Selection of Fimbriate Transductants of Salmonella typhimurium Dependent on Motility

The ability to form type 1 fimbriae (Fim(+)) was readily transduced to 159 out of 161 wild-type motile Fim(-) FIRN strains of Salmonella typhimurium with phage P22 propagated on a Fim(+) donor strain. Fim(+) clones were isolated from about 35% of tests after the fimbriate bacteria in the transduction mixture had been enriched by culture in aerobic static broth for 48 to 96 hr. A Fim(+) transductant was isolated from only 1 out of 280 tests made with 10 nonmotile recipient FIRN strains that were nonflagellate (Fla(-))- or possessed "paralyzed" flagella (Fla(+) Mot(-)), though motile variants from these strains were fully competent in yielding Fim(+) transductants. The property of motility was thought to facilitate the selective outgrowth of Fim(+) transductant bacteria by enabling them to migrate aerotactically to the surface of the broth where their fimbriae permitted them to float and grow in a pellicle stimulated by the free supply of atmospheric oxygen.     

Excretion of unassembled hook-associated proteins by Salmonella typhimurium.

Hook-associated proteins (HAPs) were excreted into the culture medium of the Fla+ strain as well as into the growth medium of the filamentless mutants of Salmonella typhimurium. This indicates that the bacteria synthesize HAPs excessively, beyond the amount required for construction of flagella. The extra HAPs are shed into the culture medium after a definite amount of each HAP has been assembled into the flagellar structure.     

Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina

Mitani, Michiko (National Institute of Genetics, Mishima, Japan), and Tetsuo Iino. Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina. J. Bacteriol. 90:1096-1101. 1965.-The arrangement of flagella was observed by dark-field and electron microscopy in three strains of Salmonella abortivoequina, namely, normal flagellar, curly flagellar, and paralyzed curly flagellar strains. With dark-field microscopy, bundled flagella could be seen in 5 to 10% of actively moving normal or curly mutant cells. Under the electron microscope, a great many bundled flagella were observed in the curly mutant strain, but in the normal strain most of the flagella were dissociated or the bundles were rather loose and irregular. Normal flagella seem to separate easily during the process of preparation, but not the curly ones. Single flagella were found to run parallel with each other and to form a bundle consisting of five or more flagella; the bundle was spirally gyrating, with the characteristic flagellar wave. It is thought that the bundle observed with the electron microscope corresponds to that observed under the dark-field microscope. Further, the marked decrease of bundle formation in the paralyzed curly mutant cells suggests that bundle formation is not caused by curly flagellar structure per se, but corresponds to the mode of locomotion of peritrichously flagellated bacteria.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.     

Serological Study of Bacterial Flagellar Hooks

Bacterial hooks were partially purified from flagella isolated from Salmonella SJ25, by treatment with heat to depolymerize flagellar filaments and with n-butanol and calcium chloride to remove membranes. Antihook serum was obtained from a rabbit inoculated with a preparation of hooks. The serum contained antibodies directed against the flagellar filament and cell membrane. These antibodies could be removed from the serum by absorption with purified flagellar filaments and cells of a nonflagellated mutant strain. It was shown by electron microscopy that anti-SJ25-hook antibody reacts with hooks from a number of strains of Salmonella which differed from SJ25 in H and O antigens, flagellar shape, and motility. Hooks possessed by various strains of Salmonella have a common antigenicity. In addition, anti-SJ25-hook cross-reacted with hooks from Escherichia coli W3110 but did not react at all which those from strains of Serratia, Proteus, Aerobacter, and Klebsiella. It is well known that bacteria stop moving upon addition of antiflagella serum to the medium. However, the addition of purified antihook was found to have little effect on motility. At physiological ionic strength and pH, flagellin (Salmonella) can polymerize into flagellar filaments only in the presence of seeds. It was shown that a crude preparation of hooks was able to initiate in vitro polymerization of flagellin.     

Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.     

