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PubMed2006

The ability of flagellum-specific Proteus vulgaris bacteriophage PV22 to interact with Campylobacter jejuni flagella in culture.

BACKGROUND: There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen. RESULTS: A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 x 10(-9) ml/min. CONCLUSION: It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.

Adsorption↗

PubMed2004

The flagella connector of Trypanosoma brucei: an unusual mobile transmembrane junction.

Throughout its elongation, the new flagellum of the procyclic form of the African trypanosome Trypanosoma brucei is tethered at its tip to the lateral aspect of the old flagellum. This phenomenon provides a cytotactic mechanism for influencing inheritance of cellular pattern. Here, we show that this tethering is produced via a discrete, mobile transmembrane junction - the flagella connector. Light and electron microscopy reveal that the flagella connector links the extending microtubules at the tip of the new flagellum to the lateral aspect of three of the doublet microtubules in the old flagellar axoneme. Two sets of filaments connect the microtubules to three plates on the inner faces of the old and new flagellar membranes. Three differentiated areas of old and new flagellar membranes are then juxtaposed and connected by a central interstitial core of electron-dense material. The flagella connector is formed early in flagellum extension and is removed at the end of cytokinesis, but the exact timing of the latter event is slightly variable. The flagella connector represents a novel form of cellular junction that is both dynamic and mobile.

Animals↗

PubMed1984

Movements and active moments of bull sperm flagella as a function of temperature and viscosity.

Detailed measurements were made of the movements and waveforms of bull sperm flagella over a temperature range of 5 to 37 degrees C and over a range of external viscosities of 1-3600 X 10(-3) Pas-1. The data obtained were used for an evaluation of the internal active moments in the flagella, using exact, large amplitude, algebra. The results of the calculations show that the axoneme in bull sperm flagella can produce the forces necessary to power the flagellar motion over the entire range of conditions. The calculations further suggest that no other structure in the bull sperm flagella has a force-producing function.

Animals↗

PubMed2004

Turnover of actin in Chlamydomonas flagella detected by fluorescence recovery after photobleaching (FRAP).

Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.

Actins↗

PubMed1987

Comparison of dark-field microscopy and a flagella stain for monitoring the effect of a Water Pik on bacterial motility.

The purpose of this examiner-blind study was threefold: to compare the microbial counts obtained by two different techniques for assessing bacterial motility, to assess the inter-rater reliability of these two techniques and to evaluate the effect of a water irrigating device (Water Pik) on bacterial motility at 3- and 6-mm probing depths. Subgingival plaque samples were taken from 10 healthy patients having at least two sites that probed greater than 6 mm, (one control, one experimental). Half of the patients were sampled at 3 mm, the other half at 6 mm, both at baseline (Day 0) and at Day 21. Two slides were prepared from each plaque sample, one for dark-field evaluation and one stained with a simplified silver-plating technique for flagella. All slides were read simultaneously by 3 observers, and the per cent motility calculated for spirochetes, motiles and all others. Strong positive inter-rater reliability correlations ranging from r = 0.95 to r = 0.99 were found for both the dark-field and flagella staining techniques. Spirochete counts obtained by both techniques were highly correlated (r = 0.91), whereas counts for motiles resulted in negative correlations between the techniques. Dark-field counts were consistently higher than the flagella stain counts for motile rods. Spirochetes were reduced, but not significantly, after irrigation of both 3-mm and 6-mm sites. Bacterial motility can be evaluated by both dark-field and flagella-staining techniques with a high degree of inter-rater reliability.

Adult↗

PubMed2002

Characterization of flagella produced by clinical strains of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an emerging nosocomial pathogen associated with opportunistic infections in patients with cystic fibrosis, cancer, and HIV. Adherence of this organism to abiotic surfaces such as medical implants and catheters represents a major risk for hospitalized patients. The adhesive surface factors involved in adherence of these bacteria are largely unknown, and their flagella have not yet been characterized biochemically and antigenically. We purified and characterized the flagella produced by S. maltophilia clinical strains. The flagella filaments are composed of a 38-kDa subunit, SM(FliC), and analysis of its N-terminal amino acid sequence showed considerable sequence identity to the flagellins of Serratia marcescens (78.6%), Escherichia coli, Proteus mirabilis, Shigella sonnei (71.4%), and Pseudomonas aeruginosa (57.2%). Ultrastructural analysis by scanning electron microscopy of bacteria adhering to plastic showed flagellalike structures within the bacterial clusters, suggesting that flagella are produced as the bacteria spread on the abiotic surface.

