Architectural Features of the Salmonella typhimurium Flagellar MotorSwitch Revealed by Disrupted C-Rings

Shahid Khan,* Rongbao Zhao,* and Thomas S. Reese†

*Department of Physiology & Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461;and †Laboratory of Neurobiology, NINDS, NIH, Bethesda, Maryland 20892

Received January 8, 1998, and in revised form March 31, 1998

The three-dimensional surface topology of rapid-frozen Salmonella typhimurium flagellar hook basalbody complexes was studied by stereo-examinationof thin-film metal replicas. The complexes containedthe extended cytoplasmic structure, composed ofthe switch complex proteins; FliG, FliM, and FliN.Distinct nanometer-scale element arrays, separatedby grooves, defined the outer surface of the cytoplas-mic (C-) ring. The number of array elements wascomparable to previously determined FliG and FliMcopy numbers in the basal body. In addition to basalbody complexes lacking C-rings, complexes contain-ing incomplete C-rings were identified. The incom-plete C-rings had lost segments of the proximalarray. Basal bodies with the distal C-ring arrayalone were not found. These findings are compatiblewith the spatial organization of the flagellar switchsuggested by previous biochemical data. r 1998

Academic Press

INTRODUCTION

The rotary motors of bacterial flagella are remark-able machines. They are reversibly energized bychemiosmotic gradients (Larsen et al., 1974; Mansonet al., 1977; Matsuura et al., 1977); reversal of thesign of the gradient reverses rotation sense (Khanand Berg, 1983). In addition, unlike unidirectionalATP-driven linear motors, they reverse rotationsense, even when the energizing chemiosmotic gradi-ent remains invariant (Silverman and Simon, 1974).

Five proteins, MotA, MotB, FliG, FliM, and FliN,form the motor machinery of the enteric bacteriaEscherichia coli and Salmonella typhimurium. Muta-tions in genes coding for these proteins give rise tothe mot (flagellate, nonmotile) phenotype (Arm-strong and Adler, 1969). This machinery is part ofthe flagellar base, a multi-subunit complex thatspans the cytoplasmic membrane and extends intothe cytoplasm. MotA and MotB are integral mem-brane proteins (Ridgeway et al., 1977), necessary for

assembly of flagellar intramembrane particle rings(Khan et al., 1988). Each ring particle may functionas a MotA/MotB ion-transporting complex consistentwith evidence that MotA functions as a protonconducting component of the flagellar motor (Blairand Berg, 1990) and interacts with MotB (Garza etal., 1996; Tsang et al., 1996).

The recently described cytoplasmic module of theflagellar basal body (Driks and DeRosier, 1990; Khanet al., 1991) contains FliG, FliM, and FliN (Francis etal., 1994; Zhao et al., 1995, 1996a,b). The nullphenotype of fliG, fliM, and fliN is nonflagellate.Nonchemotactic (che), as well as mot, mutant allelesare found in all three proteins which are thought toform an interactive structural complex, the switchcomplex (Yamaguchi et al., 1986). Study of fusionmutant strains (Francis et al., 1992; Kihara et al.,1996) and of purified protein binding interactions(Oosawa et al., 1994) has shown that FliG contactsFliF, the protein forming the MS-ring and proximalrod (Ueno et al., 1992). Genetic and biochemicalevidence also indicates that, of the three switchcomplex proteins, FliG interacts most directly withMotAMotB (Garza et al., 1995; Tang et al., 1996).FliM, but not FliG and FliN, binds to CheY in vitro.Phosphorylation of the CheY protein enhances thisinteraction (Welch et al., 1994), promoting clockwiserotation (Hess et al., 1988).

The basal cytoplasmic structure, copurified as partof hook basal body (HBB) complexes, has beenstudied extensively by negative-stain and cryoelec-tron microscopy. The structure has a bell-shapedmorphology (Khan et al., 1991), with a spatiallyextensive outer shell, or C (cytoplasmic)-ring (Fran-cis et al., 1994), and an inner rod, or C (cytoplasmic)-rod (Katayama et al., 1996; Zhao et al., 1996b). FliG,FliM, and FliN are present in 44 6 5, 34 6 5, and111 6 13 copies in column-purified basal bodies(Zhao et al., 1996a,b). The high copy numbers indi-cate that the proteins form the outer shell.

JOURNAL OF STRUCTURAL BIOLOGY 122, 311–319 (1998)ARTICLE NO. SB983999

311 1047-8477/98 $25.00Copyright r 1998 by Academic Press

All rights of reproduction in any form reserved.

Here we have examined metal replicas of quick-frozen, column-purified HBB complexes adsorbedonto mica. The mica chip technique has provedpotent for characterization of the 3D-architecture ofisolated macromolecular assemblies (e.g., Hanson etal., 1997). In common with other cryotechniques, it isfree of the staining/drying artifacts that plaguenegative-stain. Thus 3D information is inherent inthe images, and the high contrast allows examina-tion of single macromolecules. Cryoelectron micros-copy provides more complete 3D structural informa-tion, but the low contrast precludes evaluation ofsingle particles and requires image analysis to sub-tract out contributions of the microscope electronoptics and to extract 3D information from images ofthe 2D electron density projections.

We have found that nanometer-scale substructurein the C-ring can be visualized using this technique.An initial set of images is presented to document thisfact. The C-ring consists of at least two ringedarrays, each composed of discrete elements, sepa-rated by a prominent seam. We have further docu-mented examples of HBB complexes containing in-complete C-rings. The incomplete structures lackedparts of the proximal arrays, indicating that theseare distinct and separable from the distal arrayswhich remained intact. The new information sup-plied by these observations provides a structuralbasis for the previously reported lability of theC-ring.

MATERIALS AND METHODS

Sample preparation. HBB complexes from the S. typhimu-rium mutant strain SJW1031 (DfliC) were prepared as previouslydescribed (Zhao et al., 1996b). The DfliC mutant lacks flagellinand, therefore, flagellar filaments. Two-liter cultures were har-vested at late exponential phase. The cells were washed and thenlysed with EDTA/lysozyme. Cell debris was discarded and theHBB complexes purified from the resultant supernatant bymechanical shearing, followed by passage through an outermembrane protein antibody column (Zhao et al., 1996b). Afterlow-speed centrifugation (312000g, 10 min) to remove aggregatedlipoprotein, the HBB complexes were pelleted by high-speedcentrifugation (100,000g, 1 h), prior to resuspension in 0.1 ml highionic strength TET buffer (10 mM Tris–HCl, pH 8.0, 100 mMpotassium chloride, 5 mM EDTA, 0.1% (w/v) Triton X-100).

Mica flakes were prepared and pretreated with potassiumbuffer (30 mM Hepes, pH 7.2–7.4, 70 mM potassium chloride, 5mM MgCl2, 3 mM EGTA) as described (Heuser, 1989). A drop ofconcentrated mica slurry was added to the 0.1-ml HBB sampleand adsorption of the complexes onto the mica was stopped after30 s by dilution (1503) with buffer. The mica was then centrifuged(3100g) and drops of mica slurry quick-frozen in a Life Cellfreezing apparatus.

Silver-stained SDS gels and anti-FliG, FliM, and FliN ECL(enhanced chemiluminescence) immunoblots (Amersham Inc.)were performed as reported previously (Zhao et al., 1996).

Metal replication. The samples were rotary-shadowed in aBalzer’s 301 freeze-fracture machine, modified to incorporate arapid rotary stage (Wiltek Inc.) and Cressington 1202EC power

supply, as described previously (Zhao et al., 1996b). For produc-tion of Pt/Ir/Ta/C replicas, the typical Pt/C rod was replaced bymelting a tantalum (Ta) wire wound with platinum/iridium (Pt/Ir)in the hollow of the usual carbon rod. The replicas were viewed at120 kV in a JEOL 200CX electron microscope. Stereo pairs weretaken at a tilt angle of 63.5°, after establishing eucentricity.

Densitometry. The electron micrographs (372,000 or 368,000magnification) were densitometered on an Agfa Arcus-II scanner(transparency mode, 16-bit greyscale, 1200 dpi scan resolution).One pixel of the digitized image corresponded to 0.3 nm. Spatialperiodicities were accentuated by filtration subroutines availablein Adobe Photoshop (Version 3.0). Low pass gaussian filters (2-nmroll-off) were used to enhance the modularity of the structures andhigh pass filters (3- to 4-nm cutoff) to enhance substructuraldetail.

RESULTS

Cytoplasmic structure of HBB complexes on mica.Under the buffer conditions used, whole fields ofview of intact basal bodies were adsorbed onto mica.The structural modules of the HBB complex werereadily identified in the replicas. The apparentlyvariable appearance of the C-ring was due, as estab-lished by study of stereo-pair electron micrographs,to it lying in various orientations. The orientationsvaried from side-on views to severe upward or down-ward tilt. Upwardly tilted complexes revealed themouth and hollow of the belled cytoplasmic struc-ture. The MS-ring and it’s connectivity to the rod andC-ring was seen particularly well in downwardlytilted complexes. In single electron micrographssuch as the one shown (Fig. 1), the degree of tilt couldbe inferred from the apparent ellipticity of the rings.The uniformity of C-ring diameter was indicated bythe relative invariance of the elliptical long axis.

The C-ring has nanometer-scale substructure.Platinum/iridium/tantalum (Pt/Ir/Ta/C) replicas hadlow contrast, possibly reflecting increased codeposi-tion of carbon caused by a raised evaporation tem-perature (Fig. 2). Fine detail on the outer C-ringsurface was visible: a central seam was particularlyprominent and the elongated nature of the repeatelements was apparent in appropriately tilted im-ages (Fig. 2, inset). These replicas presumably havegreater spatial information content than Pt/C repli-cas, even though substructural elements were high-lighted to a lesser degree. Films with increasedcarbon content may benefit from the amorphousnature of carbon films to achieve both higher resolu-tion and more accurate representation of surfacerelief (Winkler et al., 1985).

The hook substructural elements made tracks,alternating with deep grooves, at an angle of 30 6 3°with the long axis. The subunits were seen asangular elements (ca. 3-nm-long axis; 1.5-nm-shortaxis), three to four being present per track half-turn

312 KHAN, ZHAO, AND REESE

(Fig. 3a). These features are consistent with thosedescribed for individual hook subunits visualized bycryoelectron microscopy (Morgan et al., 1993).

The thickness of Pt/C replicas limited C-ring sub-structural detail, which was obscured in tiltedsamples by increased deposition of metal on theC-ring wall. Examples of precursor basal bodies(Zhao et al., 1996b) provided en face views of basalbody periplasmic and cytoplasmic faces. These provedmore favorable for resolving substructural elements(Figs. 3b and 3d). C-ring substructural elementswere also evident in favorable side-on views in ourbest Pt/C replicas. In the example shown (Fig. 3c),the central seam divides the C-ring into proximaland distal segments, defined relative to the cellcenter. The distal and proximal segments consistedof arrays of elongated, fingerlike, and bilobed repeatelements, respectively. The element center-to-centerdistance was comparable to that for C-ring repeatelements seen in the cytoplasmic en face images.Finally, the dimensions of the various structuralmodules were comparable to those determined previ-ously in negative-stain or vitreous ice (Table I).

Topology of the hook basal-body complex. Thetopology of the HBB complex was evaluated fromexamination of stereo-pair electron micrographs. En

face views of basal body periplasmic and cytoplasmicfaces were readily distinguished in stereo (Figs. 4aand 4d). In periplasmic en face images, the proximalrod and S-ring jutted up from the C-ring. In cytoplas-mic en face images, the protrusion of the C-rod upfrom the hollow of the bell as well as the ridgedboundary between the C and MS-rings was evident.The C-ring and rod appeared unconnected by fibril-lar or other structures. This ability to distinguishsidedness from single-shot stereo-images is a particu-lar strength of this technique, as may be appreciatedby comparing these stereo-images to images of thesestructures obtained in negative stain (Fig. 6 of Zhaoet al., 1996).

Stereo-images of substantially tilted examples pro-vided, in addition, views of the inner surface. A ridgepresent on the inner surface (Fig. 4b), ca. 20 nmaway from the mouth, represented, most likely theboundary between the C and MS-rings. The ridgedfeature on the outer surface is presumably due to theoutwardly protruding lobes of the bilobed proximalarray elements seen in Fig. 3c.

Pt/Ir/Ta/C replicas, routinely provided images ofC-rings with visible central seam and substructuraldetails, in contrast to Pt/C replicas where such detailwas sensitive to orientation. Incomplete C-ringswere seen in both types of replicas, but the relativelyfeatureless nature of the C-ring wall in many Pt/Creplicated HBB complexes (e.g., Fig. 1) preventedelucidation of the nature of the structural defect.Pt/Ir/Ta/W/C replicas were therefore used to docu-ment and analyze incomplete C-rings. HBB com-plexes with incomplete C-rings lacked segments ofthe proximal array, as is evident in the stereo-example shown (Fig. 4c). It may also be appreciatedfrom examination of this stereo-image that the curvedappearance of the C-rings, commonly seen in manyimages (see Fig. 2), is due to the downward tilt ofthese structures.

