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

BIOCHIMICAET BIOPHYSICAACTA

195

BBA 35308 P O L Y M E R I Z A T I O N OF SALMONELLA F L A G E L L I N IN T H E P R E S E N C E OF H I G H CONCENTRATIONS OF SALTS

K A T S U Z O W A K A B A Y A S H I * , H I R O K A Z U H O T A N I ' " AND S H O A S A K U R A ' "

Department of Ph:vsics, Faculty of Science, Hokkaido Universily, Sapporo* and Institule of Molecular Biology, Faculty of Science, Nagoya University, Nagoya'* (Japan) (Received A u g u s t i 2 t h , I968)

SUMMARY

I. Salmonella flagellin (monomer) spontaneously polymerizes into flagellar filaments in the presence of moderately high concentrations of some kinds of salts at neutral pH. The process was followed b y viscosity measurement and the product was examined with an electron microscope. 2. Rapid and complete polymerization of flagellin was brought about by the addition of F-, CO32-, SO42-, HPO4 ~- and citrate ion to final concentrations higher than o.3 M. The initial rate of polymerization increased with increasing concentrations of these ions. On the other hand, in the presence of high concentrations of Mg 2+ and Ca 2+, flagella underwent depolymerization. These anions and cations are known as good "salting-outers" and "salting-inners", respectively. 3. Reformed filaments were indistinguishable in the overall shape from intact flagella. The average length of reformed flagella, measured after complete polymerization, decreased with increasing concentrations of the above anions and with increasing concentrations of flagellin. 4. From the dependence of the average length of reformed flagella on the initial concentration of flagellin, the number of flagellin molecules needed for the initial step of polymerization, nucleation, was estimated.

INTRODUCTION

Bacterial flagella treated with heat or acid completely depolymerize into monomeric flagellin. Recently, several authors 1 4 have reported reversible polymerization of monomeric flagellin into flagellar filaments. ASAKURAet al. 4-7 have investigated the polymerization process, using Salmonella strains, and it has been shown to be similar to crystallization. At physiological ionic strength and pH, the initial step of polymerization, nucleation, rarely happens and a monomer solution, if it contains no existing flagella, remains in a state of supersaturation for a substantially long period. The Biochirn. Biophys. Acla, 175 (1969) 195 2o3

z96

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H. H ( ) ' I A N I , S. ASAKI*I,LX

addition of fragments of flagella or "seeds" to this solution brings about rapid and complete polymerization of the monomer into flagellar filaments. On the other hand, ADa et al. 1 have found that in the presence of a high concentration of (NH4)2SO 4 at neutral pH, monomeric flagellin (Sahnonella) polymerizes into flagellar filaments without adding seeds. The present authors observed that spontaneous polymerization occurred also in the presence of a high concentration of phosphate buffer at neutral pH. In view of these circumstances, it appeared of interest to examine the effect of high concentrations of various salts on the spontaneous initiation of polymerization. We have studied this problem and found that moderately high concentrations of anions, which are known as good "salting-outers", are effective for the initiation of polymerization. This result will be reported in this paper. METHODS

Preparation of monomeric flagellin Salmonella strains SJ25 and SJ67o, which produce normal flagella with ±,2and/-antigens, respectively, were used. Cultivation of the organisms of each strain and isolation of flagella were carried out by the method described previously 4. Isolated flagella were twice washed by centrifugation at 7 ° ooo x g for I h with solvent containing o.15 M NaC1 and o.oi M phosphate buffer (pH 6.5) and further purified by that procedure described previously '5 which involves a cycle of depolymerization and reformation of flagella. Finally, reformed flagella were resuspended to concentrations between io to 15 mg/ml protein in the same solvent used above and stored at o °. Monomer solutions were obtained by heating purified flagella solutions at 65 ° fin. 3 min. Hereafter, flagellins derived from SJ25 and SJ67o will be referred to as z,2and i-flagellins, respectively. Properties of these flagellins have been reported previously a&

Viscosity measurement Polymerization of monomeric flagellin was lollowed by viscosity measurement. A viscometer of the Ostwald type was used: capacity, 0. 5 ml, flow time of water at 25 °, 21.3 sec. Usually, reduced viscosities of monomer solutions were in a range between o.I and 0.2 (dl/g).

