaccine elicits specific anti-flagella antibodies in the airways of healthy individuals.

To test the hypothesis that a Pseudomonas aeruginosa flagella (FLA) vaccine, intramuscularly administered, elicits specific antibodies in the respiratory tract, antibody titers against FLA were determined in sera and bronchoalveolar lavage (BAL) fluids of 10 healthy human adults before and after immunization. Immunization produced significantly increased anti-FLA antibody titers in all sera (mean reciprocal titers: IgG: 26,711; IgA: 2,767; IgM: 421) and on respiratory epithelial lining fluids (mean reciprocal titers: IgG: 112; IgA: 76; sIgA: 26). Significant differences in class-specific serum and BAL anti-FLA titers suggested airway antibody production. The study shows that parenteral immunization provides not only high systemic antibody titers but also local antibody production in the human respiratory tract.

Adult↗

Generation of flagella by cultured mouse spermatids.

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.

Animals↗