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PubMed1987

Production, characterization, and protective effect of monoclonal antibodies to Clostridium chauvoei flagella.

Monoclonal antibodies to flagella of Clostridium chauvoei were obtained by the fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with partially purified flagella of strain Okinawa. Enzyme-linked immunosorbent assay and Western blot analysis with partially purified flagella, flagellated cells, and nonflagellated mutants were used to show that five monoclonal antibodies are specific for the flagella. In the Western blot analysis, all five antiflagellar antibodies reacted strongly with the 56,000-molecular-weight protein, which corresponds to the flagellin. By using the ELISA-derived reactivity of monoclonal antibodies to the various clostridia and the competitive binding assay, we showed that the flagella of C. chauvoei had at least three epitopes. The three antiflagellar monoclonal antibodies (one immunoglobulin G and two immunoglobulin M) demonstrated passive protective effects in mice. These results strongly suggest that the flagella of C. chauvoei are important for protective immunity in mice.

Animals↗

PubMed1999

Induction of cytokine synthesis by flagella from gram-negative bacteria may be dependent on the activation or differentiation state of human monocytes.

We have previously demonstrated that salmonellae, but not Escherichia coli or Yersinia enterocolitica, stimulates tumor necrosis factor alpha (TNFalpha) production in the human promonocytic cell line U38. Subsequent analysis revealed that the TNFalpha-inducing activity of salmonellae was associated with flagellin, a major component of flagella from gram-negative bacteria. In the present study, we have explored the basis for the apparent specificity of action of Salmonella flagella on TNFalpha expression in U38 cells and have extended this analysis to normal human peripheral blood mononuclear cells (PBMC). Flagella from the enteropathogenic E. coli strain E2348/69, Y. enterocolitica JB580, and Pseudomonas aeruginosa PAO1, which did not induce significant levels of TNFalpha production in U38 cells, were as potent as Salmonella flagella in terms of TNFalpha and interleukin 1beta activation in PBMC. However, TNFalpha production in U38 cells was greatly enhanced when these cells were stimulated with flagella from E. coli, Y. enterocolitica, and P. aeruginosa in the presence of a costimulant, phorbol 13-myristate acetate. These findings are consistent with the hypothesis that the activation or differentiation state of a monocyte may have a substantial effect on the cell's responsiveness to flagellum stimulation of cytokine synthesis. Furthermore, these results indicate that cytokine induction in monocytes may be a general property of flagella from gram-negative bacteria.

Cell Line↗

PubMed2002

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.

Aeromonas↗

PubMed1991

Flagella number among Naegleria flagellates.

Scanning electron microscopy was used to determine the number of flagella on the flagellates of Naegleria australiensis, N. fowleri, N. gruberi, and N. jadini. Although the majority of flagellates had 2 flagella, there was considerable variation among individual cells. The number of flagella per flagellate varied from 1-8, with 2.4 being the average number per cell. For the different species, the average number of flagella per cell ranged from 2.0 in N. jadini to 3.1 for N. australiensis. The greatest amount of variation occurred in N. australiensis, with only 43% of the cells having 2 flagella. By contrast, 92% of N. fowleri cells had 2 flagella. Naegleria jadini and N. gruberi were intermediate with 80% and 74% biflagellates, respectively.

Animals↗

PubMed1971

Purification of intact flagella from Escherichia coli and Bacillus subtilis.

A procedure is described for the purification of bacterial flagella in the form of a filament-hook-basal body complex (intact flagella) free from detectable cell wall, membrane, or cytoplasmic material. Spheroplasts produced with lysozyme and ethylenediaminetetraacetic acid were lysed with Triton X-100, and the flagella were purified by (NH(4))(2)SO(4) precipitation, differential centrifugation, and CsCl gradient centrifugation. As much as 40% of the flagella were recovered, and they contained about one basal body per 4 to 6 mum of flagella. The same procedure developed for Escherichia coli was also successful for purifying intact flagella from Bacillus subtilis.

Bacillus subtilis↗

PubMed1994

Flexural rigidity of echinoderm sperm flagella.

The stiffness (flexural rigidity) of live sperm flagella, Triton-demembranated flagella (axonemes), trypsin-digested axonemes, and doublet microtubules of the axonemes in echinoderms was determined from the relationship between their deformation when a stream of medium was applied and the viscous resistance of the medium acting on the flagellum. The stiffness of the flagellum beating in seawater was 5.8 x 10(-21) Nm2 for bending in the direction perpendicular to the beating plane and 4.2 x 10(-22) Nm2 for bending within the beating plane. A similar difference in stiffness from the difference in bending directions was found in reactivated flagella with 1 mM ATP. The stiffness of live flagella immobilized in CO2-saturated seawater and axonemes in ATP-free medium was similar to that of beating flagella for bending in the direction perpendicular to the beating plane. The stiffness of motionless flagella significantly decreased with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and vanadate. The trypsin-digestion of motionless axonemes did not change their stiffness. The stiffness of doublet microtubules was 1.4 x 10(-23) Nm2 in 0.1 mM ATP medium and 6.1 x 10(-23) Nm2 in ATP-free medium. These results suggest that doublet pairs lying parallel to the beating plane of the flagellum retain fewer cross-bridges than doublet pairs lying perpendicular to the beating plane.