Animals↗

PubMed1994

[The role of flagella of Campylobacter jejuni in colonization in the intestinal tract in mice and the cultured-cell infectivity].

For analyzing the role of the bacterial flagella in colonization in the intestinal tract of mice and adhering to or invading the Intestine 407 cell, a nonflagellated, nonmotile mutant was induced by ultraviolet irradiation of a flagellated, motile wild-type strain of Campylobacter jejuni CF84-340. There was no great difference in the cellular infectivity to the Intestine 407 cells between the wild-type and the mutant strains. Cellular adherence and invasiveness were then compared by fluorescent antibody staining, and an obvious difference was found in the latter. While 21.4% of the organisms of the wied-type strain invaded the cells, only 6.1% of those of the flagella-defective mutant did so. In the experiments in mice involving oral administration, cellular invasiveness was not found with the flagella-defective mutant and no organisms were detected from the blood, although bacteremia is one of the characteristics of infection with C. jejuni. Moreover, no intestinal adherence of the mutant was detected, suggesting early elimination of the organism administered. These results indicate that the bacterial flagella are concerned in not only the cellular adherence and intestinal deposit, but also the intracellular invasiveness and invasion into the blood stream from the intestinal wall in the infected mice.

Animals↗

PubMed1978

[Flagella specific H antigenic schema of Pseudomonas aeruginosa (author's transl)].

Serogrouping of P. aeruginosa on the basis of heat-stable somatic (O-)agglutinogens is well established, but H-typing on the basis of heat-labile agglutinogens is not accepted generally because of uncertain flagella specificity. In contrast to the agglutination reaction, the indirect fluorescent antibody technique allows the differentiation of soma and flagellum of P. aeruginosa cells at the morphological and serological level simultaneously, and thereby the specific analysis of flagellar (H-)antigens, even with OH-antisera. Using this technique the complex flagella-antigen a with the partial antigens a0, a1, a2, a3, a4 and the uniform flagella-antigen b are differentiated by cross absorption experiments. As the partial factors a1-a4 are independent determinants, a flagellar antigenic schema containing 17 H-types is devised. Type strains, immunization and absorption procedures for preparing diagnostic antisera are described. O-group and H-type show free combination. Completing the extended O antigenic schema of Habs (slide agglutination) with the flagella specific H antigenic schema (indirect fluorescent antibody technique), a detailed OH typing system of P. aeruginosa is obtained, by which clinical isolates are subdivided into 99 serovars.

Antigens, Bacterial↗

PubMed1975

[On the morphology of flagella and fimbriae of Escherichia strains (author's transl)].

100 Escherichia-strains, isolated from urine and faeces, were investigated with regard to flagella and fimbriae by electron-microskopy. The flagella had a length of 3-6 mu and a diameter of ca 0.02 mu. The flagellation was different not only between to the single bacteria of the same strain, but also in the separated isolates. Of 50 strains from urine had 27 and of 50 strains from feces had 28 no flagella. Different Escherichia-strains contained fimbriae, the number of which was much higher with regard to the flagella. The length of the fimbriae differed between 0.25 and 2 mu, the diameter was constantly ca 8 nm. 21 strains of 100 (4 from 50 urine strains and 17 from 50 faeces strains) had no fimbriae.

Cell Wall↗

PubMedDate not supplied

[Interaction of bacterial flagella with myosin].

