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PubMed1994

The H-NS protein is involved in the biogenesis of flagella in Escherichia coli.

The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. In this paper, we show that motile behavior was affected in Escherichia coli hns mutants. The loss of motility resulted from a complete lack of flagella. A decrease in the level of transcription of the flhD and fliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation. Furthermore, the Fla- phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex. These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway.

Bacterial Outer Membrane Proteins↗

PubMed1995

Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7.

Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.

Amino Acid Sequence↗

PubMed2003

Genetic analysis of the AdnA regulon in Pseudomonas fluorescens: nonessential role of flagella in adhesion to sand and biofilm formation.

AdnA is a transcription factor in Pseudomonas fluorescens that affects flagellar synthesis, biofilm formation, and sand adhesion. To identify the AdnA regulon, we used a promoterless Tn5-lacZ element to study the phenotypes of insertion mutants in the presence and absence of AdnA. Of 12,000 insertions, we identified seven different putative open reading frames (ORFs) activated by AdnA (named aba for activated by AdnA). aba120 and aba177 showed homology to flgC and flgI, components of the basal body of the flagella in Pseudomonas aeruginosa. Two other insertions, aba18 and aba51, disrupted genes affecting chemotaxis. The mutant loci aba160 (possibly affecting lipopolysaccharide synthesis) and aba175 (unknown function) led to loss of flagella. The mutant bearing aba203 became motile when complemented with adnA, but the mutated gene showed no similarity to known genes. Curiously, aba18, aba51, aba160, and aba203 mutants formed biofilms even in the absence of AdnA, suppressing the phenotype of the adnA deletion mutant. The combined findings suggest that flagella are nonessential for sand attachment or biofilm formation. Sequence and promoter analyses indicate that AdnA affects at least 23 ORFs either directly or by polar effects. These results support the concept that AdnA regulates cell processes other than those directly related to flagellar synthesis and define a broader cadre of genes in P. fluorescens than that described so far for its homolog, FleQ, in P. aeruginosa.

Bacterial Adhesion↗

PubMed1989

Ultrastructure and chemical analysis of Campylobacter pylori flagella.

Flagella of Campylobacter pylori were analyzed by electron microscopy and purified, and the molecular weight of the flagellin was determined. Isolation of flagella was performed by mechanical shearing from the cell surface, sucrose density gradient centrifugation, and Sepharose CL-4B gel chromatography. The flagella of C. pylori differ from those of other Campylobacter species and of most other bacteria by the presence of a flagellar sheath. The sheath narrows at the end and is linked to a club-shaped terminal structure. The molecular weight of C. pylori flagellin was 51,000.

Campylobacter↗

PubMed1986

Fluorescent antibody and flagella stains for rapid detection of bacteria at periodontally healthy and diseased sites.

Two rapid methods of identifying bacteria associated with periodontal disease were investigated to determine their diagnostic usefulness in longitudinal or epidemiologic studies. Three nonmotile organisms were identified by fluorescent antibody stains (FA) while percentages of motile bacteria were assessed by counting all spirochetes, flagellated and nonflagellated organisms stained with a simplified silver-plating stain for flagella. Relationships between disease activity and these bacteria from subgingival plaque samples taken at 18 individual sites (12 diseased, 6 healthy) were determined by correlating the quantity of detectable bacteria with the Gingival Index (GI), Plaque Index (PLI) and probing depth (PD). The highest correlations found with the FA stains were between Bacteroides gingivalis and probing depth (rs = 0.85), GI (rs = 0.80) and PLI (rs = 0.80). Bacteroides melaninogenicus and/or Bacteroides intermedius also correlated well with the GI (rs = 0.66), PLI (rs = 0.64), and PD (rs = 0.59), but to a lesser degree than B. gingivalis. Flagella stains showed that spirochetes correlated highly with PD (rs = 0.82), as did the total motile group with PLI (rs = 0.82). Motile bacteria alone were only moderately associated with the clinical parameters measured. The results of this investigation suggest that FA- and flagella-staining methods can be valuable screening tools for the detection of bacterial species and motile organisms in longitudinal or epidemiologic studies.

Actinomyces↗

PubMed1990

Effect of D-mannose on motility, presence of flagella, and attachment of Pseudomonas aeruginosa to chicken skin cells.

