Transition of bacterial flagella from helical to straight forms with different subunit arrangements

J. Mol. Biol.

(1979) 131, 725-742

Transition of Bacterial Flagella from Helical to Straight Forms with Different Subunit Arrangements R. KAMIYA, Iwtitute

of Molecular Biology, Faculty of Science Nagoya University, Nagoya, Japan K. WAKABAYASHI

Department

S. ASAKURA

AND

K.

NAMBA

of Biophysid Engineering, Osaka University Toyonaka, Osaka, Japan (Received

3 November

1978)

We have found that several kinds of helical flagella from Salmonella and Escheri&a become straight in the presence of 0.5 M-citric acid at pH values below 4.0, while the straight flagella from a mutant SaZmoneZZu (55814) 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 55814 flagellum (O’Brien & Bennett, 1972) agrees closely with the degree of twisting predicted for the righthanded type, this flagellum has been considered to be the right-handed type. Wre have determined that t’he basic (l-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 55814 flagellum are right-handed, in agreement with the prediction. However, our optical diffraction and X-ray diffraction studies have revealed that the nearlongitudinal rows of subunits in the citric acid-induced straight flagella and in the & Yanagida, 1975) tilt at an angle straight flagella from a mutant E. coli (Kondoh of 2’ to 3’ with respect to the flagellar axis. This inclination is probably lefthanded. Thus the predict,ed presence of the tw-o types of straight flagella seems bo be proved.

1. Introduction We have reported that reconstituted flagella from Salmon&z are reversibly transformed into several helical structures when environmental factors, such as the pH value, are changed (Kamiya & Asakura, 1976,1977). These helical structures include left-handed (normal, coiled) and right-handed helices (curly, semi-coiled). The transformation can also be induced by mechanical forces (Macnab & Ornston, 1977; H. Hotani, personal communication), and its possible importance in the tactic behaviour of bacteria has been pointed out by Macnab & Ornston (1977). We report here that several kinds of helical flagella from Salmonella and Escherichia become straight in 725 u0~s-~83~/79/‘00725-1~

$02.00/0

@ 1979 Academic

Press Inc. (London)

Ltd.

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the presence of a high concentration of citric acid at pH values below 4.0, while the straight flagella from a mutant Salmonella (55814) (O’Brien $ Bennett, 1972; Asakura & Iino, 1972) are converted to a helical structure under the same conditions. Salmonella and Escherichiaflagella are composed of only one-kind of protein subunit, flagellin. The surface lattice of these flagella has been determined to comprise l-, 5-, 6- and 11-start helices, as shown in Figure l(a) (O’Brien & Bennett, 1972; Finch & Klug, 1972; Kondoh & Yanagida, 1975). On the basis of such ultrastructural evidence, models have been presented to explain the polymorphism of flagella (Asakura, 1970; Wakabayashi & Mitsui, 1972; Calladine, 1975,1976,1978; Kamiya, 1976). According to these models, all of which are based on the assumption that flagellin molecules in a flagellum are dimorphic, i.e. have bistable conformations, two discrete types of straight flagella are expected to arise: one with the maximum degree of left-handed twist and the other with the maximum degree of right-handed twist. Calladine (1975,1976) found that when the curvature of flagellar polymorphs is plobted against the twisting, the plot displays a sinusoidal, discrete vkriation. He also found that the inclinat)ion of t,he near-longitudinal rows of flagellin (the 11-start helices) to the filament axis in the straight flagellum from Salmonella 55814 (O’Brien & Bennett, 1972) agrees closely with the maximum right-handed twist expected among the polymorphs. However, Kondoh & Yanagida (1975) found a different type of straight flagella from a mutant Escherichia coli, which we suspected to be the left-handed type (Matsuura et al., 1978), although Kondoh & Yanagida considered that the twisting of the longitudinal rows in this flagellum is zero. In this study we have investigated the molecular arrangement of straight flagella by means of electron microscopy, optical diffraction and X-ray diffraction. We found that the inclination of the near-longitudinal rows to the flagellar filament axis has a value of about 2” to 3” in the citric acidinduced straight type, as well as in t’he straight flagella studied by Kondoh & Yanagida. This value of inclination is in good agreement with the prediction of the model. These models explain the curvature of flagellar helices as originating from t’he combination of two kinds of longitudinal rows of subunits with different unit lengths. Accordingly, the right- and left-handed types of straight flagella are expected to have different inter-subunit distances along the near-longitudinal rows. We attempted to detect this difference by X-ray diffraction of oriented flagellar gels. However, the results showed that the difference, if any, is less than lyb.

2. Model Asakura (1970) presented a model of flagella to explain the observed polymorphism. Since knowledge of flagellar structure was limited at that time, certain features of his original model are inconsistent with current observations. However, the assumption of two discrete states in flagellin conformation seems to explain best the stepwise occurrence of helical polymorphs, and revised models have been based on the same assumption (Wakabayashi & Mitsui, 1972; Calladine, 1975,1976,1978; Kamiya, 1976). We describe here our current model of flagella in relation to the problems that we have tried to make clear in this study. This model owes much to the considerations of Calladine (1976) and is very similar to his 1978 model, in that 12 types of polymorphs can be constructed. But our model is simpler than his model, as we have not assumed specific shapes of subunits.