The interaction of isolated flagellar filaments of Bacillus brevis var. G.-B. P+ with skeletal muscle myosin has been investigated. Bacterial flagellar filaments co-precipitate with myosin at low ionic strength (at the conditions of myosin aggregation). Addition of bacterial flagellar filaments to myosin led to inhibition of its K+-EDTA- and Ca2+-ATPase activity, but had no influence on Mg2+-ATPase. Monomeric protein of bacterial flagella filaments (flagellin) did not co-precipitate with myosin and had no influence on its ATPase activity. The flagella filaments did not co-precipitate with myosin in the presence of F-actin if it was mixed with myosin before the filaments. If the flagella filaments were added to myosin solution before the addition of F-actin the amount of filaments and actin in myosin precipitate were comparable. In this case the presence of flagella filaments decreased activation of myosin Mg2+-ATPase by actin to 25-30%. Thus the bacterial flagellar filaments are able to interact with myosin and modify its ATPase activity. Probably, these properties of filaments are caused by resemblance of flagellin and actin. For instance, the unique origin of these proteins may be the reason of such resemblance.

Adenosine Triphosphatases↗

PubMed1981

The elevation of cyclic AMP concentrations in flagella-less sea urchin sperm heads.

Cycl AMP concentrations were elevated and acrosome reactions were induced in intact sea urchin spermatozoa by Nigericin, A23187, and pH 9.0 seawater. To determine whether or not the metabolism of cyclic AMP was being altered in sperm heads, the heads were mechanically separated from the flagella, and the flagella-less heads were then isolated by differential centrifugation. The isolated heads contained 1 to 2 nmol of ATP and 1 to 2 pmol of cyclic AMP/mg wet weight and retained these concentrations for several hours if stored at 0 degrees C. The flagella-less heads also retained the mitochondria of the midpiece area. The heads retained their functional status and could be stimulated to undergo acrosome reactions (filament extension) in response to Nigericin, A23187, or pH 9.0 seawater. Furthermore, the isolated heads could activate sea urchin eggs after induction of an acrosome reaction by Nigericin or pH 9.0 seawater. The isolated heads contained appreciable adenylate cyclase, cyclic AMP phosphodiesterase, cyclic GMP phosphodiesterase, guanylate cyclase, cyclic AMP-dependent protein kinase, and calmodulin. Nigericin, pH 9.0 seawater, and A23187 caused not only the induction of an acrosome reaction but also elevations of cyclic AMP in the isolated heads, and extracellular Ca2+ was an absolute requirement for both responses. At 16 degrees C, Nigericin caused elevations of cyclic AMP within 5 s, but maximal elevations were not observed until 1 min; it induced a maximal percentage of acrosome reactions by 40 s. Incubation of cells at 0 degrees C resulted in a delay of maximal acrosome reactions until between 10 and 20 min after addition of Nigericin. Under these conditions, maximal elevations of cyclic AMP were observed by 5 min, demonstrating that cyclic AMP elevations precede the complete morphological change associated with an acrosome reaction. ATP concentrations within the sperm heads declined in response to Nigericin, pH 9.0 seawater, or A23187, and its decrease also required the presence of extracellular Ca2+. The decline in ATP concentrations was slightly more rapid in the presence of rotenone, suggestive of some ATP synthetic capabilities of the isolated head preparation. 45Ca2+ uptake was increased by Nigericin elevated pH, and A23187 but was not appreciably altered by monensin. Monensin also did not cause appreciable elevations of cyclic AMP concentrations, induction of an acrosome reaction, or decreases of ATP concentrations. Here, we describe for the first time that cyclic AMP concentrations can be increased in flagella-less heads of spermatozoa and show that these changes are associated with an acrosome reaction.

Acrosome↗

PubMed1984

Sodium and proton transport in flagella isolated from sea urchin spermatozoa.

