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PubMed1992

Flagella in prokaryotes and lower eukaryotes.

During the past year, significant advances have been made in the understanding of both prokaryotic and eukaryotic flagella. About 50 genes are dedicated to the assembly and operation of bacterial flagella. Recent discoveries have advanced our understanding of how these genes are regulated and how their products assemble into a functional, rotating organelle. The dynein arms of eukaryotic flagella are now also better understood. Several genes that are found in the mechanochemical macroassemblies have been cloned, and other loci have been identified, suggesting that there is even greater complexity than first expected.

Animals↗

PubMedDate not supplied

Differential inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine of flagella-like and cilia-like movement of Leishmania promastigotes.

Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibits axonemal dynein ATPase activity and hence the beating of sea urchin and mammalian flagella. We have found that EHNA has an unusual effect on the flagella of Leishmania promastigotes in that it alters both the waveform and polarity of the beat. We report here results which suggest that in Leishmania promastigotes there are either distinct EHNA-sensitive dyneins or different conformational states of a single dynein involved in the cilia-like and flagella-like waveforms and in the propagation of flagellar waves from tip-to-base and from base-to-tip.

Adenine↗

PubMed2003

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.

Biofilms↗

PubMed1970

Fine structure of the motile cells and flagella in a member of the Actinoplanaceae (Actinomycetales).

The motile cells (sporangiospores) of an undescribed member of the Actinoplanaceae are studied by electron microscopy as shadowed, negatively stained, and sectioned preparations. The rod-shaped spores exhibit a typically bacterial internal structure. However, a single tubular structure (rhapidosome) is positioned just inside the site of flagellar attachment of each spore and is oriented perpendicular to the direction of the flagella. Flagella arise from basal dises and pass through the plasma membrane and the two-layered cell wall to become associated with other flagella to function as a posteriorly directed unit. Each flagellum consists of a helical band or ribbon which dissociates into 5 or 6 subfibrils.

Actinomycetales↗

PubMed1977

Bacterial flagella rotating in bundles: a study in helical geometry.

Bacterial flagella are semi-rigid helices that undergo true rotation. In peritrichously flagellated bacteria (e.g., Escherichia and Salmonella) there are many flagella on each cell; during translational cell movement these operate as a coordinated bundle that actively disperses upon reversal of the rotation sense. The dynamic behavior of a set of helices originating on separate rotational axes is explored by a working model, geometrical analysis, and hydrodynamic calculations. A critical relationship exists between the interaxial separation and phase difference of parallel helices with overlapping domains; in the subcritical case the filaments are not intertwisted, whereas in the supercritical case they are intertwisted in the same sense (left-handed) as the helices, with one twist per helical turn. During counter-clockwise rotation (the sense operative in forward swimming) any preexisting twists of this kind are automatically cancelled and the helices brought progressively into phase. Hydrodynamic calculations suggest that some wrapping then occurs in a right-handed sense, opposite to that of the helices; this necessitates a distortion from true helical geometry which is minimized by maintaining a coaxial in-phase relationship. A highly coordinated helical bundle results that is capable of operating smoothly for an indefinite period, in agreement with the observed behavior of swimming bacteria. During reverse rotation, the supercritical case develops to cause jamming of the bundle, as has been observed with bacteria in high-viscosity medium. The explosive dispersal of the bundle during reversal in low-viscosity medium is a consequence of a complicating phenomenon, namely, a drastic change in flagellar quaternary structure. The overall conclusion is that bundle formation and function are perfectly compatible with a rotational mechanism for the individual flagella.

Bacterial Physiological Phenomena↗

PubMed1990

Rescue of a paralyzed-flagella mutant of Chlamydomonas by transformation.

The biflagellate alga Chlamydomonas has been used extensively in the genetic and biochemical analysis of flagellar assembly and motility. We have restored motility to a paralyzed-flagella mutant of Chlamydomonas by transforming with the corresponding wild-type gene. A nitrate reductase-deficient paralyzed-flagella strain, nit1-305 pf-14, carrying mutations in the genes for nitrate reductase and radial spoke protein 3, was transformed with wild-type copies of both genes. Two-thirds of the cells that survived nitrate selection also regained motility, indicating that they had been transformed with both the nitrate reductase and radial spoke protein 3 genes. Transformants typically contained multiple copies of both genes, genetically linked to each other, but not linked to the original mutant loci. Complementation of paralyzed-flagella mutants by transformation is a powerful tool for investigating flagellar assembly and function.

Blotting, Southern↗

PubMed1991

The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms.

A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella less than 6 microns long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions causing complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner arm row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.

