JOURNAL OF BACTERIOLOGY, Oct. 1971, p. 282-286Copyright 0 1971 American Society for Microbiology

Vol. 108, No. 1Printed in U.S.A.

Purification and Partial Characterization ofBacillus subtilis Flagellar Hooks'

K. DIMMITT AND M. I. SIMON

Department of Biology, University of California, San Diego, La Jolla, California 92037

Received for publication 7 July 1971

A method for preparing bacterial flagellar hook structures is described. Themethod involves isolating intact flagella from a mutant which makes thermally la-bile flagellar filaments and heat-treating them to disaggregate the filament prefer-entially. The resulting hook preparation can be separated and purified by velocityand isopycnic centrifugation. The purified hooks sediment at a relative S value of77. On acrylamide gel electrophoresis in sodium dodecyl sulfate, they show onemajor and a number of minor protein bands. The purified hooks can be used toimmunize rabbits, and the resulting antiserum is hook-specific. These results sup-port the notion that hooks are composed of a protein that differs from flagellin.

Bacterial flagella terminate in hooklike struc-tures which are in turn fixed into the cell mem-brane and cell wall (3-5). The hook portion of aflagellum is easily distinguished from the fila-ment by its morphology (1) and its immuno-chemical reactivity (6, 11). Koffler and his co-workers showed that the hook is more stablethan the filament under a variety of denaturingconditions. This property can be used as a basisfor the purification of small amounts of the hookstructure (2).

Although very little is known about the natureor function of the hook, this information almostcertainly is implicated directly in the growthmechanism of flagella and in motility (8). Fur-ther, we have shown that the presence of thehook stabilizes the structure of the filament (7).To characterize the hooks and the other structuresattached to the base of the flagella, we developedprocedures for isolating relatively intact, pureflagella (7). Using these as source material, wecould isolate the hook and the other structures.To enhance the difference in stability between thehook and the filament, we prepared the intactflagella from a mutant of Bacillus subtilis whichmakes heat-labile filaments. The filaments couldbe easily removed, leaving the hook structure in-tact. The hooks were purified further by differ-ential centrifugation and isopycnic gradient tech-niques. A similar method was described byKoffler and his co-workers (Xth InternationalCongress of Microbiology).

1 Presented in part at the XI" International Congress ofMicrobiology, Mexico City, August 1970.

MATERIALS AND METHODS

Bacterial strauns. The strain used for the preparationof intact flagella was B. subtilis GD1. This strain wasderived from B. subtilis BD71, which we obtained fromD. Dubnau (hisAl, ura, argC4). B. subtilis GDI car-ries a mutation in the flagellin structural gene whichresults in the synthesis of flagella which disaggregatewhen heated at 12 C lower than the temperature ofdisaggregation of wild-type flagellar filaments.

Media. The minimal medium of Spizizen and Anag-nostapoulus was used to grow the bacteria. In experi-ments in which the flagella were labeled with radioac-tive amino acids, the Casamino Acids in the mediumwas replaced by a mixture of 18 amino acids at a finalconcentration (except for the radioactive amino acid)of 10 jsg/ml.

Purification of flagellar hooks. B. subtilis GDI wasgrown in minimal medium supplemented with 9H-la-beled leucine and histidine. Specific activity of the leu-cine was 12 mCi/Mumole; that of the histidine was 3mCi/Mmole. A 30-ml amount of culture was collected,and intact flagella were prepared as previously de-scribed (7). Nonradioactive, intact B. subtilis GDI fla-gella were added as carrier so that the final concentra-tion of intact flagellar protein was about 100 ug/ml.The flagellar filaments then were sheared by passingthem through a 24-gauge needle ten times, and thesample was heated at 65 C for 30 min. The resultingsolution was layered on a gradient formed of threelayers of 38, 45, and 53% sucrose in 0.01 M tris(hy-droxymethyl)aminomethane (pH 8.0); 0.5% Brij 58(Atlas Chemical Industries); and 0.01 M ethylenedi-aminetetraacetate (TBE buffer). This layered solutionwas centrifuged at 100,000 x g in the SW27 rotor of aSpinco model L ultracentrifuge for 5 hr at 4 C. A bandformed at the 45% layer (Fig. 1). This band was re-moved and dialyzed against TBE buffer overnight at 4C. The sample then was applied to a linear 30 to 60%(v/v) renografin gradient. After the sample was centri-

282

B. SUBTILIS FLAGELLAR HOOKS

fuged at 144,000 x g for 11 to 12 hr at 10 C in theSW41 rotor, fractions were collected from the bottomof the tube.

Electron microscopy. Samples were placed directlyon carbon-coated, Formvar-covered grids. These werenegatively stained with 1% phosphotungstic acid (pH7.3). A Phillips 200 electron microscope was used withan accelerating voltage of 60 kv.