A method was developed to isolate flagella with intact membranes from sea urchin sperm to further study the mechanism of the Na+-dependent H+ extrusion which increases the intracellular pH and triggers motility. Based on cytochrome c oxidase activity and cell count, the flagellar preparation contained at most 0.05% of the sperm heads present in the initial homogenate. The isolated flagella retained a Na+-dependent H+ extrusion mechanism which, like that of intact sperm, was inhibitable by high external potassium and with an ionic selectivity in the order of Na+ greater than Li+ much greater than Rb+ approximately equal to Cs+ (essentially ineffective). Two methods were employed to measure the change in intraflagellar pH (pHi): the accumulation of [14C]methylamine and a spectrofluorimetric technique using acridine orange. Flagella isolated from sperm suspended in Na+-free seawater (pH 7.90) maintained a pHi of 6.72 +/- 0.05 which increased by 0.5 to 0.6 pH unit following a Na+ (10 mM)-dependent release of H+ (114 +/- 18 nmol/mg) and an uptake of 22Na+ (134 +/- 36 nmol/mg). Both ionic movements were inhibitable by high external K+, suggesting a coupled exchange mechanism. This study provides direct evidence for the alkalinization of the flagellar pH under a condition compatible with motility initiation and shows that the isolated flagella is a useful model system for studying its mechanism.

Animals↗

PubMed1981

Bacterial cell envelopes with functional flagella.

Our aim was to isolate from bacteria a flagellated, subcellular system whose content could be changed at will. Because the control of bacterial chemotaxis resides in the direction of rotation of the flagella, such a system would be ideal for the study of this control mechanism. By incubating bacteria with penicillin and then lysing them osmotically, we were able to isolate cell envelopes from Escherichia coli and Salmonella typhimurium. These envelopes have the same sidedness and similar shape and dimensions as the original bacteria; they are practically free of cytoplasm; they are osmotically sensitive, having intact the cytoplasmic membrane and at least part of the cell wall; and they have flagella. This preparation was used to find out what is required to restore flagellar rotation, which had been lost during osmotic lysis. By visualizing the image of individual flagella with high intensity light microscopy or by tethering the cell envelopes, we found that adding artificial electron donors as an energy source is enough to restore rotation. This seems to indicate that no cytoplasmic components are required and that the proton electrochemical potential is indeed the driving force for flagellar rotation. However, the rotation was almost entirely counterclockwise, while in intact bacteria the flagella rotate in both directions. This may indicate that a cytoplasmic component is required to allow clockwise rotation. The significance of these results for the study of chemotaxis is discussed.

Cell Fractionation↗

PubMed1976

[Biochemical and serological investigations of flagellae of Campylobacter fetus (author's transl)].

Flagellae of Campylobacter fetus group O, types 1, 2 and 7 were prepared. First they were separated from cell bodies using an ultramix. The suspension was then centrifuged for 20 mins. at 10000 rpm and the supernatant frozen at -40 degrees C. This is a simple method for the enrichment of preparations of flagellae, as they become tangled up and accumulate in the inferior third part of the frozen liquid. The physicochemical basis of this phenomenon was discussed. After thawing and spinning for 20 mins. at 5000 rpm, the sediment was suspended in 0.9% of NaC1. The purity of the preparation was checked by electron microscopy. Antibodies to this antigen showed no cross-reaction with O-Antigen, when tested by tube agglutination. The amino acid composition of flagellae from different O-antigen serotypes was different (see Tab. 1). Cysteine could not be detected and proline only in traces. After breakdown with urea followed by gel filtration on Sephadex G 200, breakdown products of diminishing molecular size were obtained (see Fig. 2). Discelectrophoresis after ultrasonic gave 8 zones (see Fig. 3). Irrespective of serotype, thin-layer chromatography of trypsin-hydrolysed flagellae always showed 9 ninhydrin-positive spots (see Fig. 1). Only breakdown products of ultrasonic reacted with antibody. After absorption of flagellar antibody with heterologous antigen, agglutination only occurred with homologous antigen (tab. 2-5). This showed that there were different flagellar antigens. Further experiments using immunoprecipitation demonstrated two common antigenic components, a and c, and a partially common antigenic factor bb (Fig. 4), and was the basis for a classification by three groups. The three antigenic components could be separated by gel electrophoresis and detected by immunoprecipitation (Figs. 5,6).

Agglutination Tests↗

PubMed1999

Relevance of zinc in human sperm flagella and its relation to motility.