Protection against Pseudomonas aeruginosa infection by passive transfer of anti-flagellar serum

The specificity of adsorbed flagellar antisera for H-antigen was demonstrated in vitro by cross-agglutination assays, motility inhibition, and an ELISA. The specific flagellar antibody was determined to be an IgG. Complete protection against burn wound sepsis was achieved with flagellar antisera. Cross-protection experiments revealed that protection was not only H-antigen dependent, but specific for the flagella antigen type. Antiserum raised against b-type flagella would only protect against homologous bacterial challenge and not against a-type flagellated strains. Results using a-type antisera were consistent, giving protection only against the homologous strain. In contrast, protective capacity was selectively removed from antisera by adsorbing with Fla+ cells. Bacteria colonized the burn wounds of passively protected mice to similar levels as seen in nonprotected animals, but the colonization remained localized and did not result in systemic infection, a pattern similar to infections with motility mutants observed in other studies. Animals rendered neutropenic prior to burning were not protected with flagellar antisera. These data suggested a role for phagocytic cells in protection. Immobilization by flagellar antiserum was observed both by microscopic studies and by inhibition of colony spreading. Antiflagellar antibody is hypothesized as exerting its protective capacity possibly in two ways; first by inhibiting the motility of invading bacteria by binding to the flagellum and immobilizing the bacteria, and secondly by acting as an opsonin, targeting either immobilized or mobile cells for phagocytosis.     

Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching.

Three flagellar genes of Salmonella typhimurium (flaAII.2, flaQ, and flaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) with counterclockwise motor bias. The distribution of Fla, Mot, and Che mutational sites within each gene was examined. Fla sites were fairly broadly distributed, whereas Mot and Che sites were more narrowly defined. Local subregions rich in sites of one type were not generally rich in sites of another type. Among Che sites, there was little overlap between those corresponding to a clockwise bias and those corresponding to a counterclockwise bias. Our results suggest that within the corresponding gene products there are specialized subregions for flagellar structure, motor rotation, and control of the sense of rotation.     

Mating in Chlamydomonas: a system for the study of specific cell adhesion. II. A radioactive flagella-binding assay for quantitation of adhesion

To measure the flagellar adhesiveness of Chlamydomonas gametes in a more quantitative manner than agglutination assays permit, a binding assay was developed which measured the binding of radioactive flagella of one mating type to unlabeled gametes of the opposite mating type. With the appropriate assay conditions, the number of [3H] flagella specifically bound was shown to be proportional to the number of cells in the incubation mixture and, therefore, to the number of binding sites that were present. The assay was used to study the effects of trypsin treatment on the loss and development of flagellar binding sites. It was shown that after trypsin treatment at least 9 h were required for the return of a full complement of binding sites to the flagellar surface; moreover, the results indicated that these sites reappeared on existing, extended flagella.     

Flagellar waveform and rotational orientation in a Chlamydomonas mutant lacking normal striated fibers

The Chlamydomonas mutant vfl-3 lacks normal striated fibers and microtubular rootlets. Although the flagella beat vigorously, the cells rarely display effective forward swimming. High speed cinephotomicrography reveals that flagellar waveform, frequency, and beat synchrony are similar to those of wild-type cells, indicating that neither striated fibers nor microtubular rootlets are required for initiation or synchronization of flagellar motion. However, in contrast to wild type, the effective strokes of the flagella of vfl-3 may occur in virtually any direction. Although the direction of beat varies between cells, it was not observed to vary for a given flagellum during periods of filming lasting up to several thousand beat cycles, indicating that the flagella are not free to rotate in the mature cell. Structural polarity markers in the proximal portion of each flagellum show that the flagella of the mutant have an altered rotational orientation consistent with their altered direction of beat. This implies that the variable direction of beat is not due to a defect in the intrinsic polarity of the axoneme, and that in wild-type cells the striated fibers and/or associated structures are important in establishing or maintaining the correct rotational orientation of the basal bodies to ensure that the inherent functional polarity of the flagellum results in effective cellular movement. As in wild type, the flagella of vfl-3 coordinately switch to a symmetrical, flagellar-type waveform during the shock response (induced by a sudden increase in illumination), indicating that the striated fibers are not directly involved in this process.     

The Bacillus subtilis catabolite control protein CcpA exerts all its regulatory functions by DNA-binding

Swimming speed (v) and flagellar-bundle rotation rate (f) of Salmonella typhimurium, which has peritrichous flagella, were simultaneously measured by laser dark-field microscopy (LDM). Clear periodic changes in the LDM signals from a rotating bundle indicated in-phase rotation of the flagella in the bundle. A roughly linear relation between v and f was observed, though the data points were widely distributed. The ratio of v to f (v-f ratio), which indicates the propulsive distance during one flagellar rotation, was 0.27 microm (11% of the flagellar pitch) on average. The experimental v-f ratio was twice as large as the calculated one on the assumption that a cell had a single flagellum. A flagellar bundle was considered to propel a cell more efficiently than a single flagellum.     