Electron microscopy Products of polymerization were examined with a JEM T 7 electron microscope with the aid of negative staining using i % phosptmtvngstate neutralized to pH 7. Lengths of reformed flagella were measuled in tile electron micr(~,.~COFe and treated statistically< 6. RESULTS

Spontaneous polymerization Monomeric flagellin dissolved in a solvent containing o.15 M NaC1 and o.oi M phosphate buffer (pH 6.5) can not repolymerize without adding seeds. In fact, the viscosity of the monomer solution only slightly changes in I day or more. When, however, a moderately high concentration of NazSO4 or (NH4)2SQ is added to this Biochim. Biophvs. Acta, 175 (19~9) t95 203

POLYMERIZATION OF FLAGELLIN

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Fig. t. A. l ' o l v m e r i z a t i o n of 1,2-flagellin i n i t i a t e d by t h e a d d i t i o n of v a r i o u s c o n c e n t r a t i o n s of Na2SO.v Various c o n c e n t r a t i o n s of Na2SO 4 s o l u t i o n s were m i x e d w i t h s o l u t i o n s c o n t a i n i n g 15 m g / m l 1,2-monomer, o.l 5 M MaC1 a n d o.o 4 M p h o s p h a t e buffer (pH 6.5) a n d p o l y m e r i z a t i o n i n i t i a t e d in each s o l u t i o n was followed by v i s c o s i t y m e a s u r e m e n t . F i n a l c o n c e n t r a t i o n s : flagellin, 4 m g / m l ; MAC1, o.o 4 M; p h o s p h a t e buffer, o.ol M, p H 6. 5 :k o.2; NaaSO 4, o.35 M (a), o.43 M (b), o.57 M (c) a n d o.64 M (d). Temp., 26 °. O r d i n a t e : specific viscosity. B. P o l y m e r i z a t i o n of i-flagellin i n i t i a t e d b y t h e a d d i t i o n of a high c o n c e n t r a t i o n of (NH4)2S() , a t v a r i o u s p H ' s . / -Monome r diss o l v e d in o. 15 M NaCI was m i x e d w i t h s a l t s o l u t i o n s c o n t a i n i n g fixed c o n c e n t r a t i o n s of (NH,)~SO 4 a n d p h o s p h a t e troffers w i t h v a r i o u s p H v a l u e s a n d p o l y m e r i z a t i o n i n i t i a t e d in each s o l u t i o n w a s followed by v i s c o s i t y m e a s u r e m e n t . F i n a l c o n c e n t r a t i o n s : flagellin, 3 m g / m l ; (NH4)2SO 4, o.6 M; MAC1, o.o9 M; p h o s p h a t e buffer, o.o 3 M, p H 5.8 (a), 6.8 (b) a nd 7.9 (c). Temp., 2~ °. O r d i n a t e : specific viscosity.

P l a t e i. F l a g e l l a r f i l a m e n t s r e f o r m e d from i-flagellin in t h e pre s e nc e of h i g h c o n c e n t r a t i o n s of s o d i u m s u l p h a t e . S o l u t i o n s c o n t a i n i n g 2 m g / m l / - m o n o m e r , o.o3 M NaC1 a nd o.o 4 M p h o s p h a t e buffer (pH 6.5) were m i x e d w i t h e q u a l v o l u m e s of conc. Na2SO ~ a nd i n c u b a t e d a t 25 ° for 4 h. F i n a l c o n c e n t r a t i o n s of Na2SO4, o. 5 M in (a) a n d o.8 M in (b). Scale: i /~.

B iochim. Bioph3's. A cta, i75 ([969) ~95 203

198

i<. WAKAI~AYASHI, I f . tl()TANI, S. ASAKURA

solution, the viscosity increases rapidly (Figs. i A and B) and soon saturates to a final level. When the viscous solution obtained was examined under the electron microscope, long flagellar filaments were observed and no structures other than this type of filament was found (Plate I). Therefore, the viscosity increase can be attributed to the polymerization of flagellin into long flagellar filaments, and the rate of viscosity increase can be used as a semiquantitative measure for the rate of polymerization. As shown in Figs. I A and B, the rate of polymerization rapidly increases b y increasing the concentration of added salt but only slightly depends on the p H of solution. Sample solutions used in the experiment of Fig. I B were left standing at 2i ° for 3 h and thereafter centrifuged at lO5 ooo x g for I tl. Upon centrifugation, the protein contained in each solution was completely sedimented in an experimental error of 5 3/o• Therefore, we can define the completion of polymerization as the complete disappearance of m o n o m e r from solutions. Each sediment obtained above was resuspended to a few nlg/ml protein in a solvent containing o.15 M NaC1 and o.oi M phosphate buffer (pH 6.5) and heated at 65 ° for 3 rain. Then, we obtained monomeric flagellin which was able to repolymerize upon the addition of seeds 4. Flagellar filaments observed after the completion of polymerization become shorter, on the average, with increasing concentrations of added salt (Plate I). It should be mentioned at this point t h a t a head-to-tail association of short flagella into longer ones rarely happens after complete polymerization (Fig. 2). A similar result has been obtained for fragments of flagella prepared by sonication< 6 A

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Fig. 2. D i s t r i b u t i o n s of l e n g t h s of s h o r t flagellar filaments. A s o l u t i o n c o n t a i n i n g 4.o m g / m l im o n o m e r a n d o .i M Tris-HC1 (pH 8.2) w a s m i x e d w i t h a n e q u a l v o l u m e of 1.8 M Na~SO 4 a n d i n c u b a t e d a t 25 ° for i o m i n (A) a n d 9o m i n (B). L e n g t h s of fl a ge l l a r f i l a m e n t s c o n t a i n e d in each s a m p l e s o l u t i o n were m e a s u r e d u n d e r t h e e l e c t r o n microscope. The n u m b e r of f i l a m e n t s used a n d t h e a v e r a g e l e n g t h s of t h e m were 475 a n d 235 m # in (A) a n d 528 a n d 240 m/~ i n (B), r e s p e c t i v e l y .

ABRAM, KOFFLER AND VATTER8 have reported t h a t when fragments of flagella (Bacillus) were observed under an electron microscope with negative staining, the two ends of each fragment seemed to be asymmetric in shape (see also ref. 6). The same asynnnetric shape was taken also b y each of the short flagella spontaneously reformed

Biochim. Biophys. Acta, ~75 (1909) 195 2o3

199

POLYMERIZATION OF FLAGELLIN

Plate a. Short flagellar~filaments reformed~-from~7,z-flagellin. A solution containing 8 m g / m l 1,a-monomer, o.o 7 M NaC1 and o.o6 M p h o s p h a t e buffer (pH 6.5) was mixed with an equal volume of 1,8 M Na2S()~ and incubated at 26 ° for io min. Scale: o.25 I~.

( P l a t e 2). T h e r e f o r e , t h e o c c u r r e n c e o f a n a s y m m e t r i c

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t h e b r e a k a g e o f p r e - e x i s t i n g flagella. When

the final concentration

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Na2SO 4 or (NH4)2SO 4 exceeds about

1.2 M, t h e s o l u t i o n b e c o m e s h i g h l y t u r b i d in a s h o r t p e r i o d . T h e e l e c t r o n m i c r o s c o p e

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Fig. 3. Polymerization of flagellin initiated b y the addition of a p r o d u c t of s p o n t a n e o u s polymerization. In the first polymerization, a solution containing 16 m g / m l z,x-monomer, o.15 M NaC1 and o.o2 M p h o s p h a t e buffer (pH 6.5) was mixed with an equal volume of 2 M Na~S()4 and left s t a n d i n g at 26 ° for 3o min. Then, a highly t u r b i d solution was obtained. I n tile second polymerization, this solution was mixed to 2o volunles of a m o n o m e r solution containing 2.0 m g / m l z,2-flagellin, o.l 5 M NaCI and o.oi M p h o s p h a t e buffer (pH 0.5) and the m i x t u r e was incubated at 26°; polymerization initiated in the m i x t u r e was followed b y viscosity m e a s u r e m e n t (a). W h e n the m o n o m e r solution used in the second polymerization was mixed with the solvent used in the first polymerization at a volume ratio of 2o: t, no polymerization took place (b). Curve c corresponds to the case in which the p r o d u c t of the first polymerization was diluted to 2o vols. of the solvent used in the second polymerization.