Incomplete C-rings. A significant fraction of HBBcomplexes (ca. 20%, n 5 200) contained incompleteC-rings. A gallery of HBB complexes with incompleteC-rings is shown in Fig. 5. Complexes in a commonorientation, viewed side-on with C-rings tilted down-wards, were choosen to enable comparison. C-ringsin precursor basal bodies were indistinguishablefrom those in HBB complexes, as assessed by inspec-tion of single images. In incomplete C-rings, theextent of the absent segment varied from pieces thesize of single substructural elements to exampleswhere almost half of the proximal array was absent.The fact that the C-ring fragmented to generatepieces of the size of the substructural elements

FIG. 1. Pt/C replica of Salmonella typhimurium flagellarHBB complexes. The mean C-ring long axis for the 4 structuresshown is 44 nm (,10% standard deviation). The bottom-mostC-ring is fragmented, revealing the cytoplasmic (C-) rod. Magnifi-cation, 300,000; Bar, 50 nm.

313ARCHITECTURE OF S. typhimurium FLAGELLAR MOTOR SWITCH

implied that these elements disassemble, or as-semble, individually from the structure. The factthat the incomplete C-rings were marked by loss of asingle segment of varying size, rather than loss ofmultiple segments, implied that the disassembly ofsingle elements is affected by contacts made withadjacent elements (i.e., the process is cooperative).Examples where part of the proximal array wasseparated from the distal array, but still remainedpart of the basal body, were also catalogued. Theseshowed that neighboring proximal array elementscan remain connected even in absence of contactswith distal elements. In a small number of cases, ,5examples in over 100 incomplete structures, theC-ring fractured transversely from top to bottom.This indicated that contacts between adjacent ele-ments could break without separation of the arrays,but that such events were rare.

Basal bodies lacking C-rings constituted another20% (n 5 200) of the population. An example isshown (Fig. 5m). Significantly, no examples of C-rings containing the distal array alone were found.The most incomplete C-rings documented (e.g., Fig.5l) still contained a third or more of the proximal

array. This implied that the distal array is either lostor unrecognizably altered when loss of the proximalarray exceeds a critical size.

DISCUSSION

Here we examined the architecture of rapid-frozen, rotary-shadowed S. typhimurium flagellarbasal bodies adsorbed onto mica. We have obtainednew information on the surface architecture of theC-ring. This information has enabled us to identifyand categorize incomplete C-ring structures. Theincomplete structures provide, as argued below, im-portant constraints on ideas about the spatial organi-zation of the switch complex.

3D-surface topochemistry of the basal body. Thedistinct architectural modules of the basal body wereidentified in the replicas from side-views of HBBcomplexes and en face views of precursor basalbodies. The images reaffirmed the previously de-scribed hollow, belled morphology of the cytoplasmicbasal structure (Khan et al., 1991; Katayama et al.,1996) and the coisolation of the C-rod as part of HBBcomplexes in our preparations (Zhao et al., 1996b).

FIG. 2. Pt/Ir/Ta/C replica of Salmonella typhimurium HBB complexes. Low magnification (3260,000) field of view. The C-ring centralseam is visible in all complexes. (Inset) High magnification (3520,000) view showing substructural details of the C-ring arrays. Theelongated repeat elements (center to center distance 5 4.8 nm) are evident in the downwardly tilted HBB complex at the bottom. Arrowdenotes central seam. D, distal array; P, proximal array; Bar, 25 nm.

314 KHAN, ZHAO, AND REESE

Surface topochemistry of protein assemblies seenin metal replicas is a complex function of surfacerelief (‘‘shadowing’’) and electrochemistry (‘‘decora-tion’’). At low shadowing angles both effects contrib-ute to the image. Metal binding sites in crystals of

lumazine synthase–riboflavin synthase complexeswere located with subnanometer resolution by goldor silver decoration (Weinkauf et al., 1990). Periodicnanometer-scale surface substructure in the C-ringwas visualized in both Pt/C and Pt/Ir/Ta/C replicas.The lack of substructural detail in C-rings visualizedin replicas of rapid-frozen bacterial cell envelopes(Katayama et al., 1996) or isolated HBB complexes(Zhao et al., 1996b), may have been due to restrictionof the angle of deposition by the attached membraneand/or to differences in thickness of the metal film. Aprominent seam separated the C-ring into proximaland distal segments. These segments were composedof distinct repeat elements.