Adenine↗

PubMed1984

Flagella and motility behaviour of square bacteria.

Square bacteria are shown to have right-handed helical (RH) flagella. They swim forward by clockwise (CW), and backwards by counterclockwise (CCW) rotation of their flagella. They are propelled by several or single filaments arising at several or single points on the cell surface. When there are several filaments a stable bundle is formed that does not fly apart during the change from clockwise to counterclockwise rotation or vice versa. In addition to the flagella attached to the cells, large amounts of detached flagella aggregated into thick super-flagella, can be observed at all phases of growth.

Bacteria↗

PubMed2005

New symbiotic hypothesis on the origin of eukaryotic flagella.

The origin of eukaryotic flagella has long been a mystery. Here we review the possibility that flagella sprouted evolutionarily from the eukaryotic cell proper seems very unlikely because it is hard to imagine what function and benefit in natural selection the flagella would have provided to the cells when they first emerged as simple buds. Lynn Margulis' 1970 spirochete hypothesis, though popular still, has never been confirmed. Moreover, the absence of tubulin and axonemal dynein in the spirochetes and the incapability of the bacterial and eukaryotic membranes' making a continuum now suggest that the hypothesis is outdated. Tubulin genes were recently identified in a new bacteria division, verrucomicrobia, and microtubules have also been found in one of these species, epixenosomes, the defensive ectosymbionts. On the basis of these data, we propose a new symbiotic hypothesis: that the mid-ancestor of eukaryotic cells obtained epixenosomelike verrucomicrobia as defensive ectosymbionts and the ectosymbionts later became endosymbiotic. They still, however, protruded from the surface of their host to play their role. Later, many genes were lost or incorporated into the host genome. Finally, the genome, the bacterial membrane, and the endosymbiotic vesicle membrane were totally lost, and fingerlike protrusions with microtubules formed. As the cells grew larger, the defensive function of the protrusions eventually weakened and then vanished. Some of the protrusions took on a new role in cell movement, which led them to evolve into flagella. The key step in this process was that the dynein obtained from the host evolved into axonemal dyneins, attaching onto the microtubules and forming motile axonemes. Our hypothesis is unproven, but it offers a possible explanation that is consistent with current scientific thought. We hope that our ideas will stimulate additional studies on the origin of eukaryotic flagella and on investigations of verrucomicrobia. Whether such studies confirm, refine, or replace our hypothesis, they should nevertheless further our understanding of the origin of eukaryotic cells.

Animals↗

PubMed1994

Movement of the F40 domain of flagellin during the morphological transition of bacterial flagella.

Flagella from Salmonella typhimurium were labeled with various amounts of fluorescein isothiocyanate. The site of labeling was identified as being predominantly in the exterior F40 domain. The fluorescence intensity decreased as the fluorescein density on the flagella increased, indicating self energy transfer between fluoresceins. The fluorescence of modified flagella was measured during the normal-to-curly morphological transition induced by alkaline pH. The morphological transition itself was simultaneously monitored by dark-field microscopy. Concomitant with the transition was a 25% increase in fluorescence for flagella heavily labeled with fluorescein. This was shown to be due to a decrease in the efficiency of energy transfer between fluoresceins on proximal flagellin subunits, implying that the F40 domains undergo relative movement apart during the morphological transition. Closer inspection of the domain movement and morphological transition as a function of pH reveals that the two processes are not exactly concomitant. This indicates the existence of intermediates during the transition. The fluorescence technique, outlined here, provides a means of directly monitoring an organizational 'switch' in the flagellin subunits during the actual morphological transition of flagella.

Flagella↗

PubMed1978

The equation of motion for sperm flagella.