STRAIGHT

BACTERIAL

FLAGELLA

727

This model is based on the following two assumptions. (1) In any type of helical flagella, the innermost or outermost line on the surface of a flagellum is always traced by a particular longitudinal row of subunits. (2) The longitudinal rows of flagellin subunits can assume two stable configurations (R or L), depending on the environmental conditions and the species of flagellin. This assumption may be regarded as an application of the allosteric model (Monod et al., 1965). R and L rows are slightly different’ in length, and are tlransformed to a different extent, To construct the model on the basis of the above principles, it is convenient to consider thesurface lattice (radial projection) of a flagellum and let the subunits be represented by two kinds of parallelograms, in either the R or L state. Figure l(a) illustrates the surface lattice of a straight flagellum after O’Brien & Bennett (1972). This flagellum is considered as consisting of 11 R rows. If A and B in this Figure are joined with A’ and B’, the two-dimensional lattice would make a cylinder, in which the near-

I

l

l l

l

(b)

l

l

l l

e l

l l

1

r/l/il

l

(Cl

FIU. 1. (a) The surface lattice of an s-flagellum. From O’Brien & Bennett (1972) with modifications. The shadowed portion represents an R parallelogram. (b) Mixt’ure of 10 R and 1 L parallelograms with the same intrinsic length. (c) Mixture of 10 R and 1 L parallelograms with different intrinsic lengths. As elastic deformations are taken int#oaccount, the R parallelograms are replaced by trapezoids.

728

R. KAMIYA

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longitudinal rows, such as the shaded portion, tilt at an angle to the axis of the cylinder. Then consider the situation when some of the 11 rows are in the other state (L state), having a different shape. First, t’o make it easy to see the effect on the twisting, the intrinsic length of the L strand is made equal to that of the R strand. In Figure l(b), ten R rows and one L row are mixed. When this lattice is closed as above, the inclination of the near-longitudinal rows to the axis would assume the weighted average value of those in the two kinds of straight flagella, which are made of 11 R rows or 11 L rows of subunits. This model represent+ the case where the twisting varies linearly with the proportion of R rows or L rows in a flagellum. Next, as illustrated by Figure l(c), the intrinsic lengths of the two kinds of near-longitudinal rows, which are considered to be elastic, are made slightly different. Such a mixed lattice forms a cylinder whose axis is wound into a helix, and the gross helical structure changes stepwise if the number of R rows (and consequently of L rows) is changed. All the R rows or L rows are expected to be clustered, since coexistence of rows of different lengths must accompany elastic deformation of subunits, and the distribution of the two kinds of rows must obey the law of lowest free energy. From a simple mechanical consideration, Calladine (1976) pointed out that the bending in such a model is proportional to sin ((v/11) x (the number of R or L rows)), and that a plot, of curvature versus twisting would show a sinusoidal, discrete variation, since the twisting varies linearly with the number of R or I, rows. He found that the flagellar polymorphs observed in our experiment (Kamiya I% Asakura, 1976) actually satisfy this relation (Fig. 2). While Calladine’s finding provides support, for this model for the first time, we have noticed that this model predicts t*he presence of two types of straight flagella made of 11 R rows and 11 L rows of subunits, which could be investigated by experiment. As is evident in Figure 2, the plot for the helical wave-forms predicts bhat one of the

-2

0

4

2

A8

6

8

(deg )

1975,1976,1978) for the polymorphs observed in FIQ. 2. Bending-twisting plot (Calladine, Bending (LY)and twisting (A 0) are given by: homopolymers (0) and copolymers (0) of flagellin. LX = 27?dD/(7raD~ + P2) and n 0 = 180dP/(7raDa + P) (deg.), where D and P are the diameter and the pitch of the large-scale helix, and d is the diameter of the flagellar fiber (Asakura & Iino, 1972). In this study, d is assumed to be 0.02 pm. The plot is for normal (l), coiled (2), semi-coiled (3), curly I (4), curly II (5) and type IV (small-amplitude) configurations (6). The original data are from Kamiya Bc Asakura (1976), Hotani (1976) and Asakuta & Iino (1972). The plot for the 2 types of straight flagella predicted from the evidence of the helical wave-forms is marked by X. Some small margin of uncertainty in the data is inevitable, due to the error in the microscopic measurements.

STRAIGHT

BACTERIAL

FLAGELLA

729

5(a-v .

'5 I .

..r 52

l

.

--

II

6

0

-5

-II.

n

I

l z

.

..I52 .

6

O-5

.