OBJECTIVE: To measure the zinc content of human sperm flagella and to analyze its relation to sperm motility. DESIGN: Prospective study. SETTING: Center of Dermatology and Andrology. PATIENT(S): Semen samples collected from 90 andrology patients and healthy donors after 3-5 days of sexual abstinence. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm morphology after Shorr staining, sperm motility, and patient age were recorded. In addition, zinc concentrations in the seminal plasma, sperm heads, and flagella were determined with the use of atomic absorption spectrometry. RESULT(S): The mean zinc concentration was 144.3 mg/L in the seminal plasma and 146.9 mg/L in the whole ejaculate and was significantly correlated with parameters of motility. The sperm heads contained only 6.7% of the zinc that was present in the whole spermatozoon. The zinc concentration in the flagella was negatively correlated with sperm motility and velocity. In addition, it was positively correlated with the percentage of abnormally blue-stained flagella and the age of the patients. CONCLUSION(S): Our results clearly demonstrate the importance of zinc elimination during epididymal sperm maturation for functional competence of the outer dense fibers and, therefore, generation of motility.

Adult↗

PubMed1993

Disturbances of sperm flagella due to failure of epididymal maturation and their possible relationship to phospholipids.

A recent classification of alterations of midpiece and flagellum describes the phenomenon of abnormal staining behaviour of human sperm flagella during Papanicolaou and Shorr staining. In these techniques, human sperm tails normally stain red; however, if epididymal function is disturbed, the flagella appear bluish, while they are devoid of other recognizable defects. Such spermatozoa were shown to be immotile, the motility disturbance being referred to as epididymal dysfunction. To define the physiological substrates of this descriptive phenomenon, caput spermatozoa from 10 epididymides of five patients who had undergone orchiectomy because of prostatic cancer were investigated. These spermatozoa showed severe motility disturbances, and almost all their flagella stained atypically. Attempts to stimulate such spermatozoa by pentoxifylline achieved a slight improvement in motility. However, addition of seminal plasma from fertile donors resulted in a significant improvement in motility, accompanied by an increase in the number of normally stained flagella. Even better results were achieved by incubation with liposomes made from soybean lipids, mainly phosphatidylcholine. It is concluded that poor motility and atypical staining behaviour of human caput sperm tails can at least partly be related to a lack of phospholipids in the flagellar membrane. Substitution of phospholipids may be a therapeutic approach in procedures using caput spermatozoa, although in addition to the improvement of sperm motility, the enhancement of fertilizing capacity remains to be established.

Aged↗

PubMed1995

Requirement for FlhA in flagella assembly and swarm-cell differentiation by Proteus mirabilis.

Swarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical rods into long, aseptate swarm cells that overexpress flagella and virulence factors, particularly haemolysin. A non-swarming Tn5phoA mutant was unable to synthesize flagella, to fully elongate or to induce high levels of the toxin. The mutation lay within a 2091 bp gene encoding a homologue of the Escherichia coli FlhA belonging to a family of proteins that are required for assembly of flagella or virulence proteins and that are suggested to act either directly in membrane translocation and/or in regulating synthesis of the export apparatus. In trans expression of multicopy flhA restored cell elongation and migration and generated differentiation-specific hyperexpression of flagellin and toxin genes to levels above those seen in the wild-type strain. Transcription of flhA was strongly induced during differentiation, from its own putative sigma 28 promoter. The results suggest a mechanistic coupling of flagella assembly and swarm-cell differentiation.

Amino Acid Sequence↗

PubMed1982

Flagella and pili of iron-oxidizing thiobacilli isolated from a uranium mine in northern ontario, Canada.

Five strains of Thiobacillus ferrooxidans, which included three recent isolates from a uranium mine, possessed flagella. Three of the strains had several pili per cell. The dimensions, fine structure, and orientation of the flagella were different. Both polar and peritrichous flagella were observed, indicating strain-dependent ultrastructural variation in acidophilic thiobacilli. Neither flagella nor pili were detected in eight other strains of T. ferrooxidans and two strains of Thiobacillus acidophilus by electron microscopy, although all of the cultures contained motile cells.

Journal Article↗