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Salmonella flagellar filaments comprise the following distinct parts connected in series: a curved hook composed of a single kind of subunit (hook protein); two short segments made up of hook-associated proteins (HAP1 and HAP3); a long helical filament composed of flagellin; and a cap composed of HAP2. In this study, a procedure was developed to isolate HAPs from the culture medium of a short-flagella mutant. We demonstrate that hook-filament complex can be formed in vitro by sequential addition of HAP1, HAP3 and flagellin to hook fragments.

Helical transformations of reconstituted Salmonella flagella were visualized by dark-field light microscopy. Flagella from SJ670 strain were lefthanded helices with a pitch of 2.3 μm at neutral pH. When, however, the pH of the solution was lowered to 4.7, they were discontinouously transformed into close-coils with a pitch of 0.5 μm and a diameter of 1.2 μm, and a further lowering of the pH converted these coiled flagella into so-called curly ones, righthanded helices with a pitch of 1.1 μm. The transformation was rapid and reversible. Two other kinds of flagella (SJ25 and SJ30) also underwent such polymorphic conversions. Thus pH is an important factor in the control of flagellar transformation. As a result of the transformation, the degree of flow birefringence of a flagellar solution depends strongly on pH. Measurements of this parameter were useful in the study of the effects on the transformation of salt concentration and temperature.

The non-motile strain W3623 ha-177 of Escherichia coli (Kondoh & Ozeki, 1976) is known to produce straight flagella as a result of a mutation in the structural gene for the flagellin. Under physiological conditions, however, flagella of this mutant undergo straight-to-helical transformation with small changes of pH. Evidence for this came from dark-field light microscope observations of reconstituted flagella. At pH values lower than 6.6 in the presence of 0.1 m -NaCl, the flagella were straight. When, however, the pH was raised above 7.3, they were transformed into left-handed helices with a pitch of 2.05 μm. The transformation was rapid and reversible. In the pH range between 6.6 and 7.3, straight and transformed flagella co-existed but no stable forms other than the two were found. Bacterial motility also depended on the pH of the medium: at pH values above 7.0, bacteria swam by means of the transformed flagella. Therefore, helically transformed flagella of the mutant strain were similar in morphology and function to normal-type flagella of the parent strain. The significance of this similarity is discussed on the basis of general considerations of polymorphism in bacterial flagella.

We have found that several kinds of helical flagella from Salmonella and Escherichia become straight in the presence of 0·5 m-citric acid at pH values below 4·0, while the straight flagella from a mutant Salmonella (SJ814) are transformed into a helical shape under the same conditions. These transformations are reversible and transitional. Current models of bacterial flagella (Calladine, 1976,1978; Kamiya, 1976) predict that the family of distinct wave-forms should include two types of straight flagella, which have either an extreme right-handed twist (about 7 ° at the surface of the flagellum) or an extreme left-handed twist (2 ° to 3 °). As the inclination of the near-longitudinal rows of subunits in the Salmonella SJ814 flagellum (O'Brien & Bennett, 1972) agrees closely with the degree of twisting predicted for the right-handed type, this flagellum has been considered to be the right-handed type. We have determined that the basic (1-start) helix in flagella is right-handed, using the method of Finch (1972). This fact, together with the selection rule (O'Brien & Bennett, 1972), strongly suggests that the near-longitudinal rows in an SJ814 flagellum are right-handed, in agreement with the prediction. However, our optical diffraction and X-ray diffraction studies have revealed that the near-longitudinal rows of subunits in the citric acid-induced straight flagella and in the straight flagella from a mutant E. coli (Kondoh & Yanagida, 1975) tilt at an angle of 2 ° to 3 ° with respect to the flagellar axis. This inclination is probably left-handed. Thus the predicted presence of the two types of straight flagella seems to be proved.

The structure of actin bundles from internodal cells of Chara australis , an algal plant, was studied by electron microscopy of negatively stained specimens and optical diffraction. Gently prepared bundles revealed paracrystalline structures resembling the Mg 2+ -induced paracrystals of rabbit skeletal muscle actin (Hanson, 1968). In addition, the algal actin bundles sometimes had transverse striations at intervals of about 130 A, as has been observed in actin bundles from sea urchin eggs (DeRosier et al. , 1977; Spudich & Amos, 1979) and sea urchin coelomocytes (De Rosier & Edds, 1980; Otto & Bryan, 1981). This finding suggests that a common mechanism might be working in a variety of cells to organize actin filaments into functional bundles.

Reconstituted Salmonella flagella were found by dark field light microscopy to undergo reversible transformations at both acidic pH (Kamiya & Asakura, 1976) and alkaline pH. When the pH was increased from 7 to 12·5, flagella from strain SJ670 were rapidly transformed from a left-handed helix with a pitch of 2·3 μm (normal type) to a left-handed close-coil (coiled type) or to two types of right-handed helices with a pitch of 1·0 or 0·9 μm (curly I and curly II types). These helical structures were similar to those observed at acidic pH, suggesting the importance in flagellar transformation of the magnitude of net electric charge and/or the numbers of salt-bridges. Two other kinds of flagella (from strains SJ25 and SJ30) also underwent similar transformations.

Flagellar filaments isolated intact from a Salmonella short-flagella mutant are unable to serve as nuclei for flagellin polymerization in vitro, whereas the filaments reconstructed in vitro from the mutant flagellin are able to do so. The inability of intact flagella to nucleate flagellin polymerization appears to be common to wild-type bacteria and thus suggests that the tip of intact flagella are generally inactivated or capped in vivo. Careful observations of the tips of intact flagella and reconstructed flagellar filaments of a wild-type species have revealed marked difference between them: the intact flagella usually have blunt ends, whereas reconstructed filaments have concave, “fish-tail” ends. Moreover, a thin structure is often observed attaching to the very end of the intact flagella. We suspect that this “capping” structure is essential to the elongation mechanism of flagellar filaments.

Scanning microcalorimetric and circular dichroism studies of the normal and mutant flagellins of Salmonella suggest that they have a multidomain structure in common. Flagellin polymers (flagella) are depolymerized irreversibly into monomers as the temperature is raised, and the monomers undergo denaturation reversibly when cooled and heated again. The calorimetric enthalpy of this reversible process is twice as large as the van't Hoff enthalpy, suggesting that flagellin monomers contain two co-operative regions that melt independently at the same temperature. In all flagellin specimens examined, the ellipticity at the same temperature. In all flagellin specimens examined, the ellipticity at 222 nm of polymers at room temperature is 1.6 times as large as that of monomers, and the dependence of ellipticity on temperature takes place in the same temperature intervals in which calorimetric effects take place. From these results, we propose that flagellin molecules consist of several domains, two of which are distinctly structured in monomers at room temperature, while the others acquire more regular structures during polymerization.

Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH4)2SO4 (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.