Some chemical and physico-chemical properties of the flagella of Proteus vulgaris
Abstract
A procedure for the preparation of flagella from Proteus vulgaris in a highly purified state has been described.
Investigations of the chemical composition of the flagella have shown an N content of 15.7 — 16.1%; only traces of phosphorus, and at the most one per cent carbohydrate (very probably a contamination) are present. Several amina acids have been found in the flagella.
Measurements of the ultraviolet absorption show that the greater part of the absorption is due to a content of 1.8 – 1.9% tyrosine. No tryptophane, purin- or pyrimidic bases are present in any appreciable amount.
These facts strongly suggest a protein nature of the flagella. The hypothesis of the polysaccharide nature of the flagella, suggested by Pijper1 can not be correct, at least not for Proteus.
The flagella are decomposed in acid medium. The split products consist of particles of rather homogeneous size with a particle weight of about 41 000 (lower limit). These particles are probably of an elongated form.
Résumé
Description d'un procédé pour la préparation des flagelles de Proteus vulgaris dans un état de pureté très poussée. L'étude de la composition chimique des flagelles montre une teneur en azote de 15.7 à 16.1%, des traces seulement de phosphore, et au plus 1% d'hydrates de carbone (constituant très probablement une impureté). Plusieurs acides aminés ont été caractérisés.
La mesure de l'absorption dans l'ultra-violet montre que la plus grande part de cette absorption est due à une teneur de 1.8 à 1.9% de tyrosine. Il n'existe pratiquement pas de tryptophane, de base purique ou pyrimidique.
Ces faits sont fortement en faveur de la nature protéique des flagelles. L'hypothèse de leur nature protéique des flagelles. L'hypothèse de leur nature polysaccharidique, émise par Pijper1 n'est pas valable, au moins dans le cas de Proteus.
Les flagelles sont décomposés en milieu acide. Les produits de décomposition consistent en particules de taille plutôt homogène, et d'un poids particulaire d'environ 41 000 (limite inférieure). Ces particules possèdent probablement une forme allongée.
Zusammenfassung
Ein Verfahren zur Bereitung von Flagella von Proteus vulgaris in hochgereinigtem Zustand wurde beschrieben.
Untersuchungen der chemischen Zusammensetzung der Flagella zeigten einen N-gehalt von 15.7–16.1%; nur Spuren, Phosphor, und höchstens 1% Kohlenhydrat (wahrscheinlich eine Verureinigung) sind vorhanden. Verschiedene Aminosäuren wurden in den Flagella gefunden.
Messungen der Absorption im Ultraviolett zeigen, dass der grössere Teil der Absorption durch einen Tyrosingehalt von 1.8–1.9% verursacht wird. Weder Tryptophan, noch Purin- oder Pyrimidinbasen sind in nennenswerten Mengen vorhanden.
Diese Tatsachen weisen sehr stark auf eine Eiweissnatur der Flagella hin. Die durch Pijper1 aufgestellte Hypothese der Polysaccharidnatur der Flagella kann also, jedenfalls bei Proteus, nicht richtig sein.
Die Flagella werden in saurem Milieu gespalten. Die Spaltprodukte bestehen aus Teilchen von beinahe homogener Grösse mit einem Teilchengewicht von ungefähr 41 000 (untere Grenze). Diese Teilchen haben wahrscheinlich eine längliche Form.
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The bacterial cell envelope - a historical perspective
1994, New Comprehensive BiochemistryThis chapter presents a historical perspective of the bacterial cell envelope. The evaluation of ideas and knowledge of the nature of universe has never ceased to fascinate and challenge physicists, astronomers and cosmologists for millennia and will without doubt continue into the for seeable future. Unraveling the complexities of biological systems has, by comparison, been a relatively recent event in human history but the “microcosmic” has proved to be equally challenging and complex but amenable to a level of direct manipulation and observation within a fairly reasonable span of time. Electron microscopy of thin sections of a variety of Gram-positive and Gram-negative bacteria provided the early key observations pointing to a fundamental difference in the organization of the surface structures of the two groups leading to the typical membrane profiles of the Gram-negative organisms compared to the thick, amorphous walls of Gram-positive bacteria with their underlying cytoplasmic membranes. Early investigations of the differences in chemical composition correlated well with the structural studies.
In vitro synthesis of phase-specific flagellin of Salmonella
1973, Journal of Molecular BiologyChromatography of Salmonella flagellin at pH 8 on DEAE-cellulose separated at least four serologically distinct kinds of flagellin, a, enx, i and 1,2, eluting in that order with increasing concentration of sodium chloride. By this chromatographic technique, the preincubated cell-free extract of Escherichia coli given saltprecipitable RNA of Salmonella was shown to synthesize flagellin characteristic of the flagellar antigen type of the cells from which the RNA was derived. Two of the in vitro synthesized flagellins specifically reacted with their corresponding antiserum.