Treatment of Pseudomonas aeruginosa with 1 to 3% d-mannose reduced cell activity indicating the flagella were affected by d-mannose but did not decrease attachment to chicken skin cells. Distance of migration of P. aeruginosa through a hollow tube filled with 1 to 4% d-mannose was reduced when compared with 0% d-mannose. This reduced movement through the tube indicated a possible affect on the flagella. Examination of P. aeruginosa with a light microscope showed that the addition of 1, 2, 3, and 4% d-mannose to a solution containing the organism removed the flagella from 27 to 40% of the cells.

Animals↗

PubMed1985

Peritrichous flagella in mesophilic strains of Aeromonas.

A total of 186 mesophilic strains of Aeromonas, comprising 151 A. hydrophila and 35 A. caviae, were tested for peritrichous flagella by Leifson's staining method. When incubated on solid medium for 18 hr at 22 C, peritrichous flagella were demonstrated in 28 (18.5%) of the 151 strains of A. hydrophila and in 6 (17.1%) of the 35 strains of A. caviae. The peritrichous flagella in these 34 mesophilic strains of Aeromonas were also observed by electron microscopy.

Aeromonas↗

PubMed2000

Spirochete periplasmic flagella and motility.

Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.

Borrelia burgdorferi Group↗

PubMed1992

[Eukaryotes devoid of the most important cellular organelles (flagella, Golgi apparatus, mitochondria) and the main task of organellology].

Comparative evidence on the lack of three important organelles (flagella, Golgi-complex, mitochondria) in cells and organisms at the cellular level of organization has been summarized for all the four eukaryotic kingdoms--Protista, Fungi, Plantae and Animalia (Metazoa). It is established that in the course of evolution these organelles may undergo the total reduction. There is no cellular organelle to be regarded as universal, indispensable. There are only three main obligatory cell components--the plasmalemma, nucleus and cytoplasm (with applied cytoskeleton, cytomembranes and ribosomes). The reduction of flagella (cilia) is occurring in different taxa independent of the transition of protists from the flagellate type of locomotion to the amoeboid, gliding of metabolizing ones, and in the number of metazoan cells. The members of Protista and Fungi, which line in microaerobic or anaerobic conditions, nearly inevitably lose their mitochondria. The tendency to lose Golgi-complex is demonstrated in protists with parasitic mode of life, especially in combination with anaerobiosis. There is so far no satisfied morphological criterium that could say with certainty whether the lacking of flagella, Golgi complex or mitochondria in the low eukaryotes may be primary or secondary (as the result of reduction). Data on the composition, structure and RNA nucleotide sequences cannot be either the straight evidence. A comparative analysis of these data shows that the ribosomes of the primary eukaryotes were, presumably, of a prokaryotic type. Their eukaryotization was carried out for a long time during the evolution of the low eukaryotes (Protista and Fungi), probably, independently in different phylogenetic lines. It is unknown at what steps and in what main phylogenetic lines the three above mentioned organelles may have appeared. It is proposed to single out a special division of cytology--organellology (organoidology)--as an individual science whose main purpose may be investigation of the origination, evolution and disappearance of organelles.

Animal Population Groups↗

PubMedDate not supplied

[Existence of an additional structural component in the basal bodies of Escherichia coli and Vibrio alginolyticus flagellae].

The structure of Escherichia coli and Vibrio alginolyticus flagella was studied using electron microscopy. An additional protein structure was shown to exist in the basal bodies of intact flagella in these organisms. It is possible that this structure involves three proteins important for the assembly of flagella, energy transduction, and a change-over in the direction of flagellar rotation.

Bacterial Proteins↗

PubMed1987

Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility.

Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein (Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J. Y., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786). The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility. It is concluded that loss of the five axonemal polypeptides upon irradiation results from a Vi-sensitized photocleavage similar to that which occurs in the alpha and beta heavy chains of outer arm dynein and that these polypeptides represent Vi-inhibitable ATPase subunits of dyneins located in the inner arms and possibly elsewhere in the flagellar axoneme.

Adenosine Triphosphatases↗

PubMed1976

Nucleotide-metabolizing enzymes in Chlamydomonas flagella.

Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts.

