Tetsuo Iino

Reconstitution of bacterial flagella in vitro

The process by which bacterial flagellar filaments are reconstituted from flagellin molecules in vitro is examined by physical measurements (flow birefringence, viscosity, sedimentation) and by electron microscopic analysis, using flagella isolated from a strain of Salmonella. It is shown to be essentially reversible and characteristic of crystallization. In the presence or absence of salt, the flagella are depolymerized into monomers by heat treatment. At neutral pH, the repoly-merization takes place only in the presence of salts. In monomer solutions, however, spontaneous nucleation rarely happens and the solutions remain in a state of super-saturation. In order to polymerize the monomers in such solutions, it is necessary to add fragmented flagella. Then, the ends of added fragments act as nuclei, resulting in rapid formation of long flagellar filaments. In this process the number of filaments contained in each solution remains unchanged, and a one-to-one correspondence holds between the added fragments and the fully grown filaments. The rate of growth of flagellar filaments in vitro is determined under various salt conditions and found to be comparable to that observed in vivo.

Salmonella flagella: in vitro reconstruction and over-all shapes of flagellar filaments

In vitro reconstitution of flagellar filaments from monomeric flagellins was carried out by the method used previously ( Asakura, Eguchi & Iino, 1964 ) using flagella isolated from Salmonella strains SJ670, SJ25, SL23 and SJ30: the former three strains possess flagella of the normal type and carry different H-antigen types, and the last one is a curly flagellar mutant obtained from SL23. Monomeric flagellins and short fragments of flagella prepared from these strains were mixed in various combinations to re-form not only homogeneous filaments but also heterogeneous ones. Kinetic studies of these processes by viscosity measurement and by electron microscope observation showed: (1) that for initiating polymerization of any kind of monomer, it is necessary in practice to add short fragments or “seeds”, which need not necessarily be homologous with the monomer; (2) that the over-all rate of polymerization is largely dependent on the nature of monomer, the order of rapidity being SJ670 > SJ25 > SJ30 > SL23; (3) that polymerization of flagellin takes place at a rate less than first order with respect to the concentration of monomer; and (4) that copolymerization of monomers of SJ670 and SJ30 proceeds at a rate markedly less than the sum of polymerization rates of the constituent monomers. These results were consistently explained by assuming trans-conformation of flagellin on polymerization. Over-all shapes of reconstituted filaments were examined by a low-magnification electron microscope. Not only homogeneous filaments but also heterogeneous ones and copolymers could be classified into either one of the two major types, namely the normal and curly types. When a small amount of short fragment was used, the shape of reconstituted filament was determined by the nature of monomer. When, however, a moderately long fragment of SJ30 flagellum (curly) was used for polymerizing the comparable concentration of SJ25 monomer (normal), the grown part of each filament was found to be curly. Copolymerization of comparable concentrations of SJ670, SJ25 or SL23 monomer with SJ30 monomer produced filament of the curlytype, irrespective of the nature of the added fragment. These experimental results show a dimorphic nature of flagellins. The normal type filament was often transformed into the curly type when it was incubated for a long time. The transformed filaments were reversed into the normal type by treatment with low concentrations of pyrophosphate or ATP. From these results it is concluded that the normal type filament possesses an intrinsic ability to transform between the normal and curly types, depending on external conditions, and that this transformation is akin to dimorphic transition in crystals.

Unidirectional growth of Salmonella flagella in vitro

In previous papers (Asakura, Eguchi & Iino, 1964, 1966), in vitro reconstitution of flagellar filaments from monomeric flagellins, using strains of Salmonella , was shown to be similar to crystallization. At physiological ionic strength and pH, reconstitution consists only of growth, that is, the polymerization of a monomer on to the ends of added fragments of flagella or “seeds”. The present study was undertaken to investigate whether growth takes place at two ends of each fragment or only at one of the two ends. For this purpose, we used two kinds of flagella, having i - and 1,2 -antigens, respectively, together with anti-flagella sera, anti- i and anti- 1,2 . Monomers and seeds derived from i - and 1,2 -flagella were cross mixed to produce heterogeneous filaments or “block copolymers”, which, after treatment with either one of the two antisera, were observed in an electron microscope with negative staining. When a product of copolymerization was treated with anti- i , for example, a part of each filment containing i -flagellin was uniformly labelled with the antibody and could be distinguished from another unlabelled part composed only of 1,2 -flagellin. In a large number of the filaments observed, the great majority were of the form of i-1,2 ; filaments of the form of i-1,2-i or 1,2-i-1,2 , were never found. On the other hand, it was shown that the addition of a small amount of seed to a mixture of i - and 1,2 -monomers results in the formation of long flagellar filments, each of which is uniformly labelled with anti- i and anti- 1,2 , respectively. In view of thes observations, it was concluded that during reconstitution flagellar filaments grow in a unidirectional manner. Abram, Koffler & Vatter (Abst. 45, 2nd Int. Cong. Biophys., Vienna , 1966) have reported that when negatively stained fragments of flagella ( Bacillus ) were observed in an electron microscope, only their distal ends appeared to be frayed. It will be shown in this paper that this end corresponds to the end where in vitro growth takes place.

Polymorphism of Salmonella flagella as investigated by means of in vitro copolymerization of flagellins derived from various strains

Abstract At physiological ionic strength and pH, two kinds of monomeric flagellins derived from different Salmonella strains may copolymerize into long filaments which, like intact flagella, assume wave-forms in electron microscope observations. In this study we carried out quantitative and statistical analyses of wave-forms assumed by various kinds of copolymers, using two kinds of normally flagellated strains SJ670 (i) and SJ25 (n), a curly flagellar mutant SJ30 (c) and a straight flagellar mutant SJ814 (s). The wave-form of copolymer was represented in terms of the contour length of filament contained in one period, L, and the wave-height, h. Usually, homogeneous polymers of i- and n-flagellins assume the large-period normal wave-form and that of s-flagellin assumes the straight form. In combinations of (i+s) and (n+s), it was found that the wave-form of copolymer became smaller in both L and h as the ratio of mixed s-monomer increased. This change of wave-form was shown to occur in a stepwise manner: between the normal and straight forms three stable intermediates appeared, which are referred to as types II, III and IV, respectively. In each stable form copolymers (i+s) and (n+s) were closely similar with respect to both L and h. These copolymers thus assumed five common stable forms depending on the ratio of constituent flagellins. Homogeneous polymers of c-flagellin assumed two types of waves, referred to as II′ and III′. Waves II′ and III′ had approximately equal values of L to waves II and III, respectively, whilst the former two waves had a larger value of h than the latter two. In the investigation of copolymers (i+c) or (c+s), it was found that when the ratio of mixed c-monomer was gradually increased, the change of wave-form from type II to II′ or type III to III′ took place in a continuous manner. For this reason, the relationship between waves II and II′ or waves III and III′ is regarded as homologous. In order to understand the polymorphism of copolymers described here, it will be necessary to consider that homogeneous polymers themselves are polymorphic and, depending on environmental conditions, capable of assuming several stable forms homologous to those which have been associated with copolymers. From this point of view, the polymorphic nature of bacterial flagella will be discussed.

A Mutant of Salmonella Possessing Straight Flagella

SUMMARY: A mutant of Salmonella typhimurium produced straight flagella in phase 2 (antigen-1,2) and normal flagella in phase I (antigen-i). The straight flagella were observed by light microscopy and electron microscopy either with or without formalin fixation. Flagellar bundles of the mutant bacteria prepared in 0.25% methylcellulose (w/v) and examined by dark-field microscopy were also found to be straight. It was shown by electron microscopy that the component flagella of the straight flagellar bundle were in most instances irregularly twisted about each other. Heteromorphous bacteria which had straight flagella and either normal or mini-small-amplitude flagella were seen at a frequency of 10–13 % among the bacterial clones in phase 2. The bacteria with straight flagella were non-motile but they were sensitive to bacteriophage X, which is known to infect motile bacteria of Salmonella species. In transduction, using phage P22 grown on a normal flagellar strain and the phase 2 straight strain as recipient, transductional clones with normal flagella in both phase I and phase 2 were obtained. The transductional clones showed the antigen of the recipient in phase I and that of the donor in phase 2. This indicated that the straight mutant originated by a mutation of the structural gene of phase 2 flagellin. In absorption-agglutination experiments with antisera prepared against flagella of either normal-1,2 or straight-1,2 no antigenic difference between normal and straight flagella could be detected.

Flagella-shape mutants in Salmonella.