When RNA was extracted from the cells of the diphasic strain propagated from a single colony, expressing either phase 1 or phase 2, the in vitro synthesized flagellin was predominantly the same as that produced by the original colony. Translation of messenger RNA specific for phase 1 flagellin was not inhibited by the presence of messenger RNA specific for phase 2. RNA extracted from the cells of a diphasic strain without any selection directed synthesis of both phase 1 and phase 2 flagellins in the ratio expected if the culture was at equilibrium with respect to phase variation. Experimental evidence is presented to support the hypothesis that phase variation is due to the alternative synthesis of phase-specific messenger RNA.
Effect of temperature on the in vitro assembly of bacterial flagella
1973, Journal of Molecular BiologyThe 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 Q10 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 ⇌ Mi is estimated as 110 kcal/mol of protein. Since the heat content of these various forms of flagella protein lies in the order Mi > F > M, by difference we estimate the enthalpy change for the conversion of monomers to polymers to be 40 kcal/mol of monomer.
Self-assembly of bacterial flagellar protein: Dielectric behavior of monomers and polymers
1972, Journal of Molecular BiologyThe temperature dependence for the dielectric dispersion of the flagellar protein of Salmonella abortus-equi in monomer and polymer form were compared over the frequency range of 50 Hz and 20 MHz. At 20 °C the critical frequency and corresponding dielectric relaxation time τ of the monomer (mol. wt. ≅ 4 × 104) are 520 kHz and 310 nsec; the values of these parameters for the polymer (mol. wt.> = 11·1 × 106) are 1·1 kHz and 140 μsec. The dielectric behavior of flagellar proteins can be related to orientation in the field of a permanent dipole on the proteins. Graphs of − log (τ) versus are linear and the Arrhenius activation energy for disorientation of polymer is 2·8 kcal./mole. Two limiting values of this parameter are observed, however, for the monomer: 6·2kcal./mole at temperatures below 26 °C and 8·4 kcal./mole above 35 °C. The two values are related to the polymerizable and inactive conformational isomers first reported by Gerber & Noguchi (1967). Nevertheless, the dimensions of the two isomers are remarkably similar. Below 26 °C flagellin can be hydrodynamically approximated by a prolate ellipsoid with an axial ratio of 9 : 1 and with a dipolar angle between 30 and 40 °; above 35 °C the axial ratio decreases slightly to a value of 8 : 1. The dipole moment of the monomer is calculated as 860 Debye; the dipole moment of a monomer in the polymer is less than 150 Debye. Furthermore, the polymer dipole is in its long axis. To account for this result a model is proposed for the helical arrangement of distally-tilted asymmetric monomers to form a tubular flagellum. Such a filament, consequently, will contain an inherent polarity in agreement with observations by others that (1) in vitro growth of the flagellum is unidirectional and (2) that the distal end of the flagellum has a notched or fish-tail appearance. Generally, the polarization of proteins is due primarily to a permanent and not an induced dipole, which arises from fixed charges on the surface of the protein. This type of dipole has two properties useful to self-assembly mechanisms: first, they are vector quantities and so specify direction (bond angle); and second, they are capable of long-range interactions. A survey of the literature shows that for all polymerizable proteins studied, the dipole moment is greatly decreased on forming a polymer. This suggests that a principle of dipole neutralization plays an important role in the self-assembly of proteins.
X-ray and optical diffraction studies of bacterial flagella
1971, Journal of Molecular BiologyFibre preparations suitable for X-ray analysis have been made from flagella of different species of bacteria. The high-angle X-ray patterns obtained with preparations from different species are all closely similar and show the presence of a 52 Å periodicity. The low-angle equatorial pattern from fibres contains reflections mainly of interflagellar origin. Intraflagellar diffraction at low-angles was obtained from virtually unoriented aqueous preparations; from this diffraction the cylindrically averaged flagellar diameter could be calculated. X-ray diffraction patterns from flagella-like filaments of certain reaggregated flagellins were found to be indistinguishable from X-ray patterns of native flagella.
The gross features of a model proposed from electron microscopy (Lowy & Hanson, 1965) for the Salmonella typhimurium flagellum are compatible with the low-angle X-ray data. Further features of the surface lattice of the S. typhimurium structure are suggested from an optical diffraction study of electron microscope images.
Bacterial Flagella
1971, Advances in Microbial PhysiologyBacterial flagella have become of great biological interest because they are useful models in studies dealing with the conversion of chemical energy to motion and with molecular aspects of morphogenesis. Electron microscopically, the flagellum can be seen to contain the following three morphologically distinct parts—namely, a basal structure that is closely associated with the cytoplasmic membrane and cell wall, a hook, and the main spiral filament— apparently a tube, the wall of which is constructed of the protein named “flagellin.” The filament is the most prominent feature of the bacterial flagellum. The hook and basal structure constitute only minor portions of the organelle, and relatively little is known about them, although the recent purification of hooks promises more tangible information regarding that region. Preparations of isolated “flagella” are predominantly filament material although, depending on the method of isolation, basal regions and hooks are also present in varying degrees.