The equation of motion for sperm flagella, in which the elastic bending moment and the active contractile moment are balanced by the moment from the viscous resistance of the surrounding fluid, is solved for a wave solution that superimposes partial solutions. Substitution of the expression for the wave solution into the equation leads to an expression for the active contractile moment. This active moment can be decomposed into two parts. The first part describes an active moment that travels over the flagellum with the mechanical flagellar wave, the second part represents a moment in phase over the entire length of the flagellum, which decreases linearly towards the distal tip. The linear synchronous moment, to which an amount of traveling moment has been added as a perturbation, leads to wave solutions that closely resemble flagellar waves. Properties such as wavelength and wave amplitudes and also the shape of the waves in sea urchin sperm flagella at different frequencies are accurately described by the theory. The change in wave shape in sea urchin sperm flagella at raised viscosity is predicted well by the theory. The different wave properties caused in bull sperm flagella by different boundary conditions at the proximal junction are explained. When only a traveling active moment is present in a flagellum, the wave solutions describe waves of a small wave length in a long flagellum. Some properties of the wave motion of sperm flagella are derived from the theory and verified experimentally.

Animals↗

PubMed1985

Investigations on the role of flagella in the colonization of infant mice with Campylobacter jejuni and attachment of Campylobacter jejuni to human epithelial cell lines.

The biochemical and biological properties of the flagella of Campylobacter jejuni have been investigated using two variants selected from a flagellate, motile clinical isolate (strain 81116): a flagellate, non-motile variant (SF-1) and an aflagellate variant (SF-2). Phenotypic and biochemical analysis of the strains and amino acid analysis of the isolated flagella suggest that the variants differed from the wild-type strain only in the absence of flagella and/or motility. The aflagellate variant poorly colonized the gastrointestinal tract of infant mice but the flagellate, non-motile variant colonized the mice as successfully as the wild-type strain. 35S-labelled organisms were used to investigate the attachment of the variants to human epithelial cell monolayers in vitro. The flagellate, non-motile strain attached more efficiently to the cells than the wild-type strain or the aflagellate strain. Differences in attachment suggest that an adhesin is intimately associated with flagella of C. jejuni and that active flagella mediate only a tenuous association with host cells. This adhesin attached most efficiently to cells of intestinal epithelial origin and was not specifically inhibited by various sugars.

Adhesiveness↗

PubMed1997

A motile but non-swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly.

A TnphoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN, a flagella gene encoding a 147-amino-acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti-sigma28 gene, flgM, flanked by the class II genes, flgA, flgBCD and flhBA, and a novel putative virulence-related gene. The flgN mutation caused a substantial reduction in cell surface-associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse-labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non-oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper-like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella-related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.

Amino Acid Sequence↗

PubMed1997

Characterization of flagella genes of Agrobacterium tumefaciens, and the effect of a bald strain on virulence.

Agrobacterium tumefaciens produces flagella that are arranged circumthecally near one end of the bacilliform cell. The flagella are required for motility to facilitate reaching the root surface, and possibly aid in orientating the bacterial cells at various sites for infection. We have identified three flagella genes designated flaA, flaB, and flaC. Mutations in flaA, flaB and flaC result in abberant swimming behaviour. Electron microscopic examination of these mutants revealed the defective flagella. A non-motile, bald mutant strain was generated by deleting all three fla genes. Nucleotide sequencing of flaA, flaB, and flaC showed that they have a potential coding capacity for polypeptides of 307, 321, and 314 amino acid residues, respectively. The predicted amino acid sequences of the A. tumefaciens FlaA and FlaB proteins are similar (66% average identity) to the FlaA and FlaB proteins encoded by flaA and flaB genes, respectively, in Rhizobium meliloti. There was no counterpart FlaC protein reported in R. meliloti, but the A. tumefaciens FlaC is similar in amino acid sequence to the R. meliloti FlaA (59.8% identity) and FlaB (66.7% identity). Distinct from FlaA and FlaB of R. meliloti is the absence of histidine and cysteine residues and their shorter length (by 88 amino acid residues fewer than FlaA and FlaB of R. meliloti). The transcriptional start sites of each fla gene determined by primer extension revealed consensus-sequence boxes representing potential binding sites for sigma 28 RNA polymerase (RNAP) upstream of the transcriptional start of each fla gene. Besides the potential sigma 28-binding site upstream of flaC, also present are additional putative conserved sequences, GC at -11 and GG at -21 from the transcriptional start, that resemble potential binding motifs for sigma 54. Because the sigma 54 promoter is associated with genes regulated by physiological changes in various bacteria, the flaC gene might be similarly regulated in response to A. tumefaciens responding to host plant stimuli. Virulence studies showed that the bald strain was consistently reduced in virulence below that of the parental wild-type strain by at least 38%. The difference is statistically significant and suggests that the flagella may play a role in facilitating virulence.

Agrobacterium tumefaciens↗

PubMed2002

Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli.