* Ii

-II

” Cd)

I%. 3. The surface lattice ((a) and (c)) and the corresponding n-5 plot ((b) and (d)) (Moody, 1967) for the R type ((a) and (b)) and the L type ((c) and (d)) straight flagella predicted by the model. In (a) and (c), the l-start (basic) and the 11 -start (near-longitudinal) helical lines are drawn.

two types (the R type) should have a right-handed twisting of about 7” and the other (the L type) a left-handed twisting of about 26” at the surface of a flagellar tube with a diameter of 200 A. Figure 3 shows the surface lattice and the corresponding n-5 plot (Moody, 1967) of these two predicted types of straight flagella. In addition to the twisting, the inter-subunit distance along the near-longitudinal rows should be different between the R and the L types of straight flagella, as is evident from the above considerations. This difference in unit length is calculated to be about 40/ the assumption that the fi agellin monomers interact at the very surface of the flage&mon but would be smaller if the interaction takes place at the inner part of the flagella; cylinder. We show in the later sections that the two predicted types of straight flagella actually occur.

730

R. KAMIYA

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3. Materials and Methods (a) Preparation

of reconstituted

flagella

Table 1 shows the 6 strains of bacteria used in this study. Salmonella strains were supplied by Dr T. Iino (Laboratory of Genetics, Faculty of Science, University of Tokyo). Escherichia strains were supplied by Dr H. Kondoh (Department of Biophysics, Kyoto University). Flagella from each strain were purified and reconstituted as described by Asakura et al. (1964), Kamiya & Asakura (lQ76) and Kondoh & Hotani (1974). TABLZ

1

The six kinds of jiagella used in this study Flagellar t YP

i n c 8 en e8

Shape at pH 7.0

Strains Salmonella SJ670 Salmonella SJ25 Salmonella SJ30 Salmonella SJ814 Escherichia W3623 Eseherichia W3623 hag177

system

References

Normal

Straight

Iino (lQ61)

Normal

Straight

Iino (1961)

Curly

Straight

Iino (1962)

Straight)

Curly

Normal

Straight

O’Brien & Bennett (1972): Asakura & Iino (1972) Matsuura et al. (1978)

Straight/ normal

Straight,

(b) Dark-Jield The optical (1976).

Shape in 0.5 M-citric acid (pH 4.0)

and the technique

Yanagida (1975) ; at cd. (1978)

light microscopy

used have been described

(c) Electron

Kondoh& Matsuura

by Kamiya

& Asakura

microscopy

Flagellar specimens prepared in the presence of high concentrations of cit,ric acid were fixed for 30 min wit,h 10% (v/v) f ormaldehyde, because unfixed flagella were readily transformed into other helical structures when washed on the electron microscope grids with solutions of low ionic strength. The normal configuration of wild-type flagella was unchanged after this fixation. Sometimes the gross morphology of flagella thus fixed was examined at low ionic strength by dark-field microscopy, prior to observation by electron microscopy. The fixed specimens were rinsed with several drops of distilled water after stained with 1% uranyl they were adsorbed to carbon-coated grids, and negatively acetate. For better preservation of the gross structure, the grids had to be dried as rapidly as possible after the addition of the negative-staining solution. A hair-dryer was often used for this purpose. The grids were always inserted in the microscope with the specimen side up, and the negative films were printed with the emulsion side facing the light source. Thus the final print represents the view seen from the specimen side. Photographs were taken at a numerical magnification of 40,000 x with a JEM 1OOC electron microscope, operated at 80 kV. The microscope was calibrated by magnesium tactoids of rabbit tropomyosin with an axial repeat of 395 A (Cohen & Longley, 1966). An anticontamination device was always used. (d) Optical

di@action

Optical diffraction of the electron micrographs was carried out by the method of Klug & Berger (1964). To visualize near-equatorial reflections, rectangular masks were often tilted to the flagellar axis.

STRAIGHT

BACTERIAL

(e) Determination

731

FLAGELLA

of the hand of the basic helix

The hand of the basic helix (the l-start helix) in es-flagella was determined by the method of Finch (1972). The principle and the technique have been described in detail by Finch (1972) and Nonomura & Kohama (1974). In brief, the filament axis of a flagellum was tilted toward or away from the direction of viewing, and the serrated-smooth asymmetry in the left and right halves of the helix in question was examined by optical diffraction. Tobacco mosaic virus (TM’V) particles were added to the specimen as a standard 1972), and examined simultaneously with flagella. of a right-handed helix (Finch, Photographs were taken at a direct magnification of 50,000 x with a JEM 1OOC microscope equipped with a side-entry type goniometer. The tilting angle was usually f 6’. (f) X-ray

diffraction

Reconstituted 8 and es-flagella were suspended in 0.15 M-NaCl, 9.02 M-potassium phosphate (pH 6.0). The suspensions were sonicated briefly, because good orientation of specimens was often achieved when short flagella were used. The flagella from n-strain acid at pH 3.7. As the n-flagella slowly were straightened by suspending in 0.5 M-CitriC underwent depolymerization under these conditions, and as the total rate of the depolymerization depended on the number of sites available, i.e. the ends of the flagella (Hotani & Kagawa, 1974), flagellar specimens with large average lengths were prepared by the method of Kamiya & Asakura (1976). Each flagellar solution was centrifuged at 100,000 g for about 10 min, to give a soft pellet of flagella, which was picked up and forced into a glass capillary (int,ernal diam. 1.0 mm). The capillary was sealed and used for X-ray diffraction. The degree of orientation within the specimen seemed to depend greatly on ehe hardness of the flagellar pellet. Diffraction patterns were recorded on Fuji Medical X-ray films, using an Elliott toroidal camera operated in wucuo to eliminate air scatterings. CuKcc radiation (h = 1.542 A) was used and diffraction spacings were calibrated with the sodium myristate powder pattern. All X-ray experiments were carried out at about 4°C.

4. Results and Discussion (a) Transformations

of wild-type

flagella

into a straight

type

Flagellar transformations induced by acidic pH (Kamiya & Asakura, 1976) depended on the species and the concentration of ions present. Although detailed experiments have not been done, the presence of salts known to have salting-out effects tended to inhibit the transformation. For instance, i-flagella assumed the normal configuration at pH 4.0 as well as at pH 7-O in the presence of 0.1 M-ammonium sulphate, while they took on curly or coiled forms when the pH value was lowered to 4.0 in the presence of 0.1 M to 0.5 M-NaCl (Kamiya & Asakura, 1976). In studying such effects of various salts on flagellar morphology, we found that high concentrations of citric acid induced the transformation of many kinds of flagella into a straight form at pH values below 4.0. These transformations were rapid and reversible processes that proceeded transitionally, as in the case previously reported (Kamiya & Asakura, 1976,1977). Citric acid was chosen as a strong salting-out agent at the initial stage of the present experiment, but the relation between these transformations and salting-out phenomena is obscure at present, since the presence of a high concentration of ammonium sulphate (up to 2 M) did not alter the flagellar shape. In a few kinds of flagella, succinic acid also could induce the transformation into the straight form, but it seemed less effective than citric acid. The effect of increased concentrations of citric acid on the shape of i-flagella at pH 4.0 is given in Figure 4 and Table 2. They show that a shape less twisted in the right-hand direction appears as the concentration of citric acid is increased, suggesting that this straight conformation might have a left-handed twisting. Due to the

R.

732

(b)

(0)

KAMIYA

ET

AL.

(cl

(e

(d)

1

FIG. 4. Morphology of i-flagella at pH 4.0 in the presence of increasing concentrations of citric acid. Dark-field light micrographs. The concentrations of citric acid were: (a) 20mM; (b) 100mM; (c) 200 mM; (d) 400 mM; and (e) 500 mix. Besides the citric acid, 100 mx-NaCl was also present. The bar represents 5 pm.

salting-out effect of the citrate ion, i-flagella formed thick bundles at pH values above 4.5 in the presence of 0.5 M-citric acid. When the pH was reduced below this value, the flagellar bundles were dissociated into individual flagellar filaments of the normal type, and the normal flagella became straight at around pH 4.0. Before this normalstraight transformation occurred, the helical diameter of the normal flagella increased continuously from about O-4 pm to 0.7 pm, while the pitch showed little change. Such a continuous change in shape has been observed in alkali-induced flagellar transformations (Kamiya & Asakura, 1977). This kind of continuous change falls outside the scope of current models. TABLE

2

EJect of citric acid at pH 4.0 on the shape of i-jlaqella Concn of citric acid (mM) 0 30 100 200 300 400 500

Flagellar

shape

Curly Curly + semi-coiled Semi-coiled Semi-coiled + normal Normal Normal + straight Straight

Besides the citric acid, 0.1 M-Nacl was present. The twisting & Asakura (1976). A positive value is assigned to a right-handed

Twisting

(deg.)

+1.s +1.0 -1.2

values are taken from twist.

Kamiya

STRAlGHT

BACTERIAL

733

FLAGELLA

Flagella from other wild-type strains (n and en) were also transformed from the normal to the straight type under apparently the same conditions as were effective for i-flagella. The continuous increase in helical diameter of the normal type was also observed in these flagella at pH values slightly higher than the transitional pH value. (b) Transformations

of mutant

jlagella

Curly flagella from Salmonella SJ30 (c-flagella) were transformed into a straight t)ype under conditions similar to those used for i-flagella (Fig. 5 (a)). In this case, the bransition in shape occurred between the right-handed curly helix and the straight form, and no other type of polymorph was observed as an intermediate. Straight flagella from Escherichia W3623 hag177 (es-flagella) (Kondoh & Yanagida, 1975),

10) FIG. 5. Morphology acid and 100 mx-N&l.

of c-flagella (a) and s-flagella The bar represents 5 pm.

tb)

(b) at pH 4.0 in the presence of 500 rmw-citric

which were known to convert at pH values above 7.0 into a left-handed helix similar t,o the normal conformation (Matsuura et al., 1978), remained straight under conditions where other types of flagella underwent the helical-straight transformations. On the other hand, another kind of straight flagella from Salmonella SJ814 (s-flagella) (O’Brien & Bennett, 1972; Asakura & Iino, 1972), in which we had failed to observe transformations even under extreme acidic or alkaline conditions, were transformed in the presence of 0.5 M-citric acid at pH 4-O into a helical form similar to the curly IT type described by Kamiya & Asakura (1977) (Fig. 5 (b)). Electron microscopic observations of fixed specimens showed that no intermediate type of configuration was present between the straight and the curly II-like helix. All these transformations were reversible. Dilution of the citric acid with distilled water or neutralization with base caused flagella to rapidly assume other helical shapes. It is remarkable that all but es-flagella underwent transformations under very similar conditions, in view of the diversity of conditions where different kinds of flagella exhibited acid or alkali-induced transformations (Kamiya BEAsakura, 1977). 2;

734

1%. KAMII’A

E’2’ AL.