Biochim. Biophvs. Acta, 175 (1909) 195 2o3

200

K. WAKABAYASHI, H. HOTANI, S. ASAKUR.\

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Time (min) Fig. 4. A. P o l y m e r i z a t i o n of 1,2-flagellin i n i t i a t e d b y t h e a d d i t i o n of v a r i o u s c o n c e n t r a t i o n s of p h o s p h a t e buffer .Final c o n c e n t r a t i o n s : x,z-flagellin, 4.2 m g / m l ; NaCI, o.o 4 M; p h o s p h a t e buffer, o.29 M (a), o.43 M (b), o.64 M (c), a n d o.74 M (d), a n d p H 6.5; T e m p . , 25 °. O r d i n a t e : specific viscosity. B. P o l y m e r i z a t i o n of z,2-flagellin i n i t i a t e d by t h e a d d i t i o n of a fixed c o n c e n t r a t i o n of p h o s p h a t e buffers w i t h v a r i o u s p H ' s . F i n a l c o n c e n t r a t i o n s : flagellin, 2.9 mg/Inl L NaC1, o.o 4 M; p h o s p h a t e buffer, o.7i M, p i t 5.5 (a), 6.0 (b), 6. 5 (c), 7.1 (d) a n d 7.6 (e); T e m p . , 25 °. O r d i n a t e : specific viscositv.

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Fig. 5. P o l y m e r i z a t i o n of z,z-flagellin i n i t i a t e d b y t h e a d d i t i o n of v a r i o u s c o n c e n t r a t i o n s of K F , Na2SO 4 a n d s o d i u m citrate. Various c o n c e n t r a t i o n s of t h e s e s a l t s were a d d e d to solutions cont a i n i n g 15. 3 m g / m l z , 2 - m o n o m e r , o.15 M NaC1 a n d o.oi M Tris-HC1 (pH 8.2) a n d p o l y m e r i z a t i o n i n i t i a t e d in each solution was followed b y viscosity m e a s u r e m e n t . F i n a l c o n c e n t r a t i o n s : flagellin, 4.4 m g / m l ; NaC1, o.o 4 M; Tris-HC1, o.oi M; a n d K F , 0.43 M (a), o.57 M (b) a n d o.71 M (c) in (A); Na2SO ~ o.29 M (a), o.43 M (b), o.57 M (c) in (B); Na3CoH.~O7 o.29 M (a), o.36 M (b), o.43 M (c) in (C). T e m p . , 26 °. O r d i n a t e : specific viscosity. B i o c h i m . B i o p h y s . .4cla, ~75 ([969) 195 2o3

POLYMERIZATION OF FLAGELLIN

20I

observation of such a solution showed that it contained only short flagellar filaments. The increased turbidity is, therefore, considered to be due to salting-out of short flagella which have been reformed in the solution. Short flagella reformed at high concentrations of Na2SO 4 and (NH4)2SO 4 are effective as seeds for initiating the polymerization of monomer at low concentrations of salts (Fig. 3)Sodium carbonate was found to be effective for the initiation of polymerization : in this respect, this salt was similar to Na2SO 4. Spontaneous polymerization occurs in the presence of moderately high concentrations of phosphate buffer (Fig. 4 A). In this case, as shown in Fig. 4 B, the rate of polymerization rapidly increases by raising the pH of added buffer; the former depends most strongly on the latter in a pH range between 6.5 and 7.1, where phosphate ion undergoes the second ionization. This result indicates that in the constituents of phosphate buffer, HP04 ~- is effective for the initiation of polymerization. In addition, flagella reformed at a fixed concentration of phosphate buffer became shorter, on the average, upon raising its pH value. Spontaneous polymerization was brought about by the addition of KF, NaF and sodium citrate (Fig. 5). On the other hand, in the presence of high concentrations of NaC1 (less than 2.5 M), spontaneous polymerization never took place. Moreover,

