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Formation of a flagella-like but straight polymer of Salmonella flagellin
J. Mol. Biol. (1974) 87, 65-62
Formation of a Flagella-like but Straight Polymer of Salmonella Flagellin RITSU KAMIYA AND SHO ASAKURA Institute of Molecdur Biology, Faculty of hience Nagoya University, Nagoya, Japan (Received 22 December 1973) Salrnor&a flag&n (monomer) polymerizes into flagellar filaments with the addition of (NH&SO4 (Ada et al., 1903; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high ooncentration of NaCl (about 1.6 M), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flag&ins derived from strains 55670, SJ25, 5530 and 55814. 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 fmgments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsucuessful. In O-16 M-N&~, 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 O-16 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 W&B concluded that in the two 6lamentous structures, flagellin molecules may be packed in different ways.
1. Introduction Salmonella flag&in (monomer) polymerizes into flagellar filaments with the addition of (NHI)$JOI at neutral pH and at room temperature (Ada et d., 1963; Wakabayashi et al., 1969). When this process was allowed to take place in the presence of a high concentration of NaCl, the product consisted of flagella-like filaments which, however, were straight. This paper reports the formation and partial characterization of this new type of flagellin polymer.
2. Materials and Methods (a) Preparakm of puri@d$4agell&a Table 1 shows four strains of Salmoaellu used in this study. The strains were supplied by Dr T. Iino (Laboratory of Genetics, Faculty of Science, University of Tokyo). In this paper, flagella or flagellins derived from strains SJ070, 5526, 5530 and 55814 will be 66
R. KAMIYA
66
APjD
S. ASAKURA
TABLE 1
Salmonella strains used in this study Abbreviation
i 12 c 8
Strain
SJ670 5526 5530 SJ814
Antigen
tYPe i 1,2 WbX
12
Flag&u shape Normal Normal Curly Straight
referred to as i, n, c and s, respectively, as in a previous paper (Asakura & Iino, 1972). Cultivation of the organisms of each strain and the isolation and purification of flagella were carried out by the method described previously (Asakura et al., 1964; Asakura & Iino, 1972). Purified flagella were suspended in 0.15 na-NaCl to give about 10 mg protein/ml and stored at 0°C. Solutions of monomeric flagellins were obtained by heating purified flagella solutions at 65°C for a few minutes, followed by centrifugation at 105,000 g for 1 h to remove heavy materials. (b) Electron microscope observations of straight Jilamenta Usually we obtained straight filaments by mixing the following solutions at appropriate volume ratios: a monomer solution, a concentrated NaCl solution and 2 M-(NH&SO, contained in 0.5 M-Tris*HCl (pH 9-O). Each mixture was left at room temperature for several hours for complete polymerization, and then diluted with a large volume of distilled water or 3 M-NaCl to give a concentration of about 0.1 mg protein/ml. A drop of the diluted solution was placed on a grid, the largest part of the drop was blotted and the grid was washed with a few drops of distilled water to remove salts. To observe overall shapes of filaments, 0.5% phosphotungstate (pH 7) was used as the negative stain. This stain, however, was inadequate for observing the substructure of filaments. For this purpose we used 1% uranyl acetate. The specimens were observed in a JEM T7 electron microscope. Straight filaments consisting of n-flagellin could be observed only when the sample solution was diluted with 3 M-NaCl, since these filaments were stable only at high ionic strengths. (c) Kinetics of depolymerktion by heat Straight filaments, formed in a solution containing high concentrations of NaCl and (NH&SO*, were sedimented by high-speed centrifugation and resuspended in a medium containing 0.16 M-N&CA and 0.01 M-phosphate buffer (pH 7.0) to give a concentration of about 3 mg protein/ml. Hereafter this medium will be called standard medium. The solution obtained was divided into several fractions, which were heated for various periods at a constant temperature, followed by high-speed centrifugation at a low temperature. The absorbance at 278 nm of the supernatant solution from each fraction was measured to determine the amount of flagella depolymerized during given periods of heat treatment. (d) Other methods Flagella and straight filaments were fragmented by sonication in a KSMlOO generator (Kubota, Tokyo). Polymerization was followed by viscosity measurements. An Ostwald type viscometer was used, which had a capacity of O-6 ml and a flow time of 15.4 s for water at 20°C. Straight filaments that were analogous to block copolymers consisting of two kinds of flagellin were prepared and examined by the antibody-labelling technique dsscribed by Asakure et al. (1968).
STRAIGHT
POLYMER
OF
FLAGELLIN
57
3. Results (a) Formation of straight jihnents The present study started from the electron microscope observation that when i-flagellin was polymerized by the method described by Wakabayashi et al. (l$Mg), the product contained a flagella-like but straight filament, though ita proportion of the total was always small (see Plate I(a)). To find the conditions required for the formation of the straight filament, we examined several factors: pH value, the concentration of added NaCl, temperature and the concentration of protein. Among these factors, only the concentration of NaCl was found to be important. Plate I(b) shows that when polymerization was brought about in the presence of 0.8 rd-NaCl and 0.5 M-(NH&SO, at room temperature, the product consisted mostly of straight filaments. Similar results were obtained when NaCl was replaced by KC1 or MgSO,. Therefore, the formation of straight filament is likely to be controlIed by ionic strength. Two roles are played by added (NH,),S04: one is to increase the ionic strength and the other is to initiate polymerization or iB initial process, nucleation. Later, it will be shown that (NH&SO4 is not necessarily required for the polymerization of flagellin into straight filaments. Straight filaments observed after complete polymerization became shorter, on the average, with an increasing concentration of added (NH&SO,. Presumably, this is due to the fact that the rate of nucleation increased more rapidly than the rate of the subsequent process, growth, as the final concentration of (NH&SO, was increased. The same phenomenon has been found for the formation of flagellar filaments in the absence of a high concentration of NaCl (Wakabayashi et al., 1969). It must be noted that solutions containing straight filaments at high ionic strengths could be distinguished, by gross observations, from solutions containing flagella at, the same ionic strengths, as only the former solutions exhibited strong, silky turbidity. This was true also for straight filaments prepared from n and c-flagellins (see below). In addition, straight filaments were often precipitated from the solutions when left at room temperature for long periods. Not only i-flagellin but also n and c-flagellins could be polymerized into straight filaments, though the ionic strengths required for n and c were higher than that required for i (Plates II and III). It must be noted at this point that, when polymerization was brought about at an intermediate ionic strength, the product was a mixture of flagella and straight filaments. (This feature is more clearly seen in Plate III than in Plate II.) For this reason, we may consider that flagella and straight filaments exist as different stable structures. Plate II shows that straight filaments formed in the presence of 2.5 M-NaCl were shorter, on the average, than those formed in the presence of 1.5 M-NaCl. In this connection, it was found that when the tw-o solutions were centrifuged at high speed, more than 90% of the total protein used was sedimented from each solution. This means that in both cases polymerization was nearly complete. These experimental results suggest that the ratio of the rate of nucleation to the rate of growth increased as the concentration of NaCl was increased from I.5 M to 2.5 Y. Depending on the concentration of added NaCl, s-flagellin polymerized into straight flagella or straight filaments on the addition of (NH,),SO,. In this case, however, the two types of filaments could not be distinguished by electron microscope observations. Later, it will be shown that the two types of filaments are distinguishable by their heat-stability.
68
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Plate IV shows electron micrographs of (a) i-flagella and (b) straight i-filaments at a relatively high magnification. These micrographs give an impression that the two types of filaments will have similar substructures, though detailed comparison has not been carried out. The bacterial flagellum exists as a tubule, the wall of which is constructed by a two-dimensional regular arrangement of flagellin molecules (Kerridge et al., 1962 ; Lowy & Hanson, 1965; Champness, 1971; O’Brien & Bennett, 1972; Finch & Klug, 1972). Presumably, the straight filament has a similar, tubular structure. It is well-known that when isolated flagella are negatively stained and observed by electron microscopy, one of the two ends of each flagellum assumes a fish-tail appearance (Abram, Kofl-ler & Vatter, 1966, Abet. 45,Znd Int. C’ong. Biophys., Vienna; Asakura et al., 1968; Bode et al., 1972; O’Brien & Bennett, 1972). A similar asymmetric appearance was frequently observed for straight filaments, when they were negatively stained with phosphotungstate. We have done experiments to determine whether straight filaments (consisting of i or c-flagellin) might be able to be transformed, without depolymerization, into normal or curly flagella under appropriate environmental conditions. Straight filaments formed in the presence of high concentrations of NaCl and (NH&SO, remained straight when the solution was left at 0 or 40°C for one day, or when it was left at room temperature after the addition of HCl or NaOH to bring the pH of the solution to about 55 or 95. Dialysis of the original solution against a large volume of 9.15 nz-NaCl or standard medium also brought about no change in the shape of the filaments. In parallel, attempts were made at direct transformation of normal or curly flagella into straight filaments : however, we also failed in this instance. Taking into account the negative results, it is unlikely that direct transformation between the two types of filamentous structures does occur. Straight filaments suspended in standard medium were depolymerized into monomeric flagellin by heating (see below), The monomer obtained was able t,o polymerize into flagella or straight filaments, depending on experimental conditions. In this respect, transformation is reversible. (b) Heat-stabilities of straight jiEaments in 0.15 M-NaCl Gerber et al. (1973) have shown, by viscosity measurements, that the heat-stabilities in 0.15 M-NaCl or standard medium of i, n and c-flagella were approximately the same. We observed that s-flagella have a similar or slightly lower heat-stability. On the other hand, it was found that straight filaments consisting of the four kinds of flagellins have markedly different heat-stabilities, all of which were lower than those of flagella. Figure 1 shows that among the three kinds of straight filaments (except n-filament), i-filaments were the most stable against heat and the order of heat-stability was i > c > s. Straight n-filaments, formed in the presence of 1.8 M-NaCl and O-65 M-(NH&SO,, seemed to depolymerize rapidly when the solution was diluted with several volumes of distilled water at room temperature or at 0°C. This filament is most unstable at low ionic strengths. (c) Polymerization initiated by seeding Monomeric flagellin dissolved in 0.15 M-Nacl at neutral pH polymerizes into flagella if the solution is seeded with fragments of flagella, or short flagella, prepared by sonic vibration (Asakura et al., 1964 ; Kuroda, 1972). We repeated this experiment using 3.5 M-NaCl. First it must be noted that, also at this concentration of NaCl,
PLATE I. Products of the polymerization of i-flapellin initiated with (NH,),SO, in the presence and absence of high concentration of’ Sa(‘l. One: vol. i-monomer solution containing 10 mg prot&n/ml, 2 vol. distilled w&or or a solution containing 1.5 M-NaCl and 0.5 M-Tris.HCl (pH 9.0) and 1 vol. solution containing 2 PI-(SH,)&), and 0.5 w-Tris.HCl (pH 9.0) were mixed, and the mist uw was kft at room trrnpwat~nw ftr 7 h for c~mqArtc polymorizat~ion. Final concns of salts: 0.5 nl-(KH,),SO, and 0.04 wXa(‘l (a): anal 0.8 M-S&C'1 (h). Negatively stained with 0.5”/, phorphotlmpsta~c at pH 7. Magnific%stiorr ~I.oOO Sot11 that straight filaments are found also in (a). Ifwiny
p. 5X
PLATE II. Products of the polymerization of n-flagellin initiated with (NH&SO4 in the presence of various concent,rations of NaCl. Two vol. n-monomer solution containing 10 mg protein/ml, 5 vol. solution containing an appropriate concn of NaCl and 0.5 iv-Tris.HCl (pH 9.0) and 4 vol. solution containing 2 M-(NH,),SO, and 0.5 M-Tris.HCl (pH 9.0) wore mixed, and the mixture was left, at room t,emperature for 6 h before observation. Final concns of NaCl were 0.03 M (a); 1 M (b); 1.5 M (c); and 2.5 M ((1). Negatively stained with 0.5:/, phosphotungst,atr at pH 7. Magnifiration, 14,000 x
I’LATE: III. I’roducts of t.hc polymerization of’c-Aagellin initiated with (NH,),HO, in the pr~w~~w~ of \ arinus coneentrat.ions of N&l. One vol. e-mononwr wlut.ion cont.aining 5.8 mg prI>tt?in/rui. 2 VIII. solution containing an appropriak concn of NaCl and 0.5 v-Tris~H(‘l (pH 9.0) and r’ x.01. 2 ~w(NH,)~SO~ and 0.5 or-Trix.HCl (pH 9.0) were mixed, ant1 thr mixture was left at room t(‘nllx:rnture for 7 h for complete polymerization. Final concns of NaCl wcr~~ 043 31 (a): 0.5 I\I (II): 141 \I (c); 1.5 11 (11). IV;c>gativcly stained with 0.5” o phl)sphr)tlUlestRtt’ nt pH 7. Xlay~riAcr~~ i<)rl. I1.lIOO
I'LATE 1V. High-magnification electron micrographs of rc:conatitut,ed (a) i-flagella and (b) straight i-filammts. Flagella were prepared by mixing i-monomer and fragment,s of i-flagella at a large protein rat,io in the presence of 0.15 M-NaCl and 0.01 M-phosphate buffer (pH 7.0) at room temperature (Asakura et al., 1966). Straight filaments were produced by spont,aneous polymerization of i-monomer in the presence of 0.5 M-(NH,),SO,, 1.0 M-NaCl and 0.13 x-Tris.HC!l (pH 9.0) at room temperature, and the product w&s dialysed against a large volume of standard medium (see Materials and Methods) to wmow KUX~HS salts. Negat)ively stainctl with 1 “/o urnnyl aortatr. Magnification. 140.000 Y
STRAIGHT
POLYMER
OF FLAGELLIN
5!,
-
0
.
0
60
120
0
IO 20 Time ( min)
0
5
IO
FIQ. 1. Heat-depolymerization of 3 kinds of straight filaments suspended in standard medium (see M8kCi8k3 and Methods). (&) Streight i-filament formed in a solution contining 3.0 mg imonomer/ml, 1.6 M-NeCl, 0.6 M-(NH&SO~ and 0.13 m-Tris.HCl (pH 9.0) W&B sedimented by high-speed centrifugation and resuspended in the same volume of standard medium. The suspension was heated at 43.6“C end the depolymerization brought about w&s followed by the method described in Materials end Methods (curve 1). At the same time, (short) i-flagella were suspended in standard medium to give 3.0 mg protein/ml and the suspension W&s heated at 43.6°C to follow depolymerization of the flagella (curve 2). In each series of experimental points, the last one was obtclined when a part of the original suepension w&5 heated 8t 66°C for 3 min for complete depolymerization: therefore, it denotes the total conoentration of protein contained in the suspension. (b) Straight c-filament formed in a solution containing 2.3 mg c-monomer/ml, 1.6 M-N&~, 0.8 M-(NH&SO, and 0.2 M-Tris.HCl (pH 9.0) was sedimented by high-speed centrifugation and resuspended in one half of the origin81 volume of standard medium. The suspension W&S heated at 4O.O”C and the depolymerization brought about Was followed (curve 1). In parallel, (short) c-flagella were suspended in at8ndard medium to give 3.9 mg protein/ml and the suspension was heated at the same temperature (curve 2). (c) Straight s-filament formed in a solution containing 2.9 mg s-monomer/ml, 2.3 M-N&I, 0.7 M-(NH&SO., and 0.17 M-Tris.HCl (pH 9.0) was sedimented by high-speed centrifugrttion and resuspended in the sa.me volume of standard medium. Depolymerization brought about in this suspension at 4O*O”C wm followed (curve 1). In parallel, (short) s-flagella were suspended in standard medium to give 1.6 mg protein/ml and the solution was heated at the same temperature (curve 2). The 3 kinds of straight filaments used in this experiment had different average lengths. Since the rate of depolymerization, measured by the present method, must depend on the concentradirectly tion of filaments, rates of depolymerization for the 3 kinds of streight filaments CaMOh be oompared using the present data. Qualitatively, however, it may be considered that the order of heat-stability was i > c > 8. The flagella used in this experiment had been shortened by sonic vibration. Therefore, straight filaments were much longer than the flagella. It must be noted that, even under these conditions, str8ight filaments depolymerized more rapidly than flagella.
neither spontaneous polymerization of monomer nor head-to-tail association of short flagella into longer ones took place at least for several hours. Curve 1 in Figure 2(a) shows that, also at 3.5 M-NaCl, polymerization was initiated by seeding. Electron microscope observations of the product showed that it consisted of normally shaped flagella, which were indistinguishable in shape from those produced at 0.15 M-NaCl. Asakura & Iino (1972) have reported that in the presence of 0.15 M-NaCl, short sflagella initiate polymerization of the i-monomer into normally shaped flagella, We obtained the same result when the concentration of added NaCl was 3.5 M. In a separate experiment, i-monomers were mixed with fragments of straight i-filaments in the presence of 0.15 M and 3.5 M-&&l, respectively (Fig. 2(b)). In O-15 M-&Cl,
60
R. KAMIYA
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_ (b)
/“y---“-
0/
/ P -.-.-.-
2
J
Time (mini
FIG. 2. Seed-inititlted polymerization of the i-monomer at high and low ionic strengths. (e) A solution of purified i-flagella was sonioated and the product WBB sedimented by high-speed centrifugetion and redissolved in standard medium (see Materiels end Methods) to give 6-O mg protein/ml. At 2VC, this solution was mixed with 9 vol. i-monomer solution oontaining 3.0 mg protein/ml and 3-Q M or 0.16 M-N&I. Thus, the mixed monomer end seed were 4.6: 1 (protein r8tiO) and the total concentration of protein was 3.3 mg/ml. The polymerization initiated in eaoh solution was followed by viscosity measurements. The ordinate denotes specific visoosity (TV&, and the abcissa denotes time after mixing monomer and seed. Final oonons of N&l were 3.6 M (curve 1) and 0.16 M (ourve 2). Note that at both smlt canons, fragments of i-flagella aoted as seeds for polymerization of the i-monomer. It was shown by eleotron miaroacopy that both polymerizations geve rise to norm81 shaped flagella. (b) A similar experiment was carried out, using fragments of straight i-filaments w seeds. In this case, straight i-filamenta formed in the presence of l-6 M-N&~, O-6 M-(NH&SO~ end O-13 M-Tria*HCl (pH 9.0) were used for the prepamtion of seed. Other experimental oouditions were the seme as desoribed in (8). Final concns of N&l were 3.6 M (ourve 1) and 0.16 M (ourve 2), respeotively. Note that when the conon of N&l was O-16 M, the i-monomer wes uneble to polymerize in the presenoe of fragments of straight i-Elements, while in the 3-6 M-NeCl solution, polymerization took plaoe repidly. The product of this polymerization consisted of straight filaments that were appreciably longer than those used as seeds.
This means that straight fragments were unable to initiate polymerization. i-filaments and s-flagella, though they are morphologically quite similar, could be distinguished. When, however, the concentration of NaCl was increased to 3.6 M, fragments of straight i-filaments were able to initiate polymerization. Electron microscopy revealed that the product consisted of straight filaments. As in the case of short flagella, short fragments of straight filaments did not join each other in head-to-tail fashion. Thus, in the presence of 3.6 M-NaCl, i-flagellin was able to polymerize into normal-type flagella and straight filaments, depending on the nature of the added seeds: the polymerization is analogous to dimorphic crystallization. It has been shown that, in the presence of O-15 M-NaCl, monomers and fragments of flagella derived from four strains listed in Table 1 can interact in any combination to produce flagella, analogous to block copolymers (Asakura et a-!., 1966; Asakura & Iino, 1972). In this study, we found that in the presence of 3.5 M-NaCl, the four kinds of monomers and fragments of straight filaments interact, in any combination, to give rise to straight Laments. Fragments of i-flaments were added to the c-monomer to initiate polymerization and the product was observed by electron microscopy after treatment with anti-i-flagella antiserum. The result is shown in Plate V. It is seen that each flament was straight and had an antibody-labelled the mixed
STRAIGHT
POLYMER
OF FLAGELLIN
61
part, corresponding to the incorporated seed, at one of the two ends. This means that during polymerization, each seed grew only in one direction. The same result has been obtained for growth of flagella in vitro (Asakura et al., 1968).
4. Discussion It has been shown that in the presence of high concentrations of NaCl or at high ionic strength, four kinds of Salmonella flagellins polymerize into flagella-like, but straight filaments with the addition of (NH&SO, or seeds. Taking into account the fact that different kinds of flagellins could be copolymerized into a single straight filament, it is likely that a common intermolecular arrangement is used for building straight filaments from the four kinds of flagellins. We have not carried out detailed structural comparison between flagella and straight filamants by high-resolution electron microscopy. However, it can be presumed that the two types of filaments have different intermolecular structures, in the light of the following experimental results. (1) Attempts unsuccessful.
at direct transformation
between the two types of filaments were
(2) Fragments of flagella (or straight filaments) were ineffective as seeds for the formation of straight filaments (or flagella). (3) At low ionic strength, straight flagella.
filaments were less stable against heat t,han
The bacterial flagellum exists as a tubular structure. Recently, O’Brien & Bennett (1972) and Finch & Klug (1972) have shown, by high-resolution electron microscopy and optical diffraction and filtering that, in Salmonella flagella, the walls of the tubule are constructed from the 11 strands of globular subunits that run approximately in the longitudinal direction. Presumably, the straight filament investigated in this study also has a similar, tubular structure. It will be of interest to determine the number of longitudinal strands of subunits involved in this tubular structure. The number might be other than 11. We examined the circular dichroism of straight i-filament suspended in standard medium, in comparison with that of i-flagella (Uratani et al., 1972). In the range of wavelengths between 299 and 250 nm, circular dichroism spectra from the two types of filament were very similar in shape and amplitude. Therefore, it is likely that flagellin molecules incorporated in the two filamentous structures have similar conformations. Flagellin polymerizes into a variety of (meta-)&able structures, which may or may not be morphologically similar to flagella (Abram & Koffler, 1964; Hotani, 1971; Asakura & Iino, 1972). Kagawa & Asakura (unpublished results, quoted in Asakura, 1970) have observed that when i-flagellin was polymerized at low temperature by the addition of (NH&SO, in the absence of a high concentration of NaC1, the product consisted of straight filaments that were morphologically quite similar to those described in this paper. However, the straight filaments formed at low temperature transformed into curved shapes (probably corresponding to the shape of normal flagella) on heating at 37°C for a few minutes, before or after replacing the medium with 0.16 na-NaCl. Therefore, the straight filament observed by Kagawa & Asakura seems to be different in structure from that described in this paper.
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REFERENCES Abram, D. & Koffler, H. (1964). J. Mol. Biol. 9, 168-185. Ada, G. L., Nossal, C. J. V., Pye, J. & Abbot, A. (1963). Nature (London), 199, 1257-1262. Asakura, S. (1970). Adwan. Biophys. (Japan), 1, 99-155. Asakura, S. & Iino, T. (1972). J. Mol. Biol. 64, 251-268. Asakura, S., Eguchi, G. & Iino, T. (1964). J. Mol. BioZ. 10, 42-56. Asakura, S., Eguchi, G. & Iino, T. (1966). J. Mol. Biol. 16, 302-316. Asakura, S., Eguchi, G. & Iino, T. (1968). J. Mol. BioZ. 35, 227-236. Bode, W., Engel, J. & Windlmair, D. (1972). Eur. J. Biochewa. 26, 313-327. Champness, J. N. (1971). J. Mol. BioZ. 56, 295-310. Finch, J. T. & Klug, A. (1972). In The Generation of SubceZZuZarStructurea (Markham, R. & Bancroft, J. B., eds), pp. 167-177, North Holland Publishing Co., Amsterdam. Gerber, B. R., Asakura, S. & Oosawa, F. (1973). J. Mol. BioZ. 74, 467-487. Hotani, H. (1971). J. Mol. BioZ. 57, 575-587. Kerridge, D., Horne, R. W. & Glauert, A. M. (1962). J. Mol. BioZ. 4, 227-238. Kuroda, H. (1972). Biochim. Biophys. Act%, 285, 253-267. Lowy, J. & Hanson, J. (1965). J. Mol. BioZ. 11, 293-313. O’Brien, E. J. & Bennett, P. M. (1972). J. Mol. BioZ. 70, 133-152. Uratani, Y., Asakura, S. & Imahori, K. (1972). J. Mol. BioZ. 67, 85-98. Wakabayashi, K., Hotani, H. & Asakura, S. (1969). Biochim. Biophys. A&, 175, 195-203.
Formation of a Flagella-like but Straight Polymer of Salmonella Flagellin RITSU KAMIYA AND SHO ASAKURA Institute of Molecdur Biology, Faculty of hience Nagoya University, Nagoya, Japan (Received 22 December 1973) Salrnor&a flag&n (monomer) polymerizes into flagellar filaments with the addition of (NH&SO4 (Ada et al., 1903; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high ooncentration of NaCl (about 1.6 M), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flag&ins derived from strains 55670, SJ25, 5530 and 55814. 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 fmgments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsucuessful. In O-16 M-N&~, 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 O-16 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 W&B concluded that in the two 6lamentous structures, flagellin molecules may be packed in different ways.
1. Introduction Salmonella flag&in (monomer) polymerizes into flagellar filaments with the addition of (NHI)$JOI at neutral pH and at room temperature (Ada et d., 1963; Wakabayashi et al., 1969). When this process was allowed to take place in the presence of a high concentration of NaCl, the product consisted of flagella-like filaments which, however, were straight. This paper reports the formation and partial characterization of this new type of flagellin polymer.
2. Materials and Methods (a) Preparakm of puri@d$4agell&a Table 1 shows four strains of Salmoaellu used in this study. The strains were supplied by Dr T. Iino (Laboratory of Genetics, Faculty of Science, University of Tokyo). In this paper, flagella or flagellins derived from strains SJ070, 5526, 5530 and 55814 will be 66
R. KAMIYA
66
APjD
S. ASAKURA
TABLE 1
Salmonella strains used in this study Abbreviation
i 12 c 8
Strain
SJ670 5526 5530 SJ814
Antigen
tYPe i 1,2 WbX
12
Flag&u shape Normal Normal Curly Straight
referred to as i, n, c and s, respectively, as in a previous paper (Asakura & Iino, 1972). Cultivation of the organisms of each strain and the isolation and purification of flagella were carried out by the method described previously (Asakura et al., 1964; Asakura & Iino, 1972). Purified flagella were suspended in 0.15 na-NaCl to give about 10 mg protein/ml and stored at 0°C. Solutions of monomeric flagellins were obtained by heating purified flagella solutions at 65°C for a few minutes, followed by centrifugation at 105,000 g for 1 h to remove heavy materials. (b) Electron microscope observations of straight Jilamenta Usually we obtained straight filaments by mixing the following solutions at appropriate volume ratios: a monomer solution, a concentrated NaCl solution and 2 M-(NH&SO, contained in 0.5 M-Tris*HCl (pH 9-O). Each mixture was left at room temperature for several hours for complete polymerization, and then diluted with a large volume of distilled water or 3 M-NaCl to give a concentration of about 0.1 mg protein/ml. A drop of the diluted solution was placed on a grid, the largest part of the drop was blotted and the grid was washed with a few drops of distilled water to remove salts. To observe overall shapes of filaments, 0.5% phosphotungstate (pH 7) was used as the negative stain. This stain, however, was inadequate for observing the substructure of filaments. For this purpose we used 1% uranyl acetate. The specimens were observed in a JEM T7 electron microscope. Straight filaments consisting of n-flagellin could be observed only when the sample solution was diluted with 3 M-NaCl, since these filaments were stable only at high ionic strengths. (c) Kinetics of depolymerktion by heat Straight filaments, formed in a solution containing high concentrations of NaCl and (NH&SO*, were sedimented by high-speed centrifugation and resuspended in a medium containing 0.16 M-N&CA and 0.01 M-phosphate buffer (pH 7.0) to give a concentration of about 3 mg protein/ml. Hereafter this medium will be called standard medium. The solution obtained was divided into several fractions, which were heated for various periods at a constant temperature, followed by high-speed centrifugation at a low temperature. The absorbance at 278 nm of the supernatant solution from each fraction was measured to determine the amount of flagella depolymerized during given periods of heat treatment. (d) Other methods Flagella and straight filaments were fragmented by sonication in a KSMlOO generator (Kubota, Tokyo). Polymerization was followed by viscosity measurements. An Ostwald type viscometer was used, which had a capacity of O-6 ml and a flow time of 15.4 s for water at 20°C. Straight filaments that were analogous to block copolymers consisting of two kinds of flagellin were prepared and examined by the antibody-labelling technique dsscribed by Asakure et al. (1968).
STRAIGHT
POLYMER
OF
FLAGELLIN
57
3. Results (a) Formation of straight jihnents The present study started from the electron microscope observation that when i-flagellin was polymerized by the method described by Wakabayashi et al. (l$Mg), the product contained a flagella-like but straight filament, though ita proportion of the total was always small (see Plate I(a)). To find the conditions required for the formation of the straight filament, we examined several factors: pH value, the concentration of added NaCl, temperature and the concentration of protein. Among these factors, only the concentration of NaCl was found to be important. Plate I(b) shows that when polymerization was brought about in the presence of 0.8 rd-NaCl and 0.5 M-(NH&SO, at room temperature, the product consisted mostly of straight filaments. Similar results were obtained when NaCl was replaced by KC1 or MgSO,. Therefore, the formation of straight filament is likely to be controlIed by ionic strength. Two roles are played by added (NH,),S04: one is to increase the ionic strength and the other is to initiate polymerization or iB initial process, nucleation. Later, it will be shown that (NH&SO4 is not necessarily required for the polymerization of flagellin into straight filaments. Straight filaments observed after complete polymerization became shorter, on the average, with an increasing concentration of added (NH&SO,. Presumably, this is due to the fact that the rate of nucleation increased more rapidly than the rate of the subsequent process, growth, as the final concentration of (NH&SO, was increased. The same phenomenon has been found for the formation of flagellar filaments in the absence of a high concentration of NaCl (Wakabayashi et al., 1969). It must be noted that solutions containing straight filaments at high ionic strengths could be distinguished, by gross observations, from solutions containing flagella at, the same ionic strengths, as only the former solutions exhibited strong, silky turbidity. This was true also for straight filaments prepared from n and c-flagellins (see below). In addition, straight filaments were often precipitated from the solutions when left at room temperature for long periods. Not only i-flagellin but also n and c-flagellins could be polymerized into straight filaments, though the ionic strengths required for n and c were higher than that required for i (Plates II and III). It must be noted at this point that, when polymerization was brought about at an intermediate ionic strength, the product was a mixture of flagella and straight filaments. (This feature is more clearly seen in Plate III than in Plate II.) For this reason, we may consider that flagella and straight filaments exist as different stable structures. Plate II shows that straight filaments formed in the presence of 2.5 M-NaCl were shorter, on the average, than those formed in the presence of 1.5 M-NaCl. In this connection, it was found that when the tw-o solutions were centrifuged at high speed, more than 90% of the total protein used was sedimented from each solution. This means that in both cases polymerization was nearly complete. These experimental results suggest that the ratio of the rate of nucleation to the rate of growth increased as the concentration of NaCl was increased from I.5 M to 2.5 Y. Depending on the concentration of added NaCl, s-flagellin polymerized into straight flagella or straight filaments on the addition of (NH,),SO,. In this case, however, the two types of filaments could not be distinguished by electron microscope observations. Later, it will be shown that the two types of filaments are distinguishable by their heat-stability.
68
It. KAMIYA
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Plate IV shows electron micrographs of (a) i-flagella and (b) straight i-filaments at a relatively high magnification. These micrographs give an impression that the two types of filaments will have similar substructures, though detailed comparison has not been carried out. The bacterial flagellum exists as a tubule, the wall of which is constructed by a two-dimensional regular arrangement of flagellin molecules (Kerridge et al., 1962 ; Lowy & Hanson, 1965; Champness, 1971; O’Brien & Bennett, 1972; Finch & Klug, 1972). Presumably, the straight filament has a similar, tubular structure. It is well-known that when isolated flagella are negatively stained and observed by electron microscopy, one of the two ends of each flagellum assumes a fish-tail appearance (Abram, Kofl-ler & Vatter, 1966, Abet. 45,Znd Int. C’ong. Biophys., Vienna; Asakura et al., 1968; Bode et al., 1972; O’Brien & Bennett, 1972). A similar asymmetric appearance was frequently observed for straight filaments, when they were negatively stained with phosphotungstate. We have done experiments to determine whether straight filaments (consisting of i or c-flagellin) might be able to be transformed, without depolymerization, into normal or curly flagella under appropriate environmental conditions. Straight filaments formed in the presence of high concentrations of NaCl and (NH&SO, remained straight when the solution was left at 0 or 40°C for one day, or when it was left at room temperature after the addition of HCl or NaOH to bring the pH of the solution to about 55 or 95. Dialysis of the original solution against a large volume of 9.15 nz-NaCl or standard medium also brought about no change in the shape of the filaments. In parallel, attempts were made at direct transformation of normal or curly flagella into straight filaments : however, we also failed in this instance. Taking into account the negative results, it is unlikely that direct transformation between the two types of filamentous structures does occur. Straight filaments suspended in standard medium were depolymerized into monomeric flagellin by heating (see below), The monomer obtained was able t,o polymerize into flagella or straight filaments, depending on experimental conditions. In this respect, transformation is reversible. (b) Heat-stabilities of straight jiEaments in 0.15 M-NaCl Gerber et al. (1973) have shown, by viscosity measurements, that the heat-stabilities in 0.15 M-NaCl or standard medium of i, n and c-flagella were approximately the same. We observed that s-flagella have a similar or slightly lower heat-stability. On the other hand, it was found that straight filaments consisting of the four kinds of flagellins have markedly different heat-stabilities, all of which were lower than those of flagella. Figure 1 shows that among the three kinds of straight filaments (except n-filament), i-filaments were the most stable against heat and the order of heat-stability was i > c > s. Straight n-filaments, formed in the presence of 1.8 M-NaCl and O-65 M-(NH&SO,, seemed to depolymerize rapidly when the solution was diluted with several volumes of distilled water at room temperature or at 0°C. This filament is most unstable at low ionic strengths. (c) Polymerization initiated by seeding Monomeric flagellin dissolved in 0.15 M-Nacl at neutral pH polymerizes into flagella if the solution is seeded with fragments of flagella, or short flagella, prepared by sonic vibration (Asakura et al., 1964 ; Kuroda, 1972). We repeated this experiment using 3.5 M-NaCl. First it must be noted that, also at this concentration of NaCl,
PLATE I. Products of the polymerization of i-flapellin initiated with (NH,),SO, in the presence and absence of high concentration of’ Sa(‘l. One: vol. i-monomer solution containing 10 mg prot&n/ml, 2 vol. distilled w&or or a solution containing 1.5 M-NaCl and 0.5 M-Tris.HCl (pH 9.0) and 1 vol. solution containing 2 PI-(SH,)&), and 0.5 w-Tris.HCl (pH 9.0) were mixed, and the mist uw was kft at room trrnpwat~nw ftr 7 h for c~mqArtc polymorizat~ion. Final concns of salts: 0.5 nl-(KH,),SO, and 0.04 wXa(‘l (a): anal 0.8 M-S&C'1 (h). Negatively stained with 0.5”/, phorphotlmpsta~c at pH 7. Magnific%stiorr ~I.oOO Sot11 that straight filaments are found also in (a). Ifwiny
p. 5X
PLATE II. Products of the polymerization of n-flagellin initiated with (NH&SO4 in the presence of various concent,rations of NaCl. Two vol. n-monomer solution containing 10 mg protein/ml, 5 vol. solution containing an appropriate concn of NaCl and 0.5 iv-Tris.HCl (pH 9.0) and 4 vol. solution containing 2 M-(NH,),SO, and 0.5 M-Tris.HCl (pH 9.0) wore mixed, and the mixture was left, at room t,emperature for 6 h before observation. Final concns of NaCl were 0.03 M (a); 1 M (b); 1.5 M (c); and 2.5 M ((1). Negatively stained with 0.5:/, phosphotungst,atr at pH 7. Magnifiration, 14,000 x
I’LATE: III. I’roducts of t.hc polymerization of’c-Aagellin initiated with (NH,),HO, in the pr~w~~w~ of \ arinus coneentrat.ions of N&l. One vol. e-mononwr wlut.ion cont.aining 5.8 mg prI>tt?in/rui. 2 VIII. solution containing an appropriak concn of NaCl and 0.5 v-Tris~H(‘l (pH 9.0) and r’ x.01. 2 ~w(NH,)~SO~ and 0.5 or-Trix.HCl (pH 9.0) were mixed, ant1 thr mixture was left at room t(‘nllx:rnture for 7 h for complete polymerization. Final concns of NaCl wcr~~ 043 31 (a): 0.5 I\I (II): 141 \I (c); 1.5 11 (11). IV;c>gativcly stained with 0.5” o phl)sphr)tlUlestRtt’ nt pH 7. Xlay~riAcr~~ i<)rl. I1.lIOO
I'LATE 1V. High-magnification electron micrographs of rc:conatitut,ed (a) i-flagella and (b) straight i-filammts. Flagella were prepared by mixing i-monomer and fragment,s of i-flagella at a large protein rat,io in the presence of 0.15 M-NaCl and 0.01 M-phosphate buffer (pH 7.0) at room temperature (Asakura et al., 1966). Straight filaments were produced by spont,aneous polymerization of i-monomer in the presence of 0.5 M-(NH,),SO,, 1.0 M-NaCl and 0.13 x-Tris.HC!l (pH 9.0) at room temperature, and the product w&s dialysed against a large volume of standard medium (see Materials and Methods) to wmow KUX~HS salts. Negat)ively stainctl with 1 “/o urnnyl aortatr. Magnification. 140.000 Y
STRAIGHT
POLYMER
OF FLAGELLIN
5!,
-
0
.
0
60
120
0
IO 20 Time ( min)
0
5
IO
FIQ. 1. Heat-depolymerization of 3 kinds of straight filaments suspended in standard medium (see M8kCi8k3 and Methods). (&) Streight i-filament formed in a solution contining 3.0 mg imonomer/ml, 1.6 M-NeCl, 0.6 M-(NH&SO~ and 0.13 m-Tris.HCl (pH 9.0) W&B sedimented by high-speed centrifugation and resuspended in the same volume of standard medium. The suspension was heated at 43.6“C end the depolymerization brought about w&s followed by the method described in Materials end Methods (curve 1). At the same time, (short) i-flagella were suspended in standard medium to give 3.0 mg protein/ml and the suspension W&s heated at 43.6°C to follow depolymerization of the flagella (curve 2). In each series of experimental points, the last one was obtclined when a part of the original suepension w&5 heated 8t 66°C for 3 min for complete depolymerization: therefore, it denotes the total conoentration of protein contained in the suspension. (b) Straight c-filament formed in a solution containing 2.3 mg c-monomer/ml, 1.6 M-N&~, 0.8 M-(NH&SO, and 0.2 M-Tris.HCl (pH 9.0) was sedimented by high-speed centrifugation and resuspended in one half of the origin81 volume of standard medium. The suspension W&S heated at 4O.O”C and the depolymerization brought about Was followed (curve 1). In parallel, (short) c-flagella were suspended in at8ndard medium to give 3.9 mg protein/ml and the suspension was heated at the same temperature (curve 2). (c) Straight s-filament formed in a solution containing 2.9 mg s-monomer/ml, 2.3 M-N&I, 0.7 M-(NH&SO., and 0.17 M-Tris.HCl (pH 9.0) was sedimented by high-speed centrifugrttion and resuspended in the sa.me volume of standard medium. Depolymerization brought about in this suspension at 4O*O”C wm followed (curve 1). In parallel, (short) s-flagella were suspended in standard medium to give 1.6 mg protein/ml and the solution was heated at the same temperature (curve 2). The 3 kinds of straight filaments used in this experiment had different average lengths. Since the rate of depolymerization, measured by the present method, must depend on the concentradirectly tion of filaments, rates of depolymerization for the 3 kinds of streight filaments CaMOh be oompared using the present data. Qualitatively, however, it may be considered that the order of heat-stability was i > c > 8. The flagella used in this experiment had been shortened by sonic vibration. Therefore, straight filaments were much longer than the flagella. It must be noted that, even under these conditions, str8ight filaments depolymerized more rapidly than flagella.
neither spontaneous polymerization of monomer nor head-to-tail association of short flagella into longer ones took place at least for several hours. Curve 1 in Figure 2(a) shows that, also at 3.5 M-NaCl, polymerization was initiated by seeding. Electron microscope observations of the product showed that it consisted of normally shaped flagella, which were indistinguishable in shape from those produced at 0.15 M-NaCl. Asakura & Iino (1972) have reported that in the presence of 0.15 M-NaCl, short sflagella initiate polymerization of the i-monomer into normally shaped flagella, We obtained the same result when the concentration of added NaCl was 3.5 M. In a separate experiment, i-monomers were mixed with fragments of straight i-filaments in the presence of 0.15 M and 3.5 M-&&l, respectively (Fig. 2(b)). In O-15 M-&Cl,
60
R. KAMIYA
AND
S. ASAKURA
_ (b)
/“y---“-
0/
/ P -.-.-.-
2
J
Time (mini
FIG. 2. Seed-inititlted polymerization of the i-monomer at high and low ionic strengths. (e) A solution of purified i-flagella was sonioated and the product WBB sedimented by high-speed centrifugetion and redissolved in standard medium (see Materiels end Methods) to give 6-O mg protein/ml. At 2VC, this solution was mixed with 9 vol. i-monomer solution oontaining 3.0 mg protein/ml and 3-Q M or 0.16 M-N&I. Thus, the mixed monomer end seed were 4.6: 1 (protein r8tiO) and the total concentration of protein was 3.3 mg/ml. The polymerization initiated in eaoh solution was followed by viscosity measurements. The ordinate denotes specific visoosity (TV&, and the abcissa denotes time after mixing monomer and seed. Final oonons of N&l were 3.6 M (curve 1) and 0.16 M (ourve 2). Note that at both smlt canons, fragments of i-flagella aoted as seeds for polymerization of the i-monomer. It was shown by eleotron miaroacopy that both polymerizations geve rise to norm81 shaped flagella. (b) A similar experiment was carried out, using fragments of straight i-filaments w seeds. In this case, straight i-filamenta formed in the presence of l-6 M-N&~, O-6 M-(NH&SO~ end O-13 M-Tria*HCl (pH 9.0) were used for the prepamtion of seed. Other experimental oouditions were the seme as desoribed in (8). Final concns of N&l were 3.6 M (ourve 1) and 0.16 M (ourve 2), respeotively. Note that when the conon of N&l was O-16 M, the i-monomer wes uneble to polymerize in the presenoe of fragments of straight i-Elements, while in the 3-6 M-NeCl solution, polymerization took plaoe repidly. The product of this polymerization consisted of straight filaments that were appreciably longer than those used as seeds.
This means that straight fragments were unable to initiate polymerization. i-filaments and s-flagella, though they are morphologically quite similar, could be distinguished. When, however, the concentration of NaCl was increased to 3.6 M, fragments of straight i-filaments were able to initiate polymerization. Electron microscopy revealed that the product consisted of straight filaments. As in the case of short flagella, short fragments of straight filaments did not join each other in head-to-tail fashion. Thus, in the presence of 3.6 M-NaCl, i-flagellin was able to polymerize into normal-type flagella and straight filaments, depending on the nature of the added seeds: the polymerization is analogous to dimorphic crystallization. It has been shown that, in the presence of O-15 M-NaCl, monomers and fragments of flagella derived from four strains listed in Table 1 can interact in any combination to produce flagella, analogous to block copolymers (Asakura et a-!., 1966; Asakura & Iino, 1972). In this study, we found that in the presence of 3.5 M-NaCl, the four kinds of monomers and fragments of straight filaments interact, in any combination, to give rise to straight Laments. Fragments of i-flaments were added to the c-monomer to initiate polymerization and the product was observed by electron microscopy after treatment with anti-i-flagella antiserum. The result is shown in Plate V. It is seen that each flament was straight and had an antibody-labelled the mixed
STRAIGHT
POLYMER
OF FLAGELLIN
61
part, corresponding to the incorporated seed, at one of the two ends. This means that during polymerization, each seed grew only in one direction. The same result has been obtained for growth of flagella in vitro (Asakura et al., 1968).
4. Discussion It has been shown that in the presence of high concentrations of NaCl or at high ionic strength, four kinds of Salmonella flagellins polymerize into flagella-like, but straight filaments with the addition of (NH&SO, or seeds. Taking into account the fact that different kinds of flagellins could be copolymerized into a single straight filament, it is likely that a common intermolecular arrangement is used for building straight filaments from the four kinds of flagellins. We have not carried out detailed structural comparison between flagella and straight filamants by high-resolution electron microscopy. However, it can be presumed that the two types of filaments have different intermolecular structures, in the light of the following experimental results. (1) Attempts unsuccessful.
at direct transformation
between the two types of filaments were
(2) Fragments of flagella (or straight filaments) were ineffective as seeds for the formation of straight filaments (or flagella). (3) At low ionic strength, straight flagella.
filaments were less stable against heat t,han
The bacterial flagellum exists as a tubular structure. Recently, O’Brien & Bennett (1972) and Finch & Klug (1972) have shown, by high-resolution electron microscopy and optical diffraction and filtering that, in Salmonella flagella, the walls of the tubule are constructed from the 11 strands of globular subunits that run approximately in the longitudinal direction. Presumably, the straight filament investigated in this study also has a similar, tubular structure. It will be of interest to determine the number of longitudinal strands of subunits involved in this tubular structure. The number might be other than 11. We examined the circular dichroism of straight i-filament suspended in standard medium, in comparison with that of i-flagella (Uratani et al., 1972). In the range of wavelengths between 299 and 250 nm, circular dichroism spectra from the two types of filament were very similar in shape and amplitude. Therefore, it is likely that flagellin molecules incorporated in the two filamentous structures have similar conformations. Flagellin polymerizes into a variety of (meta-)&able structures, which may or may not be morphologically similar to flagella (Abram & Koffler, 1964; Hotani, 1971; Asakura & Iino, 1972). Kagawa & Asakura (unpublished results, quoted in Asakura, 1970) have observed that when i-flagellin was polymerized at low temperature by the addition of (NH&SO, in the absence of a high concentration of NaC1, the product consisted of straight filaments that were morphologically quite similar to those described in this paper. However, the straight filaments formed at low temperature transformed into curved shapes (probably corresponding to the shape of normal flagella) on heating at 37°C for a few minutes, before or after replacing the medium with 0.16 na-NaCl. Therefore, the straight filament observed by Kagawa & Asakura seems to be different in structure from that described in this paper.
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REFERENCES Abram, D. & Koffler, H. (1964). J. Mol. Biol. 9, 168-185. Ada, G. L., Nossal, C. J. V., Pye, J. & Abbot, A. (1963). Nature (London), 199, 1257-1262. Asakura, S. (1970). Adwan. Biophys. (Japan), 1, 99-155. Asakura, S. & Iino, T. (1972). J. Mol. Biol. 64, 251-268. Asakura, S., Eguchi, G. & Iino, T. (1964). J. Mol. BioZ. 10, 42-56. Asakura, S., Eguchi, G. & Iino, T. (1966). J. Mol. Biol. 16, 302-316. Asakura, S., Eguchi, G. & Iino, T. (1968). J. Mol. BioZ. 35, 227-236. Bode, W., Engel, J. & Windlmair, D. (1972). Eur. J. Biochewa. 26, 313-327. Champness, J. N. (1971). J. Mol. BioZ. 56, 295-310. Finch, J. T. & Klug, A. (1972). In The Generation of SubceZZuZarStructurea (Markham, R. & Bancroft, J. B., eds), pp. 167-177, North Holland Publishing Co., Amsterdam. Gerber, B. R., Asakura, S. & Oosawa, F. (1973). J. Mol. BioZ. 74, 467-487. Hotani, H. (1971). J. Mol. BioZ. 57, 575-587. Kerridge, D., Horne, R. W. & Glauert, A. M. (1962). J. Mol. BioZ. 4, 227-238. Kuroda, H. (1972). Biochim. Biophys. Act%, 285, 253-267. Lowy, J. & Hanson, J. (1965). J. Mol. BioZ. 11, 293-313. O’Brien, E. J. & Bennett, P. M. (1972). J. Mol. BioZ. 70, 133-152. Uratani, Y., Asakura, S. & Imahori, K. (1972). J. Mol. BioZ. 67, 85-98. Wakabayashi, K., Hotani, H. & Asakura, S. (1969). Biochim. Biophys. A&, 175, 195-203.