FliG Subunit Arrangement in the Flagellar Rotor Probed by Targeted Cross-Linking

FliG is a component of the switch complex on the rotor of thebacterial flagellum. Each flagellar motor contains about 25FliG molecules. The protein of Escherichia coli has 331 aminoacid residues and comprises at least two discrete domains. AC-terminal domain of about 100 residues functions in rotationand includes charged residues that interact with the statorprotein MotA. Other parts of the FliG protein are essentialfor flagellar assembly and interact with the MS ring proteinFliF and the switch complex protein FliM. The crystal structureof the middle and C-terminal parts of FliG shows two globulardomains joined by an ${alpha}$ -helix and a short extended segment thatcontains two well-conserved glycine residues. Here, we describetargeted cross-linking studies of FliG that reveal featuresof its organization in the flagellum. Cys residues were introducedat various positions, singly or in pairs, and cross-linkingby a maleimide or disulfide-inducing oxidant was examined. FliGmolecules with pairs of Cys residues at certain positions inthe middle domain formed disulfide-linked dimers and largermultimers with a high yield, showing that the middle domainsof adjacent subunits are in fairly close proximity and puttingconstraints on the relative orientation of the domains. Certainproteins with single Cys replacements in the C-terminal domainformed dimers with moderate yields but not larger multimers.On the basis of the cross-linking results and the data availablefrom mutational and electron microscopic studies, we proposea model for the organization of FliG subunits in the flagellum.

INTRODUCTION

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Introduction

Bacterial flagella are turned by rotary motors that obtain energyfrom the membrane gradient of protons or sodium ions (2, 17,25). In many species, the motor can rotate either counterclockwise(CCW) or clockwise (CW), and cells regulate their swimming behaviorby controlling reversals in motor direction (26). In the entericspecies Escherichia coli and Salmonella enterica serovar Typhimurium,CCW flagellar rotation allows cells to swim in relatively straightpaths, whereas CW rotation causes cells to tumble (i.e., rapidlychange orientation). When a cell moves up a gradient of a chemicalattractant or other positive stimulus, the probability of CWrotation is decreased so that the run in a favorable directionis extended. Other modes of motor control are found in someother species; the motors of Rhodobacter sphaeroides alternatelystart and stop, while those of Sinorhizobium meliloti rotateat variable speeds (1). Whatever the mode of swimming, the swimmingis regulated by sensory cues to bias cell movement toward attractantstimuli and away from repellents.

The molecular mechanisms of motor rotation and direction switchingare not understood. The proteins that function in rotation havebeen identified, mainly through genetic studies of E. coli andSalmonella. The stator (nonrotating part) is formed from theproteins MotA and MotB (10-12, 33, 36). The motor contains severalindependently functioning stator complexes (5, 6), each withthe subunit composition MotA₄MotB₂ (24, 32). The stator complexesare located in the cytoplasmic membrane surrounding the basalbody (21), where they function to conduct either protons orsodium ions (depending on the motor) and couple ion flow torotation (4, 15, 37). MotB contains a conserved, functionallycritical aspartate residue (Asp32 in E. coli MotB) that appearsto have a direct role in ion conduction (50). Charge-neutralizingmutations of this aspartate induce a conformational change inthe stator complex, as evidenced by the altered protease susceptibilityof MotA (23). On the basis of these findings, it was proposedthat proton (or Na⁺) association/dissociation at Asp32 mightdrive conformational changes in the stator that act on the rotorto drive rotation. A power stroke mechanism such as this isconsistent with the torque-speed characteristic of the motoras measured by Berry and Berg (3). The motor torque was foundto be nearly constant over a wide range of speeds, which ispredicted for a mechanism in which energy from the ion gradientis used to apply force to the rotor, but not for a Brownianratchet mechanism in which the gradient is used to rectify thermallydriven movements.

The rotor proteins that function to support rotation are FliG,FliM, and FliN. Together, these proteins form the "switch complex"that is essential for assembly, rotation, and CW/CCW switchingof the flagellum (45). FliG is involved most directly in motorrotation (19, 28). Its C-terminal domain (FliG_C), in particular,is essential for rotation but dispensable for flagellar assembly(28). FliM is closely involved in CW/CCW switching (34) andinteracts with the chemotaxis signaling molecule phospho-CheY(7). The precise role of FliN is unknown, but it is essentialfor flagellar assembly and has been implicated in the processof flagellum-specific export that delivers the proteins formingthe exterior structures (the rod, hook, and filament) to theirsites of installation (39, 44). FliN also appears to be involvedin switching, but its specific role in this process is unknown(19).

The rotor contains many molecules of FliG, probably about 25(38, 47). FliG binds to the MS ring protein FliF (13, 16, 22,31), and electron microscopic studies have suggested that aportion of FliG might be located on the cytoplasmic face ofthe MS ring (14, 38, 40) (Fig. 1). It has been difficult toassign specific features in micrographs to FliG, however, andthe precise location of FliG in the rotor remains uncertain.The crystal structure of the middle and C-terminal domains ofFliG (FliG_MC) from Thermotoga maritima revealed two compact,globular domains connected by an ${alpha}$ -helix and a short segmentwith extended structure (8). The extended segment includes twowell-conserved Gly residues and might therefore be flexible.The C-terminal domain of FliG contains well-conserved chargedresidues that are important for rotation (27) and that interactwith charged residues of the stator protein MotA (48, 49). Thestructure shows that these charged residues are clustered ona prominent ridge in the C-terminal domain (8, 29). In the flagellum,FliG is presumably positioned with this ridge near the stator.FliG interacts with FliM (22, 30, 42), and sequence alignmentshave identified conserved surface features in both the middleand C-terminal domains that might function in binding FliM orother proteins in the rotor (8).

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FIG. 1. Schematic diagram of the flagellar basal body. FliG is believed to be in the upper (membrane-proximal) part of the C ring, where it can interact with the MS ring protein FliF and the stator protein MotA. Its exact location is unknown. OM, outer membrane; PG, peptidoglycan; IM, inner membrane.

While the crystal structure of FliG_MC provides a useful startingpoint, to understand the functions of FliG in the flagellum,we must determine how the $~$ 25 FliG subunits are organized. Here,we used a targeted cross-linking approach to deduce featuresof FliG subunit organization. Several positions on the surfaceof FliG_MC were mutated to cysteine, singly and in pairs, andcysteine-specific cross-linking was examined using either maleimideor disulfide-inducing oxidants. The results indicate that themiddle domains (FliG_M) are adjacent to each other in the flagellum,with their long axes pointing toward each other. The FliG_C domainsdo not appear to be in close contact, but they are able to undergodomain rotations or other movements that allow cross-linkingbetween proteins with single Cys replacements in certain positions.We propose a model for FliG organization in which the FliG_Cdomains are at an outer radius, where they interact with thestator, while the FliG_M domains are nearer the MS ring.

MATERIALS AND METHODS

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Materials and Methods

Strains, plasmids, and mutagenesis. The FliG-null strain DFB225 (28) was used for assays of mutantFliG function and for cross-linking experiments with cysteine-containingmutant proteins. To control for cross-linking that might occurindependent of flagellar assembly, parallel experiments werecarried out with strain RP3098, a flhDC mutant that does notassemble flagella (a gift from J. S. Parkinson).

Cysteine replacements were made by site-directed mutagenesisof the fliG gene in pSL27, using the Altered Sites procedure(Promega). Plasmid pSL27 is a derivative of pAlter-1 (Promega).It expresses FliG at about twice the wild-type level and complementsa fliG-null strain to wild-type function in a soft-agar swarmingassay (28). Mutagenesis reactions were carried out by usingthe manufacturer's protocol, except that the concentration ofmutagenic oligonucleotide was increased two- to fivefold insome reactions. Mutations were confirmed by dideoxy sequencing,and then mutant plasmids were transformed into the fliG-nullstrain DFB225 and the nonflagellated control strain RP3098.

The function of the mutant FliG proteins with Cys replacementswas tested in soft-agar swarming assays. Cells of strain DFB225transformed with pSL27 variants encoding the mutant proteinswere cultured to saturation in TB (1% tryptone, 0.5% NaCl) anddiluted 1:500 or 1:1,000 in fresh medium, and then 1-µlportions of the diluted cultures were spotted onto soft-agartryptone plates (1% tryptone, 0.5% NaCl, 0.30% Bacto agar [Difco]).The plates were incubated at 32°C, and once swarming commenced,the colony diameters were measured at regular (approximatelyhourly) intervals. Rates were computed from plots of swarm diameterversus time and compared to the rates for wild-type controls(strain DFB225 transformed with wild-type pSL27) included onthe same plates.

Cross-linking. Freezer stocks of DFB225 cells containing the Cys mutant pSL27plasmids were used to inoculate 1- to 2-ml cultures, which weregrown to saturation in LB (1% tryptone, 0.5% yeast extract,0.5% NaCl) containing 125 µg/ml ampicillin at 37°Cwith shaking. Cultures were diluted 100-fold in TB containingampicillin (typically 4 ml) buffered at pH 7.5 with 43 mM sodiumphosphate and incubated overnight at 32°C with shaking inculture tubes (17 by 100 mm). The optical density at 600 nmwas measured for each culture, and then the cells were pelletedby centrifugation ( $≥$ 2,000 x g, 15 to 30 min) and resuspendedin a volume of XL buffer (20 mM sodium phosphate, pH 7.4, 150mM NaCl) calculated to give an optical density at 600 nm of10. Samples (100 µl) were dispensed and chilled on icefor at least 10 min before cross-linking.

For cross-linking with 1,6-bismaleimidohexane (BMH), 100-µlsamples of cells were mixed with 2 µl of 50 mM BMH (dissolvedin dimethyl sulfoxide and stored at –20°C), whichresulted in a final BMH concentration of approximately 1 mM.Cells were incubated on ice for 30 min, and then the reactionwas quenched by adding 2.5 µl of 2-mercaptoethanol (BME).Control samples received 2 µl of dimethyl sulfoxide andthen 2.5 µl of BME. In a small number of experiments weused the longer bifunctional cross-linker 1,4-di-(2-pyridyldithio-propionamido)butane(DPDPB). The protocols were similar to those used with BMH,except that the DPDPB concentration was 2 mM and cross-linkingwas done at room temperature for 90 min.

In some cross-linking experiments we used samples solubilizedin Triton X-100. Cells were cultured as described above, exceptthat in the second culturing step we used 25 ml of bufferedTB in 125-ml flasks. Cells were collected by centrifugation,frozen at –70°C, thawed, and then resuspended in 0.5ml of 0.5 M sucrose, 10 mM Tris, pH 8.0, on ice. Lysozyme wasadded from a 20-mg/ml stock solution to a final concentrationof 1 mg/ml, and then EDTA was added from a 0.5 M stock solutionto a final concentration of 120 mM. Samples were stirred for30 min on ice and then solubilized by addition of 650 µlof a cocktail containing 127 mM MgSO₄, 6.4% Triton X-100, 840µM 4-amidino-phenylmethanesulfonyl fluoride, 8 µg/mlpepstatin, 17 µg/ml leupeptin, and 170 µg/ml DNaseI. Samples were stirred for 3 h on ice and then centrifuged(5,600 x g, 20 min) to remove unsolubilized material. Aliquots(100 µl) of the supernatant were used for cross-linkingexperiments by using the protocols described above, except thatthe final sodium dodecyl sulfate (SDS) concentration was 2%(rather than 7%) in the samples loaded on gels.

Disulfide cross-linking was induced by iodine. Cells were washedand resuspended in XL buffer as described above and then treatedwith I₂ at a concentration of 0.2 mM, added from a 20 mM workingstock solution in ethanol. Following 10 min of incubation onice, unreacted Cys residues were blocked with 20 mM N-ethylmaleimidefor 5 min on ice. In experiments to monitor disulfide rereduction,dithiothreitol was added from a 1 M stock solution (in water)to a final concentration of 20 mM.

In some of the iodine-induced cross-linking experiments (thosewith Cys at positions 297, 298, and 299 in FliG_C) we used 50mM Tris, pH 8.0, 50 mM NaCl, and the experiments were done atroom temperature. The other steps were the same as those describedabove.

Following cross-linking, cells were collected by centrifugationand resuspended in 100 µl of a loading buffer containing62.5 mM Tris, pH 6.8, 7% (wt/vol) SDS, 10% glycerol, and $~$ 0.01%bromophenol blue. For samples cross-linked with BMH, the loadingbuffer also included 5% BME. Samples were heated for 10 minat >95°C, refrigerated briefly, and either sonicatedfor 15 to 20 s (Branson model 450 with a microtip; power, 1;duty cycle, 50%) and loaded on SDS-polyacrylamide gels (typically7.5%) (10 µl/well) or frozen at –70°C for lateruse. Following electrophoresis, the gels were soaked in transferbuffer (20% [vol/vol] methanol, 48 mM Tris base, 39 mM glycine,0.0375% [wt/vol] SDS) at room temperature with slow shakingfor at least 10 min. Proteins were transferred to nitrocellulosemembranes by using a Bio-Rad semidry blotting apparatus at 13V for 48 min. After transfer, the membranes were soaked forat least 30 min in 4% dry nonfat milk in 20 mM Tris, pH 8.0,500 mM NaCl at room temperature with slow shaking. Immunoblottingwas carried out using anti-FliG (dilution factor, between 1:2,500and1:5,000) and horseradish peroxidase-conjugated goat anti-rabbitsecondary antibody. Blots were visualized using WestPico SuperSignalreagents (Pierce) and X-ray film. Bands were quantified by usingvideo densitometry and the public domain NIH Image program (developedat the National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/)

RESULTS

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Results

Cys replacement mutations. Site-directed mutagenesis was used to make cysteine replacementsat 22 positions on the surface of FliG_MC, singly and in variouspairwise combinations. The positions of the replacements onthe structure are shown in Fig. 2, which also shows the doubleCys replacements that were studied. In FliG_M, Cys replacementswere made at positions that sampled most of the surface of thedomain. In FliG_C, the substitutions were made on the faces aroundthe active site ridge, encircling the domain at approximatelyits widest part. This choice of positions was based on mutationaldata which indicated that the active site ridge interacts withthe stator protein MotA (49) and is therefore not likely toface toward adjacent FliG subunits.

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FIG. 2. Locations of Cys replