JOURNAL OF BACTERIOLOGY, Nov. 1972, p. 986-993Copyright ( 1972 American Society for Microbiology

Vol. 112, No. 2Printed in U.S.A.

Flagellar Assembly Mutants in Escherichia coli1MICHAEL R. SILVERMAN AND MELVIN I. SIMON

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

Received for publication 31 July 1972

Genetic and biochemical analysis of mutants defective in the synthesis offlagella in Escherichia coli revealed an unusual class of mutants. These mutantswere found to produce short, curly, flagella-like filaments with low amplitude(.0.06 gim). The filaments were connected to characteristic flagellar basal capsand extended for 1 to 2 pm from the bacterial surface. The mutations in thesestrains were all members of one complementation group, group E, which islocated between his and uvrC. The structural, serological, and chemicalproperties of the filament derived from the mutants closely resemble those ofthe flagellar hook structure. On the basis of these properties, it is suggestedthat these filaments are "polyhooks", i.e., repeated end-to-end polymers of thehook portion of the flagellum. Polyhooks are presumed to be the result of a de-fective cistron which normally functions to control the length of the hook regionof the flagellum.

Biochemical and genetic analysis of mutantsdefective in the assembly and functioning ofbacterial flagella has been undertaken in anumber of laboratories with the goal of usingthis system as a model for studying the mech-anism involved in gene regulation and expres-sion (14, 22), protein structure and function (6,7, 15, 20), subcellular organelle assembly (4, 16,18), and primitive behavioral systems (3, 12).Escherichia coli mutants defective in the as-sembly of flagella can be obtained easily byselecting clones resistant to the flagellotropicphage x (21, 23). However, obtaining biochemi-cal information from these mutants is usuallydifficult for two reasons: (i) most of thesemutants possess no flagella and are phenotypi-cally indistinguishable; (ii) the assembly offlagella probably involves many structural pro-teins which lack a specific activity that can beeasily measured.During the course of genetic analysis of fla

mutants in E. coli, we became aware of a classof mutants with an unusual phenotype. Al-though these mutants were nonmotile withrespect to translational motion, they showed arapid spinning motion. They produced curlyfilaments with a wavelength of 0. 14 ,m whichwere 1 to 2 Am in length. What appeared to be anormal flagellar filament was often attacheddistal to this curl filament. Four independentmutants showed this phenotype and were all'A preliminary report of these findings was presented at

the Annual Meeting of the American Society for Microbiolo-gy, 21 April 1972.

found to be members of complementationgroup E (Silverman and Simon, in manuscript).In this paper we report the results of aninvestigation of the nature of the filamentsproduced by these mutants. Our experimentsindicate that they are composed of a subunitprotein very similar to the flagellar hook pro-.tein. We suggest that a defect in a cistron whichnormally codes for a protein that terminates thehook structure results in this filament which wenow call polyhook filament.

MATERIALS AND METHODSMedia. Tryptone broth contained per liter of

distilled water: 10 g of tryptone (Difco), 5 g of NaCl,and 0.1 g of thymine. L broth contained per liter ofdistilled water: 10 g of tryptone, 10 g of NaCl, 5 g ofyeast extract (Difco), and 0.1 g of thymine. L agarplates were prepared by adding 1.5% agar (Difco) to Lbroth. Motility plates were prepared by adding 0.35%agar to tryptone broth. Minimal medium containedper liter of distilled water: K2HPO4, 11.2 g; KH2PO4,4.8 g; (NH4)2S04, 2.0 g; MgSO4.7H20, 0.25 g;Fe2(S04)3, 0.5 mg; glucose, 5 g; and thiamine, 1 mg.The MgSO4-7H2O glucose and thiamine were addedaseptically after autoclaving. Amino acids and thy-mine, if required, were added to a final concentrationof 100 mg/liter. Minimal plates were prepared byadding 1.5% agar to minimal medium.

Bacteria. The flaE mutants described in thisstudy were derived from MS1350. MS1350 is his,argE, thy, thi, sup', galU, uvrC, strR, fla+ and wasconstructed by M. Silverman in M. Simon's labora-tory from the K-12 strain AB1884 which was obtainedfrom J. Adler. Two of the flaE mutants are suppressi-ble by a F80 d sup... transducing phage supplied by J.

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FLAGELLAR ASSEMBLY MUTANTS IN E. COLI

Abelson. These two are flaE234, and flaE694. Theother two flaE mutants are flaElOll, and flaE1071.

Isolation of mutants. Mutants in flagellar assem-bly were selected for their resistance to the flagello-tropic phage x after mutagenesis with ethyl methane-sulfonate (EMS). The procedure of M. Wright wasused for EMS mutagenesis (25) except that minimalmedium was used and 0.05 ml of EMS was added to2.5 ml of cell concentrate. Phage resistance selectionwas accomplished on L agar plates at 37 C with a softagar overlay consisting of a mixture of 2.5 ml ofmotility agar, plus 0.1 ml of exponential phase cellsgrown to allow the mutations to segregate, and 0.1 mlof phage, for a final multiplicity of infection ofapproximately 1. Survivors were restreaked twice andthen tested for motility. We obtained paralyzedmutants (mot), flagellar mutants (fla), and flaEmutants (polyhook mutants) by this procedure. Glu-cose must be excluded from the media used tocultivate flagellated cells (2, 26) or cells with curlfilaments (Silverman and Simon, in preparation),since the synthesis of these structures is subject tocatabolite repression.

Electron microscopy. Exponential phase cellsgrown in tryptone broth at 37 C were washed gentlytwo times with 0.05 M tris(hydroxymethyl)aminome-thane (Tris)-hydrochloride, pH 7.8, and spread oncarbonized, Formvar-coated grids. Excess fluid wasremoved after 5 min. Antibody at 1/100 dilution, ifadded, was flooded on the grid after the cells andallowed to react for 3 min. One wash with 0.05 M Trisbuffer followed. Antibody binding can be easilyobserved as the increase in the apparent width of thefilament. Grids were stained with 2% phosphotung-stic acid and examined with the Phillips 200 electronmicroscope.

Purification of curl filaments. The filamentsobtained from flaE mutants are referred to as curlfilaments or polyhooks. Curl filaments could beremoved from cells by shearing a 100-fold concentrateof cells, harvested in late log or early stationary phase,for 1 min at 13,000 rev/min in an Omnimixer (VirtisCo., Inc., Gardiner, N.Y.). Cells were removed by twolow-speed centrifugations at 10,000 x g for 5 min. Thesuspended filaments were then collected by centrifu-gation at 100,000 x g for 90 min. The pellet wasresuspended overnight in 0.05 M Tris-hydrochloride,pH 7.8. A low-speed clearing spin removed insolublematerial. Flagellin filaments were removed from thecurl filament preparation by incubating the mixtureat 56 C for 15 min. This treatment disaggregated theflagellin filament but had no apparent effect on thecurl filament. After a low-speed spin to removedenatured protein, the curl filaments remaining werepelleted by high-speed centrifugation, leaving theflagellin subunits in suspension. Flagellin filamentscould be prepared by shearing and concentratingflagella from MS1350 grown in tryptone broth. Foramino acid analysis, it was necessary to purifyflagellin more extensively and this was done byammonium sulfate precipitation followed by isopyc-nic centrifugation in cesium chloride (8). Homoge-neity was tested by sodium dodecyl sulfate (SDS)

acrylamide gel electrophoresis and electron micro-scope examination.

Purification of hook protein. Hook structurewas purified by a modification of the procedure ofAbram et al. (1). The flagellin filaments in 20 mg of apreparation of sheared wild-type flagella were disag-gregated by heating at 56 C for 15 min. Remainingflagellar components were collected by centrifugationat 100,000 x g for 180 min and resuspended in 0.05 MTris, pH 7.8. Electron microscope examinationshowed only hook-like structures remaining. Flagellinprotein was still present, but about 50 ,ug of hookprotein enriched by more than 100-fold remained.This was used for electrophoretic comparison on SDSacrylamide gel electrophoresis with curl filamentprotein.Preparation of whole flagella. Flagella with

basal structures were obtained by following the proce-dure of DePamphilis and Adler (5).

Antisera. Six-month-old rabbits were injectedintramuscularly and in the toe pads with 0.5 mg ofantigen in complete Freund's adjuvant every week for3 weeks. One month later another injection wasadministered, and the following week the animal wasbled and serum prepared. Anti-curl filament anti-body had some anti-flagellin filament activity. Thisactivity was removed by absorption with a prepara-tion of flagellar filaments which contained less than1% contamination by hook protein as determined bygel electrophoresis. Anti-curl filament antibody un-contaminated with anti-flagellin filament activitywas also prepared by injecting curl filaments derivedfrom flaE flaF double mutants. flaF (hag) is thecomplementation group which controls the structureof the flagellin protein (Silverman and Simon, inpress).

Complement fixation assay. The thermal stabil-ity of the flagellin and curl filaments could bemeasured by following the loss of complement-fixingactivity as a function of temperature (8). The result-ing subunits did not bind complement in the presenceof specific antibody. Each time point was held at thegiven temperature for 15 min and then cooled to 0 C.The procedure of Wasserman and Levine was used forcomplement fixation (24).SDS gel electrophoresis. The curl and flagellin

filaments as well as the hook structures were disaggre-gated according to the method of Dimmitt and Simon(9). The electrophoresis was performed according tothe method of Gelfand and Hayashi (11). Gels weredialyzed overnight in 5% trichloroacetic acid andstained for about 12 hr with 0.25% Coomassie blue in5% trichloroacetic acid. Gels were then destained in7% acetic acid.Amino acid analysis. Amino acid determinations

were performed according to the method of Fuller andDoolittle (10). In order to detect tryptophan, p-toluenesulfonic acid hydrolysis was used (19).

Construction of double mutants. flaE flaF (hag)double mutants were obtained by using P1 trans-duction to contransduce a uvr+ flaF (hag) fragmentinto a uvrC flaE recipient. The hag gene is moreclosely linked to the uvrC locus than flaE (Silverman

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SILVERMAN AND SIMON

and Simon, in press), so the double mutant can beconstructed by using uvrC as the selective marker.The method of Armstrong and Adler (3) was usedto select uvr+ recombinants with the exception thatcells were plated on minimal medium agar for uvr+selection. Double mutants could be identified bytheir inability to give fla+ recombinants with eitherflaE or flaF donor lysates whereas they give fla+recombinants with wild-type or other fla lysates.

RESULTSElectron microscopic examination. All

four group E mutants displayed unusual fila-ments - 1 to 2 Aim long with a wavelength of-0.14 ,um and an amplitude of -0.06 gim. Theirwidth was slightly greater than a flagellinfilament, or about 0.02 gm (Fig. 1A, 1B). Oftena flagellin filament was attached distal to whatwe shall tentatively call a curl filament (Fig.1C). The curl filament appeared to be attached

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directly to the basal assembly (Fig. 1D, 1E).The arrangement of subunits on the curl fila-ment appeared to be helical whereas that ofthe flagellin filament appeared more like par-allel rows. The wavelength of these filamentsis much less than that reported for curly fla-gella which result from the polymerization ofaltered flagellin molecules (13). Further stud-ies were carried out on mutant flaE694.Antigenicity of curl filaments. flaE mu-

tants produce curl filaments and flagellin fila-ments. Often the flagellin filament is foundattached to the curl filament. Antibody specificfor wild-type flagella binds only the flagellinfilament and not the curl filament region. Uponelectron microscope examination, this bindingcan be seen as an enhancement of the width ofthe filament (Fig. 2A). Conversely, anti-curlfilament antibody binds only the curl filamentregion (Fig. 2B). Significantly, this anti-curl

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FIG. 1. Curl fikaments on Escherichia coli flaE694. A and B, Filaments are attached to cells. C, A flagellinfilament is seen attached to the curl filament. D and E, The curl filament is attached directly to the basalflagellar structures.

988 J. BACTERIOL.

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FIG. 2. Antibody binding to curl and flagellin filaments and whole flagellar structures. A, Anti-flagellinfilament antibody added to filaments from mutant flaE694. B, Anti-curl filament antibody added to filamentsfrom mutant flaE694. C and D, Anti-curl filament antibody added to whole flagellar structures fromEscherichia coli.

serum also binds to the hook region of wholewild-type flagella (Fig. 2C, 2D).Thermal stability of the curl filament.

These initial observations suggested that thecurl filaments are not composed of flagellin andappear to be related to the hook structures.Hooks have been shown to be more stable toheating than flagellin (1, 7, 9). We thereforetested the curl filament for its relative heatstability. Curl filaments do not disaggregatewhen heated at 46 C, which is the temperatureat which flagellar filaments disaggregate. Theybegin to lose antigenic activity at 65 C and arecompletely disaggregated after heating at 72 C(Fig. 3). Their relative thermal stability allowedthe separation of the curl filament from theflagellar filament by heating at 56 C to removeresidual flagellar filament structures.SDS acrylamide gel electrophoresis. In

order to further compare the properties offlagellin with the hook protein subunit and theprotein component of the curl filament, all were

examined by SDS acrylamide gel electrophore-sis. The mobility of curl filament protein rela-tive to flagellin indicated that it is about 20%smaller than the flagellin molecule (Fig. 4). Onthe other hand, curl filament protein had thesame mobility as hook protein derived from apreparation of wild-type flagella. The bulk ofthe other protein in the hook protein prepara-tion has the same mobility as flagellin (Fig. 5)and represents residual flagellin.Amino acid analysis. Curl filament protein

is considerably different from flagellin withrespect to its amino acid composition (Table 1).Specifically, curl filament protein has moreresidues per molecule of proline, methionine,and phenylalanine than flagellin (Table 1). Notryptophan or cysteine is present in eithermolecule. It was not possible to obtain enoughhook protein to do amino acid analysis.Double mutants. flaE flaF (hag) double

mutants were prepared to test the relationshipbetween the flagellin gene and the expression of

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the curl filament phenotype. The result ofintroducing the flaF (hag) defect (several differ-ent flaF mutants were used) was to remove theflagellin filament distal to the curl filament. Nochange in the appearance, serology, or physicalproperties of the curl filament resulted. flaEmutants show no translational motion but dospin in tryptone broth. The flaE flaF doublemutants are motionless or show slight jerkingmotions. Binding the double mutants togetherby means of anti-curl antibody results in vio-lent counter-rotation of the bound pair of cells.The question of the relative contribution of thecurl and flagellin filaments to the motion of thecells will be the subject of a subsequent report(Silverman and Simon, in preparation).Antigenic specificity. The flaE flaF double

mutant served as a source of curl filamentdevoid of flagellin filament contamination. An-tisera prepared with filaments from the flaEflaF mutant react with low concentrations ofcurl filament and also react with high concen-tration of flagellin filaments sheared from wild-type cells (Fig. 6A, 6C). This reaction couldresult from the activity of partial hook struc-tures associated with the flagellin filaments.

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FIG. 4. SDS acrylamide gel electrophoresis.Anode is at bottom. In all gels, top band is bovineserum albumin reference and bottom band is lyso-zyme reference. A, 25-pg flagellin filament proteinfrom wild-type Escherichia coli. B, 25-pg curl fila-ment protein from mutant flaE694. C, 25-pg curlprotein plus 25-pg flagellin protein.

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FLAGELLAR ASSEMBLY MUTANTS IN E. COLI

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TABLE 1. Amino acid composition of flagellin andcurl filament proteina

Flagellin CurlAmino acid (molelli filament(mole%) (mole %)

Alanine ................. 12.1 10.0Arginine ................. 3.3 2.0Aspartic ................. 17.0 17.8Glutamic ................ 8.5 8.9Glycine ................. 9.5 10.1Histidine ................ 0.4 0.6Isoleucine ............... 4.2 3.6Leucine ................. 7.6 7.7Lysine .................. 7.2 3.1Methionine .............. 1.0 2.6Phenylalanine ............ 1.4 4.4Proline .................. 1.4 2.6Serine ................... 8.6 9.1Threonine ............... 9.9 10.0Tyrosine ................ 2.3 2.9Valine ................... 5.8 4.4

aAfter 24-hr acid hydrolysis.

