Flagellin Monomers Research Articles

Photobleaching recovery studies of antigen-specific mouse lymphocyte stimulation by DNP-conjugated polymerized flagellin.

We have used fluorescence photobleaching recovery (FPR) to study the lateral diffusion of antigen-receptor complexes during stimulation of DNP-specific mouse B cells by the T-independent antigens DNP-polymerized flagellin (DNP-POL). Depending on epitope density and dose, these antigens behave either as immunogens or tolerogens. Lymphocyte DNP receptors binding DNP0.5 flagellin monomer show a diffusion constant D of 2.2 X 10(-10) cm2 sec-1 and ca 50% fluorescence recovery after bleaching. For DNP-POL bound to DNP-specific lymphocytes, the observed diffusion constants decrease monotonically with increased antigen dose and epitope density. Under optimally immunogenic conditions of DNP2.3-POL at 1 micrograms/ml, D = 1.5 X 10(-11) cm2 sec-1, some 14-fold less than for a single DNP receptor. Under tolerogenic conditions lower diffusion constants approaching 0.8 X 10(-11) cm-2 sec1 are observed. The fraction of aggregates mobile on the time scale of the experiment remains constant at about 50 to 60% in all immunogenic situations, but falls abruptly to about 24 to 32% in precisely those situations where the antigen/dose combination is tolerogenic. This might support the hypotheses that there exist critical epitope densities above which antigens and receptors form rigidly cross-linked aggregates that bring about B cell tolerance. The mobility of DNP0.5 flagellin monomer bound to receptors left unoccupied after treatment with various doses and batches of DNP-POL is independent of DNP-POL presence. Receptor aggregate diffusion is unaffected by treatment with colchicine or cytochalasin B.

Differences between surface antigenic determinants of polar monotrichous flagella of Vibrio parahaemolyticus and of related species.

Polar monotrichous flagella (M-flagella) of Vibrio parahaemolyticus have antigens in common with those of various species of Vibrio including V. cholerae and V. anguillarum, and of Beneckea, revealed by gel diffusion tests with flagelli monomers. However, antiserum against M-flagellin of V. parahaemolyticus did not agglutinate cells of V. cholerae and V. anguillarum, although it did agglutinate cells of V. parahaemolyticus. Agglutination tests after absorption of the antiserum with purified M-flagellar filaments or flagellin monomers and H-agglutination inhibition tests demonstrated that there are two different antigenic determinants in M-flagella as in lateral flagella. One is on the surface of the M-flagella (surface antigenic determinant, SA) and disappears or is buried in dissociated monomers. The other is inside the flagella (internal antigenic determinant, IA) and is exposed when the flagella are dissociated to flagellin monomers. SA of V. parahaemolyticus is different from those of V. cholerae and V. anguillarum, whereas the three species have a common IA.

Behavior of a surface antigenic determinant of lateral flagella of Vibrio parahaemolyticus.

The behavior of a surface antigenic determinant (SA) of lateral flagella of Vibrio parahaemolyticus was studied. SA, located on the surface of flagellar filaments, is responsible for H-agglutination and specific for serotype. Results of H-agglutination inhibition tests demonstrated that SA could not be detected on the flagellin molecule when the flagellar filaments were dissociated to flagellin monomers by heating or treatment with urea, sodium dodecyl sulfate, HCl, or acetone, although SA could be detected on short flagellar fragments obtained by milder heat treatment. When flagellar filaments were reconstituted from flagellin monomers, SA was detected on the surface of the filaments. These results suggest that SA is buried in the flagellin molecule with dissociation of flagellar filaments to flagellin monomers or steric configuration of SA itself is altered to a different form which cannot react with the responsible antibody.

Antigenicity of lateral flagella of Vibrio parahaemolyticus: existence of two distinct antigenic determinants in a flagellum.

Cells of Vibrio parahaemolyticus treated with Formalin agglutinated with anti-lateral flagella antiserum. On the basis of agglutination tests, antigens of lateral flagella were divided into three groups. HL1, HL2, and HL3. However, in gel diffusion tests, flagellins prepared from strains belonging to different groups showed a common antigenicity. It is assumed that these results are due to the existence of two distinct antigenic determinants in the lateral flagella. One of them exists on the surfaces of the flagella and is responsible for H-agglutination, and the other exists inside the flagella and is exposed when the flagella are solubilized to flagellin monomers.