In cryoelectron-microscopic image-averaged recon-structions of HBB complexes the C-ring is dividedinto proximal and distal lobed segments by a slenderwaist (Francis et al., 1994), which could correspondto the central seam visualized in our replicas. A 33-to 34-element repeat periodicity has been obtainedfrom averaging power spectra of azimuthally pro-jected single C-ring electron densities (Thomas et al.,personal communication). An analysis of the shape,periodicity, and registration of the array elements,as well as detailed comparison of surface substruc-ture, seen in the replicas, with the electron densitymap obtained from cryoelectron microscopy, will bepresented elsewhere.

Here we have used the additional structural infor-mation, particularly the visualization of the centralseam, to identify and characterize incomplete C-rings. These structures lacked a segment of theproximal array, the size of which varied from as littleas one repeat element to up to two-thirds of thearray. C-rings lacking multiple proximal segmentswere not observed, implying that the association ofindividual elements with the HBB complex wasstabilized by contacts with their neighbors. The

FIG. 3. Substructural elements. Original micrographs (leftpanels) were Gaussian low-pass filtered, then high-pass filtered(right-panels). The cut-off spatial frequency varied slightly (ca.3–4 nm21) from image-to-image. (a) Hook. (b) Basal body periplas-mic face. (c) Basal body side-view. Arrow denotes central seam. (d)Basal body cytoplasmic face. The center-to-center distance of thedistal repeat elements was measured to be 4.6 nm from selectedsegments in (c) and (d). The diameter of the C-ring in (d) was 49nm. Therefore, the number of elements (circumference/center-to-center distance) in the distal ring was 33. Magnification, 580,000;Bar, 25 nm.

TABLE IDimensions of Basal Body Structural Modules

Module n Diameter

Proximal rod 5 17.2 6 0.5 nmS-ring 5 26.7 6 0.8 nmC-ring 25 47.4 6 2.4 nmC-rod 3 12.0 6 0.4 nm

Note. N denotes sample size. The elliptical long axis in case oftilted images was taken as the diameter. The contribution of themetal film (ca. 1.5 nm) was not subtracted. Both side-views ofHBB complexes (n 5 22) and en face views of precurser basal bodycytoplasmic faces (n 5 3) were used to estimate C-ring diameters.The C-ring diameters of basal bodies with en face periplasmicfaces were not used, as these may have been flattened somewhatdue to interactions with the mica. The diameters of the proximal-rod and S-ring were estimated from periplasmic en face imagesand those for the C-rod for cytoplasmic en face images of precursorbasal bodies. These images were rare, hence the low n.

315ARCHITECTURE OF S. typhimurium FLAGELLAR MOTOR SWITCH

incomplete structures would have been extremelydifficult to detect in projected images of negativelystained or vitreous ice embedded basal bodies. It isnot known whether such structures are present invivo or generated during isolation.

FliG, FliM, and FliN proteins in the C-ring. Inprevious work, we showed that FliG, FliM, and FliNare the major structural proteins of the cytoplasmicbasal structure, being the only proteins present inamounts sufficient to circumscribe the C-ring (Zhaoet al., 1996b). We further found that HBB complexesisolated from fliG, fliM, and fliN mot mutants lackedFliM, FliN, and morphologically distinguishable C-rings. We argued that FliM and FliN contact FliF viaFliG based on the facts that FliM and FliN are losttogether in all fliM, fliN, and fliG mot mutant basal

bodies examined, while FliG is retained in fliM orfliN mutant basal bodies (Zhao et al., 1995, 1996b).

How might structural understanding of C-ringlability, as refined by the incomplete C-rings docu-mented in this study, be reconciled with biochemi-cally identifiable loss of the FliM and FliN proteinsfrom isolated mot mutant basal bodies? The possibil-ity that the incomplete rings result from proteolysiscan be ruled out on the basis of gel immunoblots.These showed no evidence of proteolysis of the FliG,FliM, and FliN proteins in the preparations, consis-tent with earlier results (Zhao et al., 1995). Thus, theincomplete structures must be generated by loss ofone or more of these proteins.