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F i g . 6. D i s t r i b u t i o n s of l e n g t h s of flagellar f i l a m e n t s r e f o r m e d from v a r i o u s c o n c e n t r a t i o n s o f flagellin. S o l u t i o n s c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f / - m o n o m e r and o . o 5 M p h o s p h a t e buffer (pH 6.5) were m i x e d w i t h e q u a l v o l u m e s o f 1.8 M Na2SO 4 and left s t a n d i n g a t 28 ° for i h a n d t h e n p r o d u c t s w e r e e x a m i n e d u n d e r t h e e l e c t r o n microscope. F i n a l c o n c e n t r a t i o n s o f flagellin: 2.5 m g / m l (A), 2.o m g / m l (B), 1. 5 m g / m l (C) a n d i . o m g / m l (D). T h e n u m b e r o f f i l a m e n t s used a n d t h e a v e r a g e l e n g t h o f t h e m w e r e : 476 and 113 mff in (A), 6 t 7 a n d I31 m f f in (B), 5 2 6 a n d 153 int~ in (C) a n d 6 8 4 a n d 2o 3 mff in (D), r e s p e c t i v e l y . F i g . 7. R e l a t i o n s h i p b e t w e e n t h e a v e r a g e length, ELI, of flagellar f i l a m e n t s r e f o r m e d and t h e initial c o n c e n t r a t i o n o f m o n o m e r , [m~o. T h e e x p e r i m e n t a l p o i n t s w e r e o b t a i n e d f r o m t h e experim e n t o f F i g . 6.

Biochim. Bioph3,s..4eta, 175 (1969) 195 2o3

202

1<. W A K A B A Y A S H I , It. HfYI'ANI, .':,. :\SAKUI,~A

MgC12, M g S Q and CaCI 2 had no effect on the initiation of polymerization. In the presence of I.O M MgCI,, or MgS() 4 and o.o2 M Tris -HC1 (pH 8.o) and in the presence of o. 5 M CaC12 and 0.o2 M Tris-HCl (pH 8.o), flagella underwent depolymerization. F r o m these results, it is concluded that polymerization of flagellin is initiated by the presence of anions which are known as good salting-outers. It is to be noted that, in Fig. 5, F , SO4 2- and citrate ion are effective for the initiation of polymerization in the order of Hofmeister series":°.

Dependence of the average length of reformed flagella on the initial concentration of monomer Flagellar filaments observed after the completion of polymerization become shorter, on the average, b y increasing the initial concentration of monomer. In the experiment of Fig. 6, lengths of flagella reformed from various concentrations of im o n o m e r were measured under the electron nlicroscope and analyzed statistically. The result shows t h a t the average length of reformed flagella is inversely proportional to the o.64th power of the concentration of m o n o m e r (Fig. 7). DISCUSSION

The present s t u d y has shown that spontaneous polymerization of flagellin occurs in the presence of anions which act as good salting-outers, and t h a t the reversed process, depolymerization, occurs in the presence of Mg 2+ and Ca 2+ which are regarded as good salting-inners. W h a t e v e r the detailed mechanism of the effect of these ions, spontaneous polymerization or nucleation is considered to be closely related to a general phenomenon, salting-out. Similar notions have been deduced in studies of the effect of various ions on the depolymerization of F-actin and the helix coil transition of D N A (refs. 9, IO). I n vitro polymerization of flagellin into flagellar filaments can be divided into two steps, nucleation and growth. Nucleation will occur as a result of a simultaneous collision of a few flagellin molecules, and growth will be b r o u g h t about by the addition of flagellin molecules onto pre-existing nuclei and flagellar filaments. The simplest mathematical expressions for the two processes will be as followsn : d[l:I/dl

d[m!/dl

,

1%i.z] ~

([)

/~2!iI:)[m]