Adenosine Triphosphatases↗

PubMed1982

Models for oscillation and bend propagation by flagella.

A computer program for simulating the movement of model flagella containing a four-state cross-bridge cycle provides a powerful tool for examining hypotheses about the control mechanisms involved in producing particular patterns of propagated bending. As learned previously with simpler models, a simple control of cross-bridge activity by curvature is sufficient to generate spontaneous oscillation and bend propagation, but fails to reproduce many important features of the behaviour of real flagella. The process of bend initiation can be isolated by studying the movement of demembranated sea urchin sperm flagella broken to lengths of 3-4 microns, and by studying the movement of the distal end of a flagellum when most of the length of the flagellum becomes stuck to a surface. In order to simulate the movement seen in these situations, at least one major modification of the control of cross-bridge activity appears to be necessary. When a new bend is forming, the active sliding of cross-bridges causing the bend to form can be turned off when the curvature of the bend reaches a critical value, as in the earlier models. However, the active sliding of cross-bridges in the opposite direction, which will cause propagation of the bend, does not appear to be turned on at the same time. The mechanisms which trigger this delayed activation of cross-bridges almost simultaneously throughout the length of a newly formed bend have not yet been identified. They are presumably the same as those involved in activating sliding throughout most of the length of a flagellum at the beginning of its effective stroke.

Adenosine Triphosphate↗

PubMed1980

Appearance of heat shock proteins during the induction of multiple flagella in Naegleria gruberi.

A heat shock to amebae of the amebo-flagellate Naegleria gruberi during differentiation into swimming flagellates results in the induction of heat shock proteins as well as multiple flagella. The principal heat shock proteins migrate on sodium dodecyl sulfate-polyacrylamide gels with apparent molecular weights of 96,000, 77,000, 70,000, and 68,000. These proteins are synthesized preferentially when cells at 25 degrees C are shifted to temperatures above 32 degrees C. The maximal incorporation of methionine into heat shock proteins occurs at 38.2 degrees C, the temperature at which maximal induction of multiple flagella has been reported. Synthesis of heat shock proteins requires new transcription as judged by the ability of actinomycin D to inhibit their synthesis during the first 15 min of heat shock but not thereafter. Although heat shock can induce multiple flagella only when applied during a restricted interval, heat shock proteins are induced at any time cells are shifted to 38.2 degrees C. The response to heat shock of the Naegleria heat shock proteins resembles that of Drosophila heat shock proteins, but the two groups of proteins differ in both size and number. Naegleria heat shock proteins are, however, strikingly similar in size to a group of heat-induced proteins found in chick embryo fibroblast, mouse L, and BHK cells.

Amoeba↗

PubMed1996

[Intervention of Campylobacter jejuni subsp. jejuni flagella in the adhesion to cellular cultures: bacteriological and immunological evidence].

The participation of the flagella of a virulent strain (O52) of Campylobacter jejuni subsp. jejuni in the adhesion to HEp-2 cells and their inhibition by means of homologous polyclonal antibodies, monoclonal anti-flagella antibodies and colostral natural antibodies (IgA) was studied. An aflagellated strain (T1) was used as negative control. Adhesion was observed in higher rates with O52 strain (72%) than with T1 strain (27.5%). Polyclonal, monoclonal and colostral antibodies inhibited O52 strain adhesion in more than 70% (p < 0.001). T1 strain adhesion was inhibited only by polyclonal and colostral natural antibodies. Our results suggest that the flagella of C. jejuni subsp. jejuni could participate effectively in the adhesion process. However, the inhibition of T1 strain by polyclonal and colostral antibodies suggests the existence of other binds of adhesins in the bacterial surface.

Bacterial Adhesion↗

PubMed1993

Microtubule sliding in reduced-amplitude bending waves of Ciona sperm flagella: resolution of metachronous and synchronous sliding components of stable bending waves.

Microtubule sliding associated with the bending of reactivated flagella of demembranated spermatozoa of the tunicate, Ciona, has been analyzed using a descriptive model that permits quantitation of metachronous and synchronous components of sliding. Reduced-amplitude bending waves, obtained by addition of increased salt (K acetate), lithium, or vanadate to the reactivation solutions, have been examined. Increased K acetate can decrease bend angle by as much as 70% with little change in frequency. In all cases, a decrease in the amplitude, or bend angle, of propagated bends is measured as a decrease in the metachronous component of sliding and is associated with a reduction in the growth of new bends after they begin to propagate during the second half-cycle of bend development. At higher K acetate concentrations, bend growth during the second half-cycle of bend development is very strongly reduced and may even become negative. A disparity between the rates of bend growth in the first and second half-cycles of bend development corresponds to a large amount of synchronous sliding in the distal portion of the flagellum. When the synchronous sliding component is large, the sliding velocity in a propagating bend decreases to near-0 values and may even reverse its direction as the bend propagates through the mid-region of the flagellum. Since these large perturbations of sliding velocity do not interfere with regular propagation of bends with nearly constant bend angle, the bend propagation mechanism cannot operate by metachronous control of the velocity of sliding, and is unlikely to operate by local monitoring of either the amount or velocity of sliding. These observations therefore argue against models in which active sliding is regulated by shear or sliding velocity, and make curvature-controlled models relatively more attractive. In many cases, a reduction in sliding during bend initiation (the first half-cycle of development of new bends) also contributes to the decreased amplitude of propagated bends. These changes in bend initiation are similar in both full-length flagella and in flagella shortened by breakage. The amount of sliding that occurs during bend initiation is relatively independent of the distribution of sliding between metachronous and synchronous components in the distal part of the flagellum. These observations therefore provide additional evidence that bend initiation and bend propagation are independent and separable processes.

Acetates↗

PubMed1994

Speract receptors are localized on sea urchin sperm flagella using a fluorescent peptide analog.

In two species of sea urchins, Strongylocentrotus purpuratus and Lytechinus pictus, the egg jelly-associated decapeptide, speract, binds to specific sperm surface receptors resulting in increased sperm motility and respiration rate. Previously, a peptide analog, GGG[Y2]-speract, was used to identify a 77-kDa receptor on intact sperm cells using chemical cross-linking. In this paper we describe the synthesis and characterization of a fluorescent derivative of GGG[Y2]-speract for use as a probe for the sperm receptor. Fluorescein isothiocyanate (FITC) was conjugated to the amino terminus of GGG[Y2]-speract and the resulting analog (FITC-GGG[Y2]-speract) was purified by size exclusion chromatography and reverse-phase HPLC. Competition binding studies with the fluorescent peptide and intact spermatozoa yielded IC50 values which were indistinguishable from native speract and GGG[Y2]-speract (approximately 20 nM). FITC-GGG[Y2]-speract half-maximally stimulated sperm respiration at a concentration nearly identical to that of the native peptide (EC50 approximately 50 pM). Using digitally enhanced video imaging fluorescence microscopy, FITC-GGG[Y2]-speract was used to localize the speract receptor on the flagella of intact sperm. Excess concentrations of both unlabeled speract and GGG[Y2]-speract abolished the binding of the fluorescent analog, yet unrelated peptides did not. Further, results of cross-linking experiments using 125I-GGG[Y2]-speract and purified sperm flagella and heads were consistent with the fluorescent labeling results on whole cells. The finding that the speract receptor is localized exclusively to the sperm flagella may reveal its role in the regulation of flagellar motility.

Amino Acid Sequence↗

PubMed1979

Starting transients in sea urchin sperm flagella.

Live sea urchin spermatozoa were rendered immotile by lowered pH; Triton-extracted spermatozoa were rendered immotile by either lowered pH or by deprivation of ATP. The spermatozoa began to beat after an increase in pH or as ATP was supplied, and the first bends were recorded on ciné film. Triton-extracted spermatozoa deprived of ATP retained a partially formed basal bend which could be either principal or reverse, and which resumed its development and propagation as ATP was supplied. Both live and tritonated flagella straightened at low pH. As the pH was increased, a series of principal bends formed near the base and propagated to the tip. Reverse bends began to develop as the pH continued to increase. The principal and reverse bends thus exhibited different sensitivities to pH, which suggests differences in the mechanisms that produce them. Straight flagella began to move by synchronous sliding all along the flagellum, thus forming principal bends. Flagella that contained a basal bend began to move by primarily metachonous sliding within that bend.

Adenosine Triphosphate↗