Citric acid at low pH values might be affecting a general principle governing the architecture of various kinds of bacterial flagella. Ss stated in later sections, we consider that the straight form occurring in t)he presence of citric acid is the lefthanded extreme among flagellar polymorphs. If this is t,rue, the effect of high concentrations of citric acid at low pH values is t,o bring about a flagellar polymorph that is twisting more in the left-handed sense. From this point of view, it is natural that the es-flagella, also having the extreme left-handed twist, could not be transformed by citric acid. (c) Preservation

of jlagellur

gross shapes in electron microscopy

It was our experience that, although use of uranyl acetate as a negative stain in electron microscopy gives good information about flagellar substructure, it’ more or less distorts the gross shape: for example, straight flagella often become flexuous and helical flagella tend to be straightened. As we thought it essential for the study of the substructure of straight flagella to preserve the gross straight morphology, we devised a method for minimizing the distortion. This method (Materials and Methods), consisting of fixation with 10% formaldehyde and rapid drying of the negative stain (1 o/0 uranyl acetate), was found to be satisfactory. The fixation of flagella with formaldehyde was originally employed by Leifson (1960) for light microscopy. We found by dark-field microscopy that this treatment destroys the ability of flagella to undergo polymorphic transitions and would be useful for electron microscopic observation of various types of flagellar configurations. For instance, this procedure prevented i-flagella that were curly at low pH values from converting to the normal form even after the pH was neutralized. (d) Optical diffraction A number of electron micrographs were taken of s, n, es and en-flagella and subjected to optical diffraction. Among them, s and es-flagella were naturally straight mutations, and n and en-flagella were rendered straight by exposure to high concentrations of citric acid. The two kinds of wild-t’ype strains, n and en, were chosen because their flagellin subunits are akin to s and es-flagellin subunits, respectively. Although systematic analyses have not been done, the diffraction patterns of straight i or c-flagella were quite similar to those of straight n-flagella. First, we re-examined es-flagella, because the selection rule (I = 2n -1 llnz) presented by Kondoh & Yanagida (1975) implies that the twisting of the near-longitudinal rows (the 11-start helices) is zero, disagreeing with the prediction of the model. Figure 6(b) shows a typical example of optical diffraction patterns of es-flagella. Besides a prominent layer-line at a spacing of about 52 A, layer-lines at about 26 8-l and near the equator are also evident, which were shown to have the 1st and 11th Bessel orders, respectively (Kondoh $ Yanagida, 1975). What has not been pointed which was often weak, was seldom posiout is that the near-equatorial layer-line, tioned strictly on the equator, suggesting that the 11 -start helix tilts slightly with respect to the filament axis. The distance of this layer-line from the equator in 43 diffraction patterns gave an average spacing of 1770 f 72OA. From this value, the twisting of the near-longitudinal rows of subunits at, the surface of the flagellum (the diameter was assumed to be 200 A) was calculat~ed to be 2.2” + 0.9”. On the other hand, Kondoh & Yanagida measured the ratio of the tlist,ances of the 26A and 52 A layer-lines from the equator and obtained a value of 1.975 -& 0.025. From this ratio

STRAIGHT

BACTERIAL

735

FLAGELLA

1 26 1 E-

0

1 52

0

i-1

FIG. B. Electron micrographs of 3 kinds of straight flagella and their optical diffraction (a) Micrograph of an es-flagellum; (c) an en-flagellum; and (e) a side-by-side aggregate of All these samples were fixed with 10% (v/v) formaldehyde and negatively stained uranylacetate. Magnification 240,000 x . (b), (d) and (f) Optical diffraction patterns and (e), respectively.

patterns. n-flagella. with 1% of (a), (c)

736

H. KAMIYA

ET

AL.

t*hey calculat’ed the twisting t*o be 1.4” ;t_ 1.4”, which they approximated t,o zero as sbated above. We also measured this ratio, but obtained a much smaller value of 1.941 f O-018 (average of 36 measurements), which gave the value of the twisting as 3.7” f 1-O”. The origin of this discrepancy is not certain, but we thought a value for the ratio as large as l-975 to be unlikely, since our measurements of this ratio using the optical diffraction patterns presented by Kondoh & Yanagida (1975) gave values between l-92 and 1.95. Thus we concluded that the 11-start helix in an e.sflagellum tilts at an angle of about 2” to 3” with respect to the flagellar axis. Optical diffraction patterns of the citric acid-induced straight flagella (n and en) were similar to those of es-flagella in that the near-equatorial layer-line was slightly off the equator (Fig. 6 (d) and (f)). The layer-line at a spacing of about 26 A was evident only in E. coli flagella, as noted by Kondoh & Yanagida. As in the case of es-flagella, twisting of the near-longitudinal rows to the flagellar axis was calculated from the height of the near-equatorial layer-line (n = 11) and from the ratio of the heights of 52 A and 26 d layer-lines in the case of en-flagella. The results are summarized in Table 3 and show that the near-longitudinal rows in all these three kinds of straight TABLB 3