"Cap" on the tip of Salmonella flagella

Flagellar filaments isolated intact from a Salmonella short-flagella mutant are unable to serve as nuclei for flagellin polymerization in vitro, whereas the filaments reconstructed in vitro from the mutant flagellin are able to do so. The inability of intact flagella to nucleate flagellin polymerization appears to be common to wild-type bacteria and thus suggests that the tip of intact flagella are generally inactivated or capped in vivo. Careful observations of the tips of intact flagella and reconstructed flagellar filaments of a wild-type species have revealed marked difference between them: the intact flagella usually have blunt ends, whereas reconstructed filaments have concave, "fish-tail" ends. Moreover, a thin structure is often observed attaching to the very end of the intact flagella. We suspect that this "capping" structure is essential to the elongation mechanism of flagellar filaments.     

Minimal requirements for rotation of bacterial flagella.

An in vitro system of cell envelopes from Salmonella typhimurium with functional flagella was used to determine the minimal requirements for flagellar rotation. Rotation in the absence of cytoplasmic constituents could be driven either by respiration or by an artificially imposed chemical gradient of protons. No specific ionic requirements other than protons (or hydroxyls) were found for the motor function.     

Experimental dissection of flagellar surface motility in chlamydomonas

Experiments have explored the possible relationships between the flagellar surface motility of chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol. 15:983-989), and the capacity of gametic flagella to participate in the mating reaction. While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those of gametic flagella exhibit no such preference. This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, J. Cell Biol. 79:680-693). Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-β-tubulin antiserum are all able to inhibit the binding of beads to the flagellar suface. Trysin digestion and an antiserum directed against whole chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile. These results suggest that at least two flagellar activities participate in surface motility: (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e.g. an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signal-to-fuse from the flagellar tips to the cell body. Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

Basal bodies and associated structures are not required for normal flagellar motion or phototaxis in the green alga Chlorogonium elongatum

The interphase flagellar apparatus of the green alga Chlorogonium elongatum resembles that of Chlamydomonas reinhardtii in the possession of microtubular rootlets and striated fibers. However, Chlorogonium, unlike Chlamydomonas, retains functional flagella during cell division. In dividing cells, the basal bodies and associated structures are no longer present at the flagellar bases, but have apparently detached and migrated towards the cell equator before the first mitosis. The transition regions remain with the flagella, which are now attached to a large apical mitochondrion by cross-striated filamentous components. Both dividing and nondividing cells of Chlorogonium propagate asymmetrical ciliary-type waveforms during forward swimming and symmetrical flagellar-type waveforms during reverse swimming. High-speed cinephotomicrographic analysis indicates that waveforms, beat frequency, and flagellar coordination are similar in both cell types. This indicates that basal bodies, striated fibers, and microtubular rootlets are not required for the initiation of flagellar beat, coordination of the two flagella, or determination of flagellar waveform. Dividing cells display a strong net negative phototaxis comparable to that of nondividing cells; hence, none of these structures are required for the transmission or processing of the signals involved in phototaxis, or for the changes in flagellar beat that lead to phototactic turning. Therefore, all of the machinery directly involved in the control of flagellar motion is contained within the axoneme and/or transition region. The timing of formation and the positioning of the newly formed basal structures in each of the daughter cells suggests that they play a significant role in cellular morphogenesis.     

New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy.

The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella.     

Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla(+) but Mot(-) phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5' region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.     

Transposon tnPhoA mutagenesis as a method of obtaining mutants of Azospirillum brasilense by motility and flagellation

Three A. brasilense strains (S27, SpBr14, and KR77) did not hydrolyze the chromogenic substrate of alkaline phosphatase (PhoA), X-phosphate, in situ, and were used as recipients in experiments on TnphoA mutagenesis. KMR transconjugates were obtained only for A. brasilense S27, 85% of them were also PhoA+. About 12% TnphoA mutants of A. brasilense S27 had reduces capacity to swarming and 3% of mutants neither swam nor swarmed. These totally immotile clones were examined under transmission electron microscope and were classified as Fla-Laf-, Fla-leakyLaf-, and Fla-Laf+ mutants. In Fla-Laf+ TnphoA mutants of S27, the expression of their lateral flagella (Laf) retained the wild-type inducibility. The presence of intact polar flagellum (Fla) did not seem to be obligatory for controllable expression of Laf in A. brasilense S27. The data suggest that A. brasilense S27 Fla and Laf systems have common structural and/or regulatory components. The PhoA+ phenotype of S27 Fla- mutants suggested a periplasmic and/or membrane localization of the hybrid proteins, the formation of which blocks the flagellar assembly or functioning. Immunochemical analysis with antibodies to alkaline phosphatase will identify these proteins.