Quorum sensing is a cell-to-cell signalling mechanism in which bacteria secrete hormone-like compounds called autoinducers. When these auto-inducers reach a certain threshold concentration, they interact with bacterial transcriptional regulators, thereby regulating gene expression. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 as well as E. coli K-12 produces the autoinducer-2 (AI-2), which is synthesized by the product of the luxS gene, and previous work from our laboratory has shown that genes encoding the EHEC type III secretion system were activated by quorum sensing. Recently, by hybridizing an E. coli K-12 gene array with cDNA synthesized from RNA extracted from EHEC strain 86-24 and its isogenic luxS mutant, we observed that other potential virulence-associated factors, such as genes encoding the expression and assembly of flagella, motility and chemotaxis, were also activated by quorum sensing. The array data also indicated that several genes encoding putative E. coli regulators were controlled by quorum sensing. In this report, we describe a two-component system regulated by quorum sensing that shares homology with Salmonella typhimurium PmrAB, which we have named quorum sensing E. coli regulator B and C (QseBC). The qseBC genes, previously identified only as open reading frames b3025 and b3026, are organized in an operon in the E. coli chromosome, with qseB encoding the response regulator and qseC the sensor kinase. We confirmed the regulation of qseBC by quorum sensing using qseB::lacZ transcriptional fusions and characterized the phenotypes of an isogenic qseC mutation in EHEC. This mutant expressed less flagellin and had reduced motility compared with the wild-type and complemented strains. Transcription of flhD, fliA, motA and fliC::lacZ fusions was decreased in the qseC mutant, suggesting that qseBC is a transcriptional regulator of flagella genes. A qseC mutant was also generated in E. coli K-12 strain MC1000 that showed the same phenotypes as the EHEC mutant, indicating that qseBC regulates flagella and motility by quorum sensing in both EHEC and K-12. QseBC activates transcription of flhDC, which is the master regulator for the flagella and motility genes and, in the absence of flhD, QseBC failed to activate the transcription of fliA. Motility of a luxS, but not of a qseC, mutant can be restored by providing AI-2 exogenously as preconditioned media, suggesting that the qseC mutant is unable to respond to AI-2. However, QseC has no effect on the expression of other quorum sensing-controlled genes such as those encoding for the type III secretion system. These data indicate that QseBC is one component of the quorum-sensing regulatory cascade in both EHEC and K-12 that is involved in the regulation of flagella and motility genes, but that additional regulators in this cascade remain to be characterized.

DNA-Binding Proteins↗

PubMed2000

Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions.

Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

Bacterial Adhesion↗

PubMed2000

A novel pH2 control on the expression of flagella in the hyperthermophilic strictly hydrogenotrophic methanarchaeaon Methanococcus jannaschii.

The methanarchaeon, Methanococcus jannaschii, a hyperthermophilic, autotrophic, and strictly hydrogenotrophic inhabitant of submarine hydrothermal vents, was cultivated in a reactor at two hydrogen partial pressure (p(H(2))) values, 178 kPa (high) and 650 Pa (ultralow), and the cells were subjected to a comparative proteome analysis. From these studies, it was discovered that, when p(H(2)) was high and the cell density was low (a combination representing a hydrogen-excess condition), the cells possessed very low or undetectable levels of four flagella-related polypeptides (FlaB2, FlaB3, FlaD, and FlaE); electron microscopic examination showed that most of these cells were devoid of flagella. Flagella synthesis occurred when hydrogen became limiting either at high cell density under high p(H(2)) or at low cell density under low p(H(2)). The results from a p(H(2))-shift experiment corroborated the above observations. The p(H(2))-dependent changes in the levels of two methanogenic enzymes (MTD and HMDX) were as expected, and thus they served as internal controls. To our knowledge, this is the first example for the regulation of expression of flagella by hydrogen in any domain of life and for a control of any kind on flagella synthesis in the archaea. Our work also provides the only known example for each of the following: (i) the pure culture cultivation of a methanogen at an ultralow, near ecologically relevant p(H(2)); (ii) experimental functional genomics for M. jannaschii; and (iii) the use of proteomics with M. jannaschii.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2000

Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in chlamydomonas.

In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.

Amino Acid Sequence↗

PubMed2004

Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella.

Regulation of motile 9+2 cilia and flagella depends on interactions between radial spokes and a central pair apparatus. Although the central pair rotates during bend propagation in flagella of many organisms and rotation correlates with a twisted central pair structure, propulsive forces for central pair rotation and twist are unknown. Here we compared central pair conformation in straight, quiescent flagella to that in actively beating flagella using wild-type Chlamydomonas reinhardtii and mutants that lack radial spoke heads. Twists occur in quiescent flagella in both the presence and absence of spoke heads, indicating that spoke--central pair interactions are not needed to generate torque for twisting. Central pair orientation in propagating bends was also similar in wild type and spoke head mutant strains, thus orientation is a passive response to bend formation. These results indicate that bend propagation drives central pair rotation and suggest that dynein regulation by central pair--radial spoke interactions involves passive central pair reorientation to changes in bend plane.

Animals↗