Parameters obtained by optical diffraction Flagellar species Number of measurements Spacing (A) of the 52 A layer-line (a) Spacing (il) of the 26 A layer-line (b) Spacing (A) of the nearequatorial layer-line (c) a/b The inclination (deg.) of the near-longitudinal rows at the surface (i) calculated from c (ii) calculated from a/b The diameter

of the flagellar

es

en

43 51.5 & 0.5

42 53.1 & 0.5

26.6 3 0.4

27.2 & 0.4

1770 & 720

1530 f

l-941 $ 0.018

1.957 !~ 0.017

2.2 & 0.9 3.7 + 1.0

2.5 & 0.9 2.7 .t 1.1

560

rc -.-.____ 37 52.2 & 0.9

8 30 56.7 k 0.9

1230 & 250

410 f

2.8 f

8.0 :k 0.6

0.6

30

fiber is assumed to be 200 A.

flagella tilt at an angle of about 2” to 3” to the flagellar axis. On the other hand, we have confirmed the result reported by O’Brien & Bennett (1972), that the twisting of the near-longitudinal rows in s-flagella is about 8” at the surface of the flagellum (Table 3). In diffraction patterns of es, en and n-flagella, one, usually strong, layer-line was found around a spacing of about 52 A, where two layer-lines with the Bessel orders 5 and 6 are expected to appear at slightly different heights (Fig. 3). Although we could not determine which Bessel order this strong layer-line has, it would be reasonable to assume this t,o be t,he 5th, for the layer-line with 5th Bessel order has been reported to be far stronger than that of the 6th in s-flagella (O’Brien C%Bennett, 1972). If this assumption is adopted, the fact, that the ratio of the heights of the 52 A and 26 A layerlines is smaller than 2.0 means that) the 1 l-start, helices (the near-longitudinal rows) twist in the sense opposite to that of the basic helix (the l-start helix) in es and en.flagella. Furthermore, this means t,hat, the I l-start helices in these flagella are opposite

STRAIGHT

BACTERIAL

FLAGELLA

737

in hand t,o t$hat in s-flagella, if the hand of the basic helix is assumed to be const,ant among different kinds of flagella. These features about handedness are also illust’rated by X-ray diffraction studies (see below). (e) The handedness of the basic helix We t#ried bo determine the hand of the near-longitudinal rows (the ll-start helices) in straight) flagella by electron microscopy using shadow-casting with tungsten or with platinum/carbon; however, these experiments were unsuccessful. However, the hand of the 11-start helix in s-flagella could be determined if the hand of the basic helix is known, since the selection rule (1 = 15n + 82m (O’Brien & Bennett, 1972)) indicates that the hand of the two kinds of helices is the same. Furthermore, it would be reasonable to assume the hand of the basic helix to be common to both Salmonella and Escherichia flagella, because their flagellins can be copolymerized into hybrid flagella (Kondoh & Hotani, 1974). We t,hen attempted to determine the hand of the

1 26jr

0

lb) E’ra. 7. Determination of the hand of the basic (l-start) helix. Electron micrographs of an esflagellum and the optical diffraction patterns from the left (L) and the right (R) halves. In (a), the top end was tilted by 6” toward the direction of the viewer, and the direction of tilting is reversed in (b). A strong meridional reflection at about 26 k1 appears from the left half of the flagellum in (a) and from the right half in (b). These situations indicate that the basic helix is right-handed.

738

1%. RAMIYA

ET

AL.

basic helix in the es-flagella, using the method of Finch (1972). We did not try this method for s-flagella because there is JW near-meridional reflection at l/26 8-l. In Figure 7(a), the t’op end of the flagellum was tilt,ed toward the direction of view. Although the left-right,, serrat,ed-smooth asymmetry in the basic helix was not evident by visual inspection, the asymmetry was clearly revealed when left and right, halves of t)he particle image were subjected t)o optical diffraction (Fig. 7L and R). Figure 7(a) L and R demonstrate that the left half gave a strong layer-line on t,hc meridian at a spacing of about 26 & while in the optical transform of the right half, the 26 A layer-line was weakened and situat,ed off the meridian. When this flagellum was tilted to the opposite direction, i.e. the t’op end was tilted away from the direction of view, the asymmetric profiles in the left and right halves were reversed and only the right half gave a strong meridional reflection (Fig. 7(b)). In this and many other experiments, the tilt,ing direction determined which side of a flagellum gave bhc strong meridional reflection at 26 A-l. We considered that, the strong meridional reflection resulted from the serrated half of the basic helix, and concluded that the basic helix is right-handed, from the above relation between the tilting direction and the profile of the asymmetry. To check our system, TMV particles were added to the flagellar specimens and examined simultaneously with flagella. These experiments, which confirmed that the helix in TMV is right-handed (Finch, 1972), ensured that, we did not commit trivial errors in the experimental procedures. The conclusion that the basic helix in a flagellum is right-handed strongly suggests that the near-longitudinal rows of subunits in an s-flagellum are also right,-handed, in agreement with the prediction of the model. (f) X-ray

diffraction

The model described above predicts that’ the inter-subunit distance along the near-longitudinal rows is different between t’he two t,ypes of straight flagella. To ascertain this and to confirm furt’her the results of optmica diffraction stu
STRAIGHT

FIG.

flagella

8. X-ray ((e) and

BACTERIAL

739

FLAGELLA

diffraction patterns from oriented gels of s ((a) and (f)). The 4.7 if layer-line is arrowed.

(b)),

n ((c) and

(d)) and

es-

It. KANIYA

740

ET AL.

Burge, 1974). From comparison with optical diffraction studies, it is evident that the 26 A layer-line is the reflection from the basic helix (n = l), and the 52 L$ layer-line arises from unresolved combination of the two layer-lines that have Bessel orders of 5 and 6. In optical diffraction patterns, the two layer-lines with Bessel orders of 5 and 6 appeared separately in s-flagella, while they did not separate in n or es-flagella. This feature is seen also in the X-ray diffraction patterns: in the s-flagella only, the 52 d layer-line is broad and gives an impression that it consists of two adjacent layer-lines The doublet appearance of the 26 L% layer-line should have originated from the principal and subsidiary maxima of the layer-line, which were arced due to imperfect orientation within the specimen. As the arc of the principal (inner) maximum of this layer-line is sharp in each diffraction pattern, we could measure the meridional intercept with considerable accuracy (Table 4). W e regarded this intercept as the ZTABLE

Parameters Flagellar

species

Spacing (d) of the 26 !L layer-line (P) Spacing (A) of the 4.7 A layer-line (h) Plh Inclination (deg.) of the near-longitudinal helix at the surface The diameter

of the flagellar

4

obtained by X-ruy

diffructiot,

8

n

es

25.37

25.78

25.80

4.641

4.678

4.681

5.466 8.3

5.511 -2.76

5.513 -2.95

fiber is assumed to be 200 .k.

co-ordinate of t,he layer-line and calculat’ed t’he pitch (P) of the basic helix for each kind of flagellum. Table 4 shows that t’he pitch of the basic helix in s-flagella is 1.7% smaller than that in n and es-flagella. Because t’he 26 Li layer-line is not truly meridional, the spacing calculated from the intercept might, be slighbly smaller than t,he actual spacing. This error was estimated t’o be about l’yO when t’he basic helix was approximated by a continuous helix with a diameter of 100 A. Although this error set limits to the determination of ultrastructural parameters, t,he relative difference between different kinds of flagella would still be meaningful, since the diameter of these flagella was similar. Gonzalez-Beltran & Burge (1974) pointed out that, in X-ray diffraction patterns of flagella, the layer-line at a spacing of 4.7 d should be the truly meridional reflection that represents the axial separation of subunits (h) along the basic helix (Cochran et al., 1952). This layer-line was observed in the moderate-angle diffraction pattern of each kind of flagella (Fig. S(b), (d) and (f)). Here again, a slight difference in spacing was observed between s-flagella and the other types (Table 4). As GonzalezBeltran BE Burge (1974) noted, n = P/h is the number of subunits per turn of the basic helix. They found that n is very close to 5.50 in many kinds of flagella, and concluded that the number of the near-longitudinal rows of subunits is commonly 11. In this respect it should be noted that the handedness of the near-longitudinal rows is correlated with whether n is larger t)han 6.50 or not : when t.hrl helix has the same hand as the basic helix, n should be less than 5.50 and vice versa. The inclination (LB)

STRAIGHT

of the near-longitudinal

BACTERIAL

rows at a radius

r of a flagellar

tan LIB = Srr(llt

741

FLAGELLA

cylinder

is given by:

- 2)/11Pt,

where t = l/n is the unit twist of the helix and P the pitch of the basic helix, and a positive value is assigned to a helix with the same hand as the basic helix. The results of this calculation are also listed in Table 4. Because of the intrinsic error in the measurement of P and of the diffuseness of t)he 4.7 A layer-line, these results are not completely conclusive, but show good agreement with the results of t’he optical diffraction st’udies. Especially in the case of s-flagella, t,he coincidence of the X-ray results and the value obtained by the selection rule (O’Brien & Bennett, 1972) is remarkable; the selection rule implies that the number of subunits per turn of the basic helix is 5.467. Furthermore, these results suggest’ that the near-longitudinal helix is right,-handed in s-flagella and left-handed in n and as-flagella, since the basic helix is right-handed (see section (e), above). With the data of P and n, we could calculate the inter-subunit dist,ance (q) along the near-longit,udinal rows by: q2 = (llPt)2 The results

(Table

5) show that

+ (2r sin n (llt

the difference

- 2))2.

in q is small, especially

at, a larger

TABLE 5 Inder-.subunit

&dance along the near-longitudinub radii of the jlagellar cylinder

Flagellar species

Relative

Distance Radius

(A) =:

30

rows at diflerent

(A)

65

89

100

8

51.10

51.30

rl

51.46

51.48

51.51 51.51

51.63 51.52

0.7

0.4

0.0

0.2

difference

(%)

radius. As the relative difference in q is almost free from t’he int,rinsic error in the value of P, and as the inclination of t!he near-longitudinal rows is measured also by optical diffraction with good agreement’, we estimated the experimental error in this comparison to be less than 0.5% in view of t,he sharpness of the 26 a layer-line. Thus, only when bhe int,eractions between flagellin subunits are assumed to take place at radii as small as 30 pi does the model presentled seem to be compatible with this result. The possibility that flagellin monomers can interact- with each other at such small radii has been suggested by Fourier analysis of the equatorial reflections (K. Wakabayashi, K. Namba & R. Kamiya, unpublished resulbs) and by the recent, st)udy of three-dimensional image reconstruction of s-flagella (Shirakihara & Wakabayashi, 1979) but the act,ual mechanism by which flagella assume discrete curvatures must await, further studies. We are grateful to Drs T. Iino (University of Tokyo) and H. Kondoh (Kyoto University) for providing bacterial strains, to Dr M. Taniguchi (Nagoya University) for the gift of TMV and to Dr H. Neimark (State University of New York) for a critical reading of our manuscript during his stay in Nagoya. We also thank Mr Iwata of JEOL Co. for carrying out tungsten shadowing experiments. This work was supported in part by a grant from

742 the Ministry acknowledges

R. KAMIYA of Education, a fellowship

ET

AL.

Scioncc and (‘rllture OF Japan. One of the authors (R-K.) from t,he Toyoda Physical and Chemical Research Institut>c. REFlmlmC’b’S

I 4

Asakura, S. (1970). Adwaw f?io&s. (Japan), 1, 99.-15.5. Asakura, S. & Iino, T. (1972). J. Mol. Biol. 64, 251-268. Asakura, S., Eguchi, G. & Iino, T. (1964). .J. Nol. Bid. 10, 42-56. Burge, R. E. & Draper, J. C. (1971). J. Blol. HioZ. 56, 21--34. (London), 255, 121-124. Calladine, C. R. (1975). Nature Calladine, C. R. (1976). J. Theoret. BioZ. 57, 469-489. Calladine, C. R. (1978). J. Mol. BioZ. 118, 457-479. Champness, J. N. (1971). J. Mol. BioZ. 56, 296-310. Cochran, W., Crick, F. H. C. & Vand, V. (1952). Actn Crystallogr. 5, 581-586. Cohen, C. & Longley, W. (1966). Scierace, 152, 794--796. Finch, J. T. (1972). J. Mol. BioZ. 66, 291 ~294. of Sub-cellularstructures (Markham, R. Finch, J. T. & Klug, A. (1972). In The GenerntiotL & Bancroft, .J. B., cds), pp. 167-177, North-Holland Publishing Co., Amsterdam. Gonzalez-Beltran, C. & Burgc, E. E. (1974). ./. ;bToZ. BioZ. 88, 711-716. Hotani, H. (1976). J. L\foZ. HioZ. 106, 151-166. Hotani, H. & Kagawa, H. (1974). J. NoZ. HioZ. 90, 169-180. Iino, T. (1961). Genetics, 46, 1471-1474. Iino, T. (1962). J. Gen. l%ficrobioZ. 27, 167~.175. Kamiya, R. (1976). Ph.D. thesis, Nagoya University. Kamiya., R. & asakura, S. (1976). J. il!1/>Z. B%oZ. 106, 167-186. Kamiya,, R. & Asakura, S. (1977). .J. Mol. BioZ. 108, 513-518. Klug, A. & Berger, J. E. (1964). J. il!1oZ. BioZ. 10, 565-569. Kondoh, H. & Hotani, H. (1974). Biochim. Biophys. Acta, 336, 117-139. Kondoh, H. & Yanagida, M. (1975). J. Mol. BioZ. 96, 614--652. Leifson, E. (1960). Atlas of Bacterial P’ZageZZution, Academic Press, New York c%London. Macnab, R. M. & Omston, M. K. (1977). J. dlol. BioZ. 112, l-30. 9. Matsuura, S., Kamiya, R. & Asakura, k (1978). -1. I~oZ. BioZ. 118, 431-440. 3 .-P. (1965). ,I. XoZ. BioZ. 12, 88-118. Monod, J., Wyman, J. & Changeux, Moody, M. F. (1967). J. Mol. Bzol. 25, 167~~200. Nonomura, Y. & Kohama, K. (1974). J. ~1301. Sol. 86, 621~-626. O’Brien, E. J. & Bennett, P. M. (1972). J. J/oZ. BioZ. 70, 133-152. Shirakihara, Y. & Wakabayasbi, T. (1979). .J. Mol. BioZ. in the press. Wakabayashi, K. & Mitsui, T. (1970). .I. Mol. RioZ. 53, 567-570. Wakabayashi, K. Sr, Mitsui, T. (1972). ,I. l’kys. Sot. (,laparz), 33, 175-182.