Similar reactions were observed between anti-sera prepared against B. subtilis hook struc-tures and sheared flagella (9). The purifiedflagellin filaments were heated to 56 C for 15min, and this heating abolished the reaction ofsheared flagellin filaments with anti-flagellarantibody (Fig. 6B) but had no effect on the re-action with anti-curl filament antibody (Fig.6C). The anti-curl filament antibody reactionwith sheared flagellar filaments is apparentlydue to residual hook structures.

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FIG. 5. SDS acrylamide gel electrophoresis.Anode is at bottom. In all gels, top band is bovineserum albumin reference. Bottom band (not visible in

B and C) is lysozyme reference. A, 25-jig curl proteinfrom mutant flaE694. B, 50-jig protein from hookpreparation enriched from wild-type Escherichia coli

DISCUSSIONSeveral explanations for the nature of the curl

filament and the genetic defect which producedit were entertained: (i) the curl filament arosefrom the polymerization of an altered flagellinmolecule; (ii) the curl filament arose from thepolymerization of a defective hook component;(iii) the curl filament might be the result ofdefective termination of the hook structure.

It is unlikely that the curl filament resultsfrom the polymerization of an altered or mutantflagellin for several reasons. The curl filamentdefect does not map with the flagellin gene nor

does its complementation behavior make it a

member of the flaF (hag) group (Silverman andSimon, in preparation). The curl filament oftenhas a normal flagellin filament attached distalto it so the capacity to produce flagellin re-

mains unaltered. The curl filament is antigeni-cally dissimilar to the flagellin filament andshows greater thermal stability than the flagel-

flagella. C, 10-,ug curl filament protein and 20-pig hookprotein preparation.

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SILVERMAN AND SIMON

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FIG. 6. Reactivity of specific anti-curl filamentantibody. A, Curl filament plus anti-curl filamentantibody (0); curl filament 56 C for 15 min plusanti-curl filament antibody (0). B, Sheared flagellinfilament plus anti-flagellin filament antibody (0);sheared flagellin filament 56 C for 15 min plusanti-flagellin filament antibody (0); curl filamentplus anti-flagellin filament antibody (A). C, Shearedflagellin filament plus anti-curl filament antibody(0); sheared flagellin filament 56 C for 15 min plusanti-curl filament antibody (0).

lin filament. It is also 20% smaller than theflagellin protein. The amino acid analysisshowed more residues of proline, methionine,and phenylalanine per molecule than flagellinprotein. The curl protein could not, therefore,be derived from the flagellin molecule in anysimple manner. Furthermore, the presence ofcurl filaments on flaE flaF double mutantsdemonstrate that native flagellin molecules are

not necessary for the expression of the curlfilament phenotype.There are considerable similarities between

curl filament proteins and hook proteins in

serological behavior and on SDS acrylamide gelelectrophoresis. Furthermore the curl filamentalso shows higher thermal stability and greaterresistance to disaggregation at low pH (Silver-man and Simon, in preparation) than theflagellar filament. The hook structures havebeen reported to have similar properties (1, 6,8). In addition, the curl filament is situatedbetween the basal assembly and the flagellinfilament, the place where the hook structure isalways found. The hook structure appears iden-tical to a one-half wavelength section of curlfilament. Furthermore, it is unlikely that thecurl filament protein is polymerized from analtered hook gene product since two of the fourflaE mutants are ambers and might be expectedto produce no recognizable protein rather thana protein so similar to the hook protein. Thefour flaE mutants all have the same phenotype,and it is improbable that four independentmutations in the same gene should all producesuch a specific effect.These arguments lead us to believe that the

curl filament results from an abnormal termi-nation of the hook structure. Thus, the curlfilament is a continuous polymer of hook pro-tein molecules, i.e., this filament is a polyhook.We conclude that the group E gene codes for theproduction of a protein which controls thetermination of the hook structures, and a defectin this gene results in a polyhook.Mutants of bacteriophage T4 form poly-

sheaths from the polymerization of sheath pro-tein in the absence of core protein (17). We arecurrently examining the possibility that thereexists in the hook structure a minor componentwhich controls termination and is defective ingroup flaE mutants. Isolation of basal body-hook complexes and basal body-curl filamentcomplexes may answer this question.

ACKNOWLEDGMENTSWe wish to thank Marcia Hilmen and Michael Kaiser for

their excellent assistance and advice during this work.This investigation was supported by the National Science

Foundation research grant GB-15655. M.R.S. was supportedon the Public Health Service genetic training grantGM-00702 from the National Institute of General MedicalSciences.

LITERATURE CITED

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3. Armstrong, J. B., and J. Adler. 1969. Location of genesfor motility and chemotaxis on the Escherichia coligenetic map. J. Bacteriol. 97:156-161.

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FLAGELLAR ASSEMBLY MUTANTS IN E. COLI

4. Asakura, S., and T. Iino. 1972. Polymorphism of Sal-monella flagella. J. Mol. Biol. 64:251-269.

5. DePamphilis, M. L., and J. Adler. 1971. Purification ofintact flagella from Escherichia coli and Bacillussubtilis. J. Bacteriol. 105:376-383.

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7. DePamphilis, M. L., and J. Adler. 1971. Attachment offlagellar basal bodies to the cell envelope: specificattachment to the outer, lipopolysaccharide membraneand the cytoplasmic membrane. J. Bacteriol.105:396-407.

8. Dimmitt, K., and M. I. Simon. 1970. Purification andthermal stability of intact Bacillus subtilis flagella. J.Bacteriol. 105:369-375.

9. Dimmitt, K., and M. I. Simon. 1971. Purification andpartial characterization of Bacillus subtilis flagellarhooks. J. Bacteriol. 108:282-286.

10. Fuller, G. M., and R. F. Doolittle. 1971. Studies ofinvertebrate fibrinogen. Biochemistry 10:1305-1311.

11. Gelfand, D. H., and M. Hayashi. 1969. Electrophoneticcharacterization of 4474-specific proteins. J. Mol.Biol. 44:501-516.

12. Hazelbauer, G. L., and J. Adler. 1971. Role of galactosebinding protein in E. coli. Nature (London)230:101-104.

13. lino, T. 1967. Curly flagellar mutants in Salmonella. J.Gen. Microbiol. 27:167-175.

14. lino, T., and M. Enomoto. 1966. Genetical studies ofnon-flagellate mutants of Salmonella. J. Gen. Microbi-ol. 43:315-327.

15. lino, T., and M. Enomoto. 1970. Motility, p. 145-163. InJ. R. Norris and D. W. Ribbons (ed.), Methods in

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microbiology, vol. 5A. Academic Press Inc., New York.16. Kerridge, D. 1966. Flagellar synthesis in Salmonella

typhimurium: factors affecting the formation of theflagellar e-N-methyllysine. J. Gen. Microbiol.42:71-82.

17. J. King. 1968. Assembly of the tail of bacteriophage T4. J.Mol. Biol. 32:231-262.

18 Koffler, H., and R. W. Smith. 1970. Production andisolation of flagella, p. 165-172. In J. R. Norris and D.W. Ribbons (ed.), Methods in microbiology, vol. 5A.Academic Press Inc., New York.

19. Lui, T. Y., and Y. H. Chang. 1971. Hydrolysis of proteinwith p-toluenesulfonic acid. J. Biol. Chem.246:2842-2848.

20. Martinez, R. J., A. T. Ichiki, N. P. Lundh, and S. R.Tronick. 1968. A single amino acid substitution re-

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21. Meynell, E. W. 1961. A phage, 4X, which attacks motilebacteria. J. Gen. Microbiol. 25:253-290.

22 Pearce, U. B., and B. A. D. Stocker. 1967. Phase variationof flagellar antigens in Salmonella: abortive transduc-tion studies. J. Gen. Microbiol. 45:335-349.

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25. Wright, M. 1971. E. coli mutants lacking endonuclease I,ribonuclease I, or ribonuclease II. J. Bacteriol.107:87-94.

26. Yokota, T., and J. S. Gots. 1970. Requirement of adeno-sine 3',5'-cyclic phosphate for flagella formation inEscherichia coli and Salmonella typhimurium. J. Bac-teriol. 103:513-516.

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