Conditional surface structure mutants of Caulobacter crescentus temperature-sensitive flagella formation due to an altered flagellin monomer

Two classes of temperature-sensitive surface structure mutants of Caulobacter crescentus have been isolated. Mutants in one class were unable to form flagella at the restrictive temperature (37°C) and mutants in the second class were unable to form a stalk structure at 37°C. All of the mutants appeared normal at the permissive temperature (23°C). Analysis of the conditional flagella mutant revealed that the temperature-sensitive lesion resided in the structural flagellin monomer, since (1) extracts of mutant cells grown at the restrictive temperature contained material cross-reacting with anti-flagellin antibody although flagella were not assembled. (2) Mutant flagellin prepared from cells grown at the permissive temperature was heat-denatured to a different conformation than wild-type flagellin, as demonstrated by lateral shifts in microcomplement fixation assays. (3) Amino acid analysis showed the presence of tyrosine in the mutant flagellin, whereas none was detected in the wild-type control. (4) Chymotryptic peptide maps of mutant and wild-type flagellin showed the appearance of two new peptides in the former case. (5) A spontaneous revertant regained both motility and wild-type flagellin amino acid composition and chymotryptic peptide maps. An alteration in the primary structure of the mutant flagellin therefore results in a temperature-dependent alteration in flagellin conformation, perhaps accounting for the inability to assemble a flagellar structure at the cell surface. Comparison of immunoprecipitates of intracellular and extracellular mutant flagellin, as well as flagellin concentrations, further showed that the inability to assemble a flagellar structure interfered with the regulatory processes controlling both the amount and structural integrity of the intracellular flagellin at the restrictive temperature.

Electric birefringence of bacterial flagellar protein filaments: Evidence for field-induced interactions

The time course for the build-up and decay of birefringence induced by a rectangular voltage pulse was measured on solutions of flagellar filaments from Salmonella equi-abortus (strain SJ25). These filaments are tubular polymers of protein (degree of polymerization ≈ 10 3) constituted by non-covalent linkage of flagellin monomers of molecular weight 4 × 10 4. The effect on electro-optical properties of solutions of filaments due to variations in temperature, concentration and mean length of protein filaments, and the duration and intensity of the applied electric field is described. Analysis of the field intensity dependence of the birefringence and comparison of the build-up and decay processes indicate that orientation in the field is due primarily to the existence of a permanent dipole moment in the filaments. At 18 °C the following values were obtained for a solution of filaments with mean length and standard deviation of 0.39 and 0.30 μm: specific Kerr constant ( K sp) = 6.14 × 10 −3 electrostatic units; optical anisotropy factor ( g 1 — g 2) = 5.66 × 10 −3; dipole moment (μ) = 1.01 × 10 5 Debye units; and mean relaxation time ( \\ ̄ gt) = 9.20 ms. At temperatures below 20 °C there is a marked increase in the optical anisotropy factor of the filaments which may be due to a change in their flexibility. The large values of K sp obtained indicate the highly responsive nature of these filaments to an electric field. The birefringence decay curves were decomposed by computer into a specified number of exponential terms from which both the mean length and the size distribution of these polydisperse filaments were calculated. The results obtained were in substantial agreement with the values of these parameters observed by electron microscopy. A cumulative field effect dependent on field intensity and filament concentration was observed. Repeated pulsing of electric field, above threshold values of field intensity and filament concentration, produced decreases in the birefringence near 60% of its initial value. The effect was reversible with a time constant greater than two minutes. No appreciable change in the relaxation time for decay of birefringence was observed on multiple pulsing of these solutions. These results are interpreted consistently to arise from the sidewise aggregation of filaments induced by electrical impulses of sufficient intensity and duration. These properties appear relevant to bacterial motility: variations in electric potential along the membrane of the bacterium might serve first to orient these organelles and then to induce their coalescence to “bundles” of filaments. The latter structures are commonly observed in vivo. In this way the activity of flagella might be co-ordinated.

Formation of a flagella-like but straight polymer of Salmonella flagellin

Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH 4) 2SO 4 (Ada et al., 1963; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high concentration of NaCl (about 1.5 m), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flagellins derived from strains SJ670, SJ25, SJ30 and SJ814. When the straight filament, suspended in 0.15 m-NaCl, was heated, it depolymerized to the monomer, which could in turn be polymerized into flagellar filaments by the addition of short fragments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsuccessful. In 0.15 m-NaCl, straight filaments prepared from the four kinds of flagellins had markedly different heat stabilities, which were much lower than that of any kind of flagella. When monomeric flagellin dissolved in 3.5 m-NaCl was seeded with short fragments of straight filaments, the monomer polymerized onto the ends of the short fragments, which consequently grew into long straight filaments. In this type of experiment, monomers and seeds derived from the four strains were able to interact in any combination, suggesting that straight filaments consisting of the four kinds of flagellins have the same substructures. Whether the concentration of added NaCl was 0.15 m or 3.5 m, fragments of flagella (or straight filaments) were unable to act as seeds for the formation of straight filaments (or flagellar filaments). From this and other experimental results, it was concluded that in the two filamentous structures, flagellin molecules may be packed in different ways.

Assembly of Salmonella flagellin in vitro and in vivo.

AbstractFormation of a flagellar filament of Salmonella from its component protein, flagellin, is in principle a self‐assembly process, which proceeds by the addition of flagellin monomers one by one to the distal end of the filament. Upon their polymerization, a conformational change of flagellin molecules occurs, and it confers polarity to the filament. For the initiation of in vitro flagellin assembly in a solution of physiological ionic strength and pH, it is essential to add fragments of flagellar filaments, which work as seeds for the polymerization of flagellin monomers. When an appropriate concentration of some anion known as a good salting‐outer is added to the solution, the polymerization begins without addition of seed. Assembly of flagellins in vivo begins at the distal end of each hook. The distal ends of the hooks on the cells of a flagellin‐less mutant were shown to initiate the assembly of exogenous flagellin in vitro, although the efficiency was not as high as that of the in vivo initiation. A flagellar filament elongates in vitro at a constant rate as long as a sufficient amount of flagellin is supplied, and the elongation terminates by an error occurring at the growing end of the filament. On the contrary, the rate of in vivo elongation decreases exponentially with increase of the length of the filament. Initial rate of the in vivo elongation depends on growth condition of the bacteria, while decrease of the rate per unit filament length is little affected by the growth condition. The observed limit in length of the flagellar filaments on growing bacteria is expected from the exponential decrease of their rate of elongation. The decrease of the in vivo elongation is correlated with the lowering of the transportation efficiency of flagellin monomers on their passage from the cell body through the central canal of a flagellar filament to the tip.

Effect of temperature on the in vitro assembly of bacterial flagella

The temperature dependence for the rate of reconstitution or polymerization ( k +) at neutral pH of the protein, flagellin, to flagella was measured using Ostwald-type viscometers. Similarly, the kinetics for the reverse process, the thermally-induced depolymerization of flagella filaments to the flagellin monomer ( k −) was measured. The temperature at which k − equals zero was used to define the thermal dissociation temperature or melting point of flagella filaments. The remarkable similarity of melting points obtained (36.8 ± 0.2 deg. C) for flagella isolated from three Salmonella strains (SJ670, SJ25 and SJ30 bearing H-antigen types i, 1.2 and e, n, x, respectively) suggests that the structural stability of these different protein filaments is also similar. On increasing the temperature between 12 and 28°C, k + increased smoothly and had a Q 10 of 1.8. Above 28.0, k + decreased rapidly and fell to zero at a temperature near 37°C, its precise value varying with the bacterial strain. This result supports the prior hypothesis (Gerber & Noguchi, 1967) that on heating, a reversible co-operative transconformation occurs between different states of the protein; in one state, flagellin (M) can polymerize to flagella, whereas its conformational isomer(s) may do so with difficulty or not at all. For strains SJ25 and SJ30 the rates of polymerization and depolymerization both fall to zero near 37°C. Therefore, mixtures of monomer and flagella fragments (short polymers or “seeds”), in all ratios, appear to be in equilibrium at temperatures near this critical temperature, and neither polymerization of flagellin to flagella nor melting of polymers is apparent. Measurements made on flagella from strain SJ670 showed that k + and k − approached zero at 45 and 37°C, respectively. Within this temperature range the conc entration of monomer in equilibrium with filaments was determined. By a null -point type experiment, solutions of monomer and seed were mixed to find the ratio that showed neither increases (polymerization) nor decreases (depolyme rization) in viscosity with time. An unexpected finding was that the temperature defines a critical monomer concentration, which exists in equilibrium with any concentration of filaments (and not the ratio of monomer-to-filament concentrations). Thus, the polymerization of fiagellin to flagella corresponds to a phase change akin to either crystallization or condensation. Application, of the Clapeyron-Clausius equation to the results obtained yields a heat of condensation of 70 kcal/mol of monomeric protein. The enthalpy change associated with M ⇌ M i is estimated as 110 kcal/mol of protein. Since the heat content of these various forms of flagella protein lies in the order M i > F > M, by difference we estimate the enthalpy change for the conversion of monomers to polymers to be 40 kcal/mol of monomer.

A Phenomenological Theory of Polymorphism of Salmonella Flagella. I. A Model

A model theory has been proposed, which explains the polymorphism of Salmonella flagella observed by Asakura. Relation between the overall shapes of the helical flagellar filaments and the shapes of their constituents, i.e. , the flagellin monomer units is derived with the aid of two parameters, the curvature and the torsion of the helix. The model proposed is based upon the assumptions that the flagellin molecules in the flagellum assume two states R and T depending upon circumstances, as discussed by Asakura, that the flagellar filament consists of eight strands of the flagellin monomer units, that the five polymorphs have one, three, five, seven and eight T-type strands, respectively. Agreement is obtained between the observed and calculated values of curvatures and torsions of the helical flagellar filaments, and the parameters are determined, which characterize the shapes of the flagellin monomer units.

Reconstruction of Salmonella flagella attached to cell bodies.

THE reconstitution in vitro of flagellar filaments from their component flagellin monomers in Salmonella has shown that the filaments have structural polarity and grow at an end distal to the cell body1; flagella in vivo also grow from their tips2,3. This suggests that even when flagella are attached to living cells, filaments may be reconstituted from exogenous flagellin monomers at the tips in appropriate conditions. In spite of some negative results4, we have been encouraged5–10 to re-examine the question.

Polymerization of salmonella flagellin in the presence of high concentrations of salts

1. 1. Salmonella flagellin (monomer) spontaneously polymerizes into flagellar filaments in the presence of moderately high concentrations of some kinds of salts at neutral pH. The process was followed by viscosity measurement and the product was examined with an electron microscope. 2. 2. Rapid and complete polymerization of flagellin was brought about by the addition of F −, CO 3 2−, SO 4 2−, HPO 4 2− and citrate ion to final concentrations higher than 0.3 M. The initial rate of polymerization increased with increasing concentrations of these ions. On the other hand, in the presence of high concentrations of Mg 2+ and Ca 2+, flagella underwent depolymerization. These anions and cations are known as good “salting-outers” and “salting-inners”, respectively. 3. 3. Reformed filaments were indistinguishable in the overall shape from intact flagella. The average length of reformed flagella, measured after complete polymerization, decreased with increasing concentrations of the above anions and with increasing concentrations of flagellin. 4. 4. From the dependence of the average length of reformed flagella on the initial concentration of flagellin, the number of flagellin molecules needed for the initial step of polymerization, nucleation, was estimated.

Disintegration of flagella by acid

During the disintegration of flagella from cells of Proteus vulgaris, hydrogen ions are taken up; thus one can determine the rate of this disintegration by measuring the number of hydrogen ions consumed at a constant pH. The rate of disintegration was studied in this manner over the pH range 2.5–3.4. In all cases the time course of the process within experimental error obeyed simple pseudo first-order kinetics. The pH dependence of the pseudo first-order rate constant corresponded to an uptake ( q ∗ ) of 2.4 hydrogen ions at 12 °C. Upon disintegration the total uptake of hydrogen ions per monomer unit ( q) is about 4. The similarity of q ∗ and q suggests that the activation process may involve the modification or liberation of perhaps only a single or no more than a few subunits, or both. The pseudo first-order character of the process, together with the failure to find evidence by ultracentrifugation for the existence of intermediates having particle weights between those of flagella and flagellin monomers or dimers, represent evidence in favor of a fully cooperative breakdown of flagella.

In vitro formation of flagella-like filaments and other structures from flagellin

The in vitro reaggregation of flagellin monomers, prepared by acid disintegration of isolated native flagella of Bacillus pumilus , was studied. Flagellin reassembles in at least three forms, two of which are highly organized in a linear fashion, and appear as straight structures and flagella-like filaments. The latter closely resemble native flagella in their morphology and in other properties. The rate and the extent of the reaggregation reaction, and the structure of the aggregates produced, are affected by the concentration of flagellin, pH, salt and temperature. Reaggregation at 2°C and in frozen samples makes it unlikely that the reaction is enzymic. Straight aggregates can be transformed to flagella-like filaments if they are suspended at an appropriate pH. On the other hand, neither flagella-like filaments nor native flagella can be changed to the straight forms by incubation at a pH at which the latter assemble from monomers. The results indicate that the primary structure of the flagellin molecules determines their configuration assumed under various conditions and the manner in which they reassemble to larger polymers in a specific environment. Flagella-like filaments and probably native flagella as well constitute the most stable organized state in which flagellin can aggregate.