Cell Movement↗

PubMed1996

PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

Algal Proteins↗

PubMed1966

Basal bodies of bacterial flagella in Proteus mirabilis. II. Electron microscopy of negatively stained material.

This paper investigates further the question of whether the flagella of Proteus mirabilis emerge from basal bodies. The bacteria were grown to the stage of swarmer differentiation, treated lightly with penicillin, and then shocked osmotically. As a result of this treatment, much of the cytoplasmic content and also part of the plasma membrane were removed from the cells. When such fragmented organisms were stained negatively with potassium phosphotungstate, the flagella were found to be anchored-often by means of a hook-in rounded structures approximately 50 mmicro wide, thus confirming Part I of our study. In these rounded structures a more brilliant dot was occasionally observed, which we interpret as being part of the basal granule. A prerequisite for the demonstration of the basal granules within the cells was, however, the removal of both the cytoplasm and the plasma membrane from their vicinity. In some experiments, the chondrioids were "stained" positively by the incorporation into them of the reduced product of potassium tellurite. The chondrioids were here observed to be more or less circular areas from which rodlike structures extended. The chondrioids adhered so firmly to the plasma membrane that they were carried away with it during its displacement by osmotic shocking, while the basal bodies were left behind. This observation disproves our previous suggestion that the flagella might terminate in the chondrioids. The basal bodies often occur in pairs, which suggest that they could be self-reproducing particles.

Cell Differentiation↗

PubMed1972

Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes.

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of approximately 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively

Acrylamides↗

PubMed1975

[A functional flagella with a 6 + 0 pattern].

The male gamete of the Gregarine Lecudina tuzetae has been studied with transmission electron microscopy and microcinematography. It is characterized by a flagellar axoneme of 6 + 0 pattern, a reduction of the chondriome, and the abundance of storage polysaccharide or lipid bodies. The movements of the flagella are of the undulating type and they are performed in the three dimensions of space. They are very slow, with a cycle time of about 2s. The structure of the axoneme components are similar to those of flagella with a 9 + 2 pattern. Each doublet has overall dimensions of 350 x 220 A; the space between the adjacent doublets is about 160 A. The A subfiber bears arms like dynein arms. The diameter of the axoneme is about 1,000 A. The basal body consists of a cylinder of dense material 2,500 A long and 1,300-1,400 A in diameter; a microtubule 200 A in diameter is present in the axis. This study shows that a 6 + 0 pattern can generate a flagellar movement. The mechanism of the flagellar movement of the male gamete of L. tuzetae does not require the presence of central microtubules and it would include molecular interactions of the dynein-tubulin type between the adjacent peripheric doublets. The slowness of the movements is discussed in terms of the axoneme's structure and its energy supply. Finally, the phylogenetic significance of this flagella is examined on the basis of the morphopoietic potentialities of the centriolar structures.

Animals↗

PubMed1976

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.

Binding Sites↗

PubMed2004

Localization of the blue-light receptor phototropin to the flagella of the green alga Chlamydomonas reinhardtii.

Blue light controls the sexual life cycle of Chlamydomonas, mediated by phototropin, a UV-A/blue-light receptor that plays a prominent role in multiple photoresponses. By using fractionation experiments and immunolocalization studies, this blue-light receptor, in addition to its known localization to the cell bodies, also was detected in flagella. Within the flagella, it was completely associated with the axonemes, in striking contrast to the situation in higher plants and the Chlamydomonas cell body where phototropin was observed in the plasma membrane. Its localization was not perturbed in mutants lacking several prominent structural components of the axoneme. This led to the conclusion that phototropin may be associated with the outer doublet microtubules. Analysis of a mutant (fla10) in which intraflagellar transport is compromised suggested that phototropin is a cargo for intraflagellar transport. The blue-light receptor thus seems to be an integral constituent of the flagella of this green alga, extending the list of organisms that harbor sensory molecules within this organelle to unicellular algae.

Animals↗

PubMedDate not supplied

Purified flagella from Treponema phagedenis biotype Reiter does not induce protective immunity against experimental syphilis in rabbits.

Thirteen rabbits were immunized three times at weekly intervals with 50 micrograms of flagella purified from Treponema phagedenis biotype Reiter. Fourteen rabbits were inoculated in the same way with a placebo preparation. Rabbits immunized with the flagella developed an immune response to the flagella but showed no statistically significant prolongation of incubation time or diminution of lesion severity when challenged intradermally with 4 X 10(3) Treponema pallidum organisms.

Animals↗

PubMed1988

Temperature-dependent expression of flagella of Listeria monocytogenes studied by electron microscopy, SDS-PAGE and western blotting.