We had previously assigned FliM and FliN to theC-ring and FliG to the ‘‘extended’’ MS-ring (Zhao et

FIG. 4. Stereo-topology of the Salmonella typhimurium HBB complex. The tilt (z)-axis is oriented vertically in these stereo-pairs. (a)En face view of the basal body periplasmic face. (b) HBB complex with upwardly-tilted C-ring. (c) HBB complex with incomplete C-ring(asterisk). (d) En face view of the basal body cytoplasmic face. Magnification, 300,000.

316 KHAN, ZHAO, AND REESE

al., 1996b), based on the facts that: (i) Basal bodieslacking C-rings were labeled with anti-FliG antibodyto an extent comparable to C-ring containing basalbodies (Francis et al., 1994; Zhao et al., 1995), (ii)Basal body preparations lacking C-rings purified

from a fliN100LP mot mutant contained FliG inamounts similar to that found in wild-type HBBcomplexes (Zhao et al., 1996b). (iii) The MS-ringappeared thickened in C-ring containing basal bod-ies relative to acid-treated basal bodies lacking

FIG. 5. Incomplete C-rings. (a–d) Intact C-rings in HBB complexes (a,d) and precursor basal bodies (b,c). Absence of the distal-array ofthe C-ring can range from 5 nm or less (e,f) sized segments to examples where half or more of the array is absent (g–j, n); (k,m) provideexamples where still-intact 5–10 nm distal array segments have separated from the proximal array; (m) also shows an HBB complexlacking the extended cytoplasmic structure (C-ring and rod). Magnification, 375,000.

317ARCHITECTURE OF S. typhimurium FLAGELLAR MOTOR SWITCH

C-rings (Francis et al., 1994). If so, loss of proximalarray segments should be coupled to loss of theconstitutent protein, either FliM or FliN. This inher-ent lability should have been accentuated by muta-tion: fliM and/or fliN mot mutant basal bodies shouldhave been found that lacked FliM or FliN alone. Thisprediction does not agree with the above notedresults of our screen of basal bodies purified fromspontaneously isolated fliM and fliN mot mutants(Zhao et al., 1996).

Alternatively, FliG could form the distal, and FliMthe proximal, C-ring. FliN could form part of theproximal array, together with FliM, or an innerbacking that depends on the presence of both arraysfor its integrity. In such a case, association of FliMand FliN with FliF would depend on FliG, and bothproteins would be lost when incomplete C-rings aregenerated by loss of the proximal array, as observed(Zhao et al., 1996b). The C-ring array elementsevident in our replica images need not correspond toindividual protein subunits, but this is the simplestpossibility. Thus, the fact that the arrays are com-posed of ca. 35 discrete elements, comparable to thepreviously determined number of FliG and FliMcopies in the basal body (Zhao et al., 1996b), alsosupports this assignment.

Why, then, is the distal C-ring array not seen inHBB complexes containing FliG but not FliMFliN(Francis et al., 1994; Zhao et al., 1995). It may be thatindividual FliG subunits coalesce to bond exten-sively with FliF, the MS-ring protein, in lieu ofinteractions with the distal array. The resultantthickened MS-ring would have a diameter more-or-less indistinguishable from that of FliG-minus MS-rings in intact HBB complexes (see Fig. 13 of Heuser,1989). Synchronous stabilization of the FliG sub-units in a defined orientation enabling either clock-wise or counter-clockwise rotation by contacts madewith FliM subunits in an adjacent array is a keytenet of the model of the flagellar switch, recentlyproposed (Khan, 1997). This postulate explains theoccurrence of fliM/fliN mot mutations and, in addi-tion, their rescue by overexpression of the mutantproteins (Lloyd et al., 1996). Our failure to find HBBcomplexes containing distal C-ring arrays alone isconsistent with the scenario envisaged above whereFliG subunits comprising the proximal array coa-lesce with FliF in absence of FliMFliN. This loss ofstructural organization being as predicted by themodel.

Importantly, it should now be possible to test theseideas. The composition of the C-ring arrays may beestablished by examination of metal-replicated, anti-body-decorated basal bodies and effects of switchcomplex mot and che mutations on their structuralorganization subsequently clarified.

We thank Jennifer Peterson for expert technical assistance.Supported, in part, by Grant GM36936 from the National Insti-tutes of Health.

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319ARCHITECTURE OF S. typhimurium FLAGELLAR MOTOR SWITCH