Antigens, antibodies, and complement fixation. Anti-hook antibody was prepared by concentrating the frontpart of the renografin gradient, mixing the proteinwith incomplete Freunds adjuvant, and injecting it in-tramuscularly and into the toepads and footpads of aNew Zealand White rabbit. Three weeks later the rab-bit was injected again. One week later the animal wasbled. The total amount of hook protein injected wasderived from 90 mg of intact flagellar protein.

Complement fixation was performed by the proce-dure of Wasserman and Levine (12).

Sodium dodecyl sulfate-acrylamide gel electropho-resis. The procedure for sodium dodecyl sulfate-acryl-amide gel electrophoresis as reported by Gelfand andHayashi (10), used 7.5% cross-linked gels. The hooksample was concentrated by centrifugation. The pelletwas suspended in 1% sodium dodecyl sulfate, 1% s-mercaptanethanol, and 0.01 M tris(hydroxymethyl)aminomethane (pH 9.1) in 12% sucrose, and this sus-pension was heated at 100 C for 3 min. The samplewas cooled and applied immediately to the gel. 3H-la-beled hooks and 3H-labeled flagellin samples were co-run with 14C-labeled XX-174 proteins (a gift from M.Hayashi) as marker. The marker preparation con-tained four proteins (molecular weights 54,000, 45,000,21,000, and 8,000 daltons). These proteins were suffi-cient to obtain clearly the positions of the unknownbands.

RESULTSShearing and heating intact flagella (7) re-

sulted in a mixture of hooks, flagellin, and somemembrane fragments. This mixture was layeredonto a sucrose gradient and centrifuged to sepa-rate the hooks from the flagellin. Figure 1 showsthe distribution of labeled flagella on the gra-dient. Most of the particulate material formed aband in the middle of the gradient. This materialwas purified further by isopycnic gradient cen-trifugation in renografin. Figure 2 shows thedistribution of the radioactive material on reno-grafin. When samples were removed from dif-ferent regions of the gradient and examined byelectron microscopy, it was found that the mate-rial at the highest density was mostly bare hookswithout basal structures (Fig. 3A, B, and C). Atintermediate densities, hooks bearing basal struc-tures were most abundant. In the least densefractions, most of the hooks were attached torelatively large pieces of membrane, and most ofthe particulate material appeared to be mem-brane-like. Subsequent work was done with theleading fractions from the renografin gradient.

This material could be further purified by sedi-mentation. More than 50% of the radioactivityderived from the renografin gradient sedimentsin a relatively homogeneous band with an ap-proximate S value of 77 (Fig. 2).

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FRACTION NUMBER

FIG. 1. Sucrose layer centrifugation of sheared andheated flagella. Brackets indicate the region of the gra-dient collected for subsequent purification procedure.

4200

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FRACTION NUMBERFIG. 2. (A) Distribution of radioactive label after

renografin gradient centrifugation. The band removedfrom the sucrose layer was dialyzed, applied to therenografin gradient, and centrifuged for 12 hr at144,000 x g. (B) Distribution of purified hooks aftersucrose gradient centrifugation. Fraction nine, takenfrom the renografin gradient, was dialyzed, mixedwith "4C-labeled MS-2 bacteriophage, layered onto a15 to 30% sucrose gradient, and centrifuged at 180,000x gfor 90 min. The dotted line represents the marker.

283VOL . 108 , 1 971

DIMMITT AND SIMON

FIG. 3. Electron micrographs of materialfrom renografin gradient. Samples were removedfrom fractions 6 (A),9 (B), and 13 (C) taken from the renografin fractionation (Fig. 2 (A)) and stained with 2% phosphotungsticacid (pH 7.0). The diameter of the hooks is about 17 nm.

284 J. BACTERIOL.

B. SUBTILIS FLAGELLAR HOOKS

Antihook antibodies. The purified hook mate-rial could be used to prepare antihook antisera.It is known that antihook antibodies can bind tohooks (6); in electron micrographs, the hookswere covered with antibody protein, but the fila-ments were not affected. Results of complementfixation using antibodies prepared against reno-grafin-purified hooks are shown in Fig. 4. Theantisera reacted strongly with intact flagella.There was much less reaction with flagellar fila-ments obtained by shearing. No reaction wasdetected with flagellin or with flagellin repoly-merized to form flagellar filaments.The antibody also was used to measure the

thermal denaturation of the hook structure. Thehook protein maintained complete antigenic ac-tivity until it was heated above 72 C (Fig. 5).Heating at 72 C lead to a loss of about 20% ofthe antigenic activity in 15 min. The loss of thedistinct hook structure was observable by elec-tron microscopy. After heating at 72 C for 15min, the preparation retained many structureswith the characteristic hook morphology; afterheating at 80 C, no such structures were found.Acrylamide gel electrophoresis. The hook

structures were disaggregated by heating in de-tergent, and the constituents were studied byacrylamide gel electrophoresis. The pattern inFig. 6 contains a major peak which includesabout 35% of the radioactivity applied to the gel.The major peak is clearly different from fla-gellin. Its estimated molecular weight is 33,000daitons as compared to 41,000 daltons for fla-gellin.

DISCUSSIONThere is increasing evidence which suggests

that the hook portion of the flagellum is com-posed of proteins that differ from the filament

100 _ __------

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60 I

L. 40 /

c, /Se 20, -

ANTIGEN ( ug)FIG. 4. Reaction of antihook antibody measured by

complement fixation. Antihook antibody was used at1/4,000 dilution. Symbols: 0, intact flagella; M,sheared flagella; A, reaggregated flagellar filaments;x, flagellin.

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O 0060 64 68 72 76 80 84

TEMPERATURE (°C)FIG. 5. Thermal stability of purified hooks after

suspension in 0.01 M tris(hydroxymethyl)aminometh-ane (pH 7.3) and 0.05 M NaCI buffer. A sample washeated at the temperature indicated for 15 min. Aftercooling, the antihook antiserum was used to measureresidual antigentic activity.

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FIG. 6. Sodium dodecyl sulfate-acrylamide gelelectrophoresis of purified hooks. The material re-maining in fractions 5 to 10 of the renografin gradient(Fig. 2A) was pooled and collected by high-speed cen-trifugation. The pellet was suspended and dissolved insodium dodecyl sulfate buffer; half the sample wasmixed with flagellin. Broken line (x ) indicates the fla-gellin marker.

subunit. Koffler and his co-workers originallyshowed the marked heat stability of the hooks.Lawn (11) showed that antibody to the flagellarfilament will coat the filament but will not coatthe hook. More recently, Koffler and his co-workers showed that tryptic digests of purifiedhook protein give patterns similar to flagellin butalso contain specific differences, further evidenceof a major hook protein which differs from fla-gellin. Finally, we have been able to purify fla-gellar hooks that also contain parts of the fla-gellar basal structure. Antisera prepared withthis material specifically coat the hook structure

285VOL. 108, 197 1

DIMMITT AND SIMON

and react with intact flagella but not with fla-gellin. Upon disaggregation, the hooks containeda major protein constituent with a molecularweight 20% lower than that of flagellin.These results strongly suggest that the hooks

are composed of a protein that is not flagellin.However, there are a number of possible expla-nations for the relationship between flagellin andthe hook subunit protein. (i) The hook cistronmay have been derived from a flagellin precursorcistron by gene duplication and mutation (H.Koffler, personal communication); (ii) the hooksubunit may be a specifically modified form offlagellin; (iii) flagellin and hook subunit proteineach may be derived from independent cistrons.The purification procedures described above,together with specific genetic crosses, shouldallow us to distinguish among these possibilities.The minor components found on acrylamide

gel electrophoresis may result from other partsof the hook structure, e.g., the basal discs andthe core. Experiments designed to identify theseproteins and to assign them to specific flagellarstructures are in progress.

ACKNOWLEDGMENT

This investigation was supported by grant no. GB-15655from the National Science Foundation.

LITERATURE CITED1. Abram, D., H. Koffler, and A. E. Vatter. 1965. Basal

structure and attachment of flagella in cells of Proteusvulgaris. J. Bacteriol. 90:1337-1354.

2. Abram, D., J. R. Mitchen, H. Koffler, and A. E. Vatter.1970. Differentiation within the bacteriol flagellum andisolation of the proximal hook. J. Bacteriol. 101:250-261.

3. Abram, D., A. E. Vatter, and H. Koffier. 1966. Attach-ment and structural features of flagella of certain bacilli.J. Bacteriol. 91:2045-2068.

4. Cohen-Bazire, G., and J. London. 1967. Basal organellesof bacterial flagella. J. Bacteriol. 94:458-465.

5. DePamphilis, M. L., and J. Adler. 1971. Fine structureand isolation of the hook-basal body complex of flagellafrom Escherichia coli and Bacillus subtilis. J. Bacteriol.105:384-395.

6. Dimmitt, K., and M. I. Simon. 1970. Antigenic nature ofbacterial flagellar hook structures. Infec. Immun. 1:212-213.

7. Dimmitt, K., and M. Simon. 1971. Purification andthermal stability of intact Bacillus subtilis flagella. J.Bacteriol. 105:369-375.

8. Doetsch, R. V., and G. J. Hageage. 1968. Motility in pro-caryotic organisms. Biol. Rev. Cambridge Phil. Soc. 43:317-362.

9. Emerson, S. E., K. Tokuyasu, and M. I. Simon. 1970. Bac-terial flagella: polarity of elongation. Science 169:190-192.

10. Gelfand, D., and M. Hayashi. 1969. Electrophoretic char-acterization of OX 174-specific proteins. J. Mol. Biol. 44:501-516.

11. Lawn, A. M. 1967. Simple immunological labellingmethod for electron microscopy and its application tothe study of filamentous appendages of bacteria. Nature(London) 214:1151-1152.

12. Wasserman, E., and L. Levine. 1961. Quantitative micro-complement fixation and its use in the study of antigenstructure. J. Immunol. 87:290-296.

286 J. BACTERIOL.