Summary: Seven flagella-shape mutants were isolated from a curly-flagella strain of Salmonella abortusequi. The motility in broth and spreading ability on semi-solid medium of these mutants as well as flagellar morphology were examined by dark field and electron microscopy. They were classified into the following five mutant types, heteromorphous, small-amplitude, para-curly, short and hooked-curly.nEach of these mutants manifests its characteristic flagellar shape and motility, and the spreading ability of these mutants on semi-solid medium decreases in the order: normal, heteromorphous, small-amplitude, para-curly, short, hooked-curly, curly.nExcept in the small-amplitude and short mutants, the shape of a flagellar bundle of living bacteria under the dark-field microscope corresponded in each mutant to a spiral of the flagella observed under the electron microscope. In the small-amplitude mutant, transconformation of the flagellar bundle from small-amplitude to curly was observed in organisms suspended in 0.5% (w/v) methylcellulose solution. In the short mutant, the flagellar bundle was not seen in the dark-field microscope.nDifference of antigenic specificities between the flagella of the parental curly and each of the mutants were not detected when examined by absorption-agglutination tests.nFrom transduction analyses with P 22 phage, it was found that the traits of each of the four mutants heteromorphous, small-amplitude, para-curly, short were transferred with the structural gene of phase-2 flagellin and had no effect on the flagellar shape and motility in phase-1. From this, it is inferred that the characteristic flagellar shape and motility of these mutants are primarily to be attributed to the conformation of the flagellin which composes the mutant flagella.

Genetical studies of non-flagellate mutants of Salmonella.

SUMMARY: Transductions were carried out with 22 non-flagellate mutants originating spontaneously from Salmonella typhimurium LT2. Taking the productions of swarm and trail as the criteria of recombination and complementation respectively, the mutants were classified into seven groups, namely flaA, flaB, flaC, flaD, flaF, flaJ and flaK. Each of them may correspond to a cistron. Among them, flaA, B, C, D and J were co-transduced with the structural gene of phase-1 flagellin, HI. Based on the frequency of recombination between two non-flagellate mutants, and that of HI-co-transduction when available, linearly arranged maps of mutant sites in flaA, flaC and flaF were constructed. Co-linearity was demonstrated between the recombination map and complementation map in flaA and flaC, but with some exceptions in flaF. Partial complementation was frequently observed in combination of two mutants in a cistron.nTransductions from thirteen non-flagellate mutants investigated by Joys & Stocker to the above mutants indicated that, among the five complementation groups assigned by them on their mutants, four correspond to our flaA, flaB, flaC and flaD; the remaining one, flaE, is missing in our mutants. Similarly, among eleven mutant sites of the non-flagellate strains of Salmonella abortusequi SL23, ten are distributed in flaA, flaF and flaC. The remaining one, assigned to a mutant of flaG, does not belong to any fla cistrons described above. The recovery of flagellation in the flaG strain by transduction was unsuccessful even when a flagellate strain was used as a donor.nRabbits were immunized with each of the twenty-one stable non-flagellate mutant clones, and their ability to elicit antiserum specific to flagellar protein was examined by absorption-agglutination test. Among the clones examined, only one belonging to flaG was found to elicit antiserum specific in flagellar antigen of the parental flagellate strain. It was inferred that the flaG mutant can synthesize flagellin but cannot construct flagellar fibres. The remaining fla mutants were presumed to be unable to synthesize flagellin.

In vitro synthesis of phase-specific flagellin of Salmonella.

Abstract Chromatography 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.

Genetics of Salmonella

The genetics of Salmonella can be traced back to the discoveries and descriptions of several remarkable phenomena of antigenic variation in this genus, namely O-H variation, S-R variation, form variation and phase variation (reviewed by Kauffmann, 1954).

Electron Microscopy of Salmonella Flagella in Methylcellulose Solution

SUMMARY: When peritrichously flagellated cells such as normal, curly, paralysed-curly and small-amplitude strains of Salmonella abortusequi are suspended in 0.5% methylcellulose solution, flagellar bundles can be clearly seen by electron microscopy. In a bundle, five or more of the component flagella are tightly united in parallel with each other, and the bundle formed into a helix with a shape characteristic for each strain. These figures reveal the structural detail of the flagellar bundle observed under a dark-field microscope. Ten minutes after the cells were suspended in methylcellulose solution, bundled flagella could be seen in approximately 70% of the cells of normal, curly and paralysed-curly strains; the remaining 30% were dispersed. At this time among the normal cells, some were single-bundled and others were multi-bundled. The fraction of single-bundled cells was larger in both motile and paralysed curly-flagellated cells than in normal cells. The fraction of normal cells having single bundles increased with time. Methylcellulose was therefore presumed to enhance aggregation of flagella and/or to inhibit the dispersion of the aggregated flagella.nIn the small-amplitude strain, transformation of flagellar shape to curly has been previously observed. In methylcellulose solution, this transformation occurs in bundled flagella but not in dispersed flagella. It is inferred that the tight association of the component flagella in methylcellulose solution enhances the stress among the flagella, thus causing the transformation.