Washed cells of Listeria monocytogenes serotype 4b, grown in broth culture at 20 degrees C and at 37 degrees C, were examined by electron microscopy for the presence of flagella. Many flagella were seen in cells grown at 20 degrees C, whereas at 37 degrees C very few were expressed. Flagella sheared from the cell surface were partially purified by differential centrifugation. Using SDS-PAGE and Western blotting two distinct protein bands were seen in this preparation, both with an apparent molecular mass of approximately 29 kDa. Further purification of these proteins was achieved by gel filtration and ion-exchange chromatography. Whole organisms grown at 20 degrees C and 37 degrees C were examined in Western blots using an affinity-purified polyclonal antibody, and a monoclonal antibody, both directed against 29 kDa putative flagellin. Bacteria grown at 20 degrees C expressed abundant flagellin, whereas only trace amounts could be detected in organisms grown at 37 degrees C. It is concluded that organisms grown at 20 degrees C both produce and assemble flagellin at the cell surface, and that flagellin production is a less marked feature of organisms grown at 37 degrees C.

Blotting, Western↗

PubMed1999

Effect of pH, temperature and surface contact on the elaboration of fimbriae and flagella by Salmonella serotype Enteritidis.

Survival of enteric pathogens exposed to various environmental stresses depends upon a number of protective responses, some of which are associated with induction of virulence determinants. Flagella and fimbriae are putative virulence determinants of Salmonella spp. and ELISAs specific for the detection of flagella and SEF21, SEF14 and SEF17 fimbriae were used to assess the effect of temperature and pH upon their elaboration by isolates of Salmonella serotype Enteritidis in planktonic growth and on the surface of two-dimensional gradient agar plates. For three phage type 4 isolates of Enteritidis of comparative clinical provenance, similar phenotypes for the elaboration of these surface antigens were observed. SEF14 fimbriae were elaborated in planktonic growth at 37 degrees C, but not 20 degrees C, at pH 4.77 and above but not at pH 4.04; whereas on agar gradient plates SEF14 fimbriae were elaborated poorly but with best yields at pH 4.04. SEF17 fimbriae were elaborated in planktonic growth at 20 degrees C, but not at 37 degrees C, at pH 6.18 and above but not at pH 5.09 or below; whereas on agar gradient plates SEF17 fimbriae were elaborated well even at pH 4.65. SEF21 fimbriae were expressed very poorly under all conditions tested. Planktonic growth at 37 degrees C induced least flagella whereas growth at 20 degrees C, and particularly surface growth at lower pH values, induced a 'hyper-flagellate' phenotype. Single colonies allowed to form on gradient agar plates were shown to generate different colonial morphologies which were dependent on initial pH. These results demonstrate that the physicochemical environment is an important determinant of bacterial response, especially the induction of putative virulence factors.

Animals↗

PubMed1983

Morphological differences in flagella in Campylobacter fetus subsp. intestinalis and C. fetus subsp. jejuni.

Intact flagella were isolated from human pathogenic strains of Campylobacter, C. fetus subsp. intestinalis and C. fetus subsp. jejuni, by the method of DePamphilis and Adler and examined by electron microscopy. The isolated flagella were composed of a filament, a hook, a basal body, and a large disk associated with the end of the hook region covering the basal body. The width of the hook was approximately 28 nm, somewhat greater than that of the filament (20 nm in diameter). The hook region of C. fetus subsp. intestinalis was curved, but it was straight in C. fetus subsp. jejuni. The structure of the basal body of the two subspecies was similar to that reported for other gram-negative bacteria. The large disk detached from the flagella showed concentrically arranged circular structures. This structure was more clearly observed in the disk of C. fetus subsp. jejuni than in C. fetus subsp. intestinalis. Observations of thin-sectioned profiles at the attachment site of the flagellum revealed that the large disk is located on the inner side of the outer membrane. The role of the large disk in bacterial movement is not clear, but it is assumed that it acts as an organ to protect the flagellar insertion site from vigorous rotation of the polar end inflicted during bacterial movement.

Campylobacter fetus↗

PubMed1984

Demonstration of protective antigen carried by flagella of Clostridium chauvoei.

The protective antigen present on the flagella of Clostridium chauvoei was studied by the mouse protection test. A partially purified flagella preparation (PPF) showed protective antigenicity after two intraperitoneal injections of 2 micrograms as protein, while the protective antigenicity of nonflagellated mutants (NFM) was 100-fold less than that of the flagellated parent strain. Although the protective effect of antisera against the whole cells and PPF, in terms of ED50 values, was mostly lost after absorption with the parent strain, that of antisera after absorption with NFMs showed no appreciable loss. These results suggest that the flagella of Cl. chauvoei play some role in inducing protective immunity in mice.

Animals↗