{-,)

where EmJ is the n u m b e r concentration of flagellin molecules at time t, [F~ is the n u m b e r concentration of pre-existing nuclei and flagellar filaments at time t, and k 1 and/e 2 are rate constants. The constant s in Eqn. I corresponds to the n u m b e r of flagellin molecules whose simultaneous interaction leads to nucleation. It was assumed in Eqn. 2 t h a t nuclei and flagellar filaments grow at a constant rate independent of theil sizes. According to the present formulation, [mj tends toward zero upon increasing t toward infinity, and an end-to-end association of short filaments into longer ones does not occur. Solving Eqns. I and 2, we obtain the n u m b e r concentration, [ F ]~, of flagellar filaments (including nuclei) after the completion of polymerization as follows : [FI~

(2 kl/s/%)1/,a [m]0.s/2

where Ira]0 denotes the initial concentration of monomer. Biochim. Biophys. Acla, 175 ( t 9 0 9 ) 195 2o3

(3)

P O L Y M E R I Z A T I O N OF F L A G E L L I N

203

According to a model of Salmonella flagella presented by LowY AND H A N S O N 12, the filament is constructed from four helical or eight longitudinal parallel strands of spherical subunits, 50 A in diameter, which possibly correspond to flagellin molecules having an approximate molecular weight of 4 ° ooo. In view of this model, nuclei and flagellar structures at the earliest stages in growth will not be observed under the electron microscope at low magnifications. However, the proportion of them in the total concentration, IFjoo, will be small when nucleation occurs slowly as compared with growth and, therefore, the value of Em]o/[F]o~ is much greater than unity. In this case, the average length, [Ln, of flagellar filaments observed willbe approximately proportional to Imo/IF!oo : ILl. (s k,_,/~I¢:)~/~[m]o~- ~/2 On the other hand, the experiment of Fig. 6 has given the following relationship : [L!~ ;n]o 0.~4 Combination of this result with Eqn. 4 leads to s -- 3.3. This means that a simultaneous interaction between three flagellin molecules or more is necessary for nucleation. At physiological ionic strength and pH, flagellin molecules are successively added to existing flagella during polymerization. In this respect, monomeric flagellin can be regarded as a kinetic unit of polymerization4,L In the present case, however, we have no information about kinetic unit of polymerization. It might be possible t h a t on adding moderately high concentrations of anions (S042- for example), monomeric flagellin instantaneously and completely associates into an oligomeric intermediate, which subsequently polymerizes as a kinetic unit. Oligomeric intermediates have been found in the polymerization of TMV protein in vitro 13. I f the above assumption is correct, the number of flagellin molecules needed for nucleation becomes a small multiple of 3.3REFERENCES I 2 3 4 5 0 7 8 9 Jo iI 12 13

G. L. ADA, G. J. v . NOSEL, J. PYE AND A. ABBOT, Nature, i 9 9 (1963) i257. l). ABRAM AND U. [(OFFLER, J. Mol. Biol., 9 (I964) i68. J- t , o w v AND M. \V. MCDONOUGH, Nature, 2o 4 (1964) 125. S. ASAKURA, G. EGUCH1 AND T. IINO, J. Mol. Biol., i o ( i 9 6 4 ) 42. S. AS.~.KURA, G. EGUCHI AND T. [INO, J. Mol. Biol., L6 ( i 9 6 6 ) 3o2. S. ASAKURA, G. EOUCH1 AND T. IlNO, J. Mol. Biol., 35 (1908) 227. S. ASAKURA, J. 3"[01. Biol., 35 (r968) 237. 1). ABRAM, H. KOFVLER AND A. E . VATTER, Abslr. Intern. Congr. Biophys. 2n& ['ienna, x~)66, P-45. B. NAGY .aND W. i'. JENCKS, .]. Am. Chem. Soc., 87 (1965) 2480. P. H. VON HIPPEL AND K . - Y . WONG, Science, 145 (1904) 577. F. ()OSAVVA AND M. KASAI, J. Mol. Biol., 4 (I962) lO. J. L o w Y AND J. HANSON, J. AIol. Biol., I I (1965) 293. l). L. 1). CASPER, .4dvan. Protein Chem., IS (t963) 37-

Biochim. Bioph3~s. Acta, J75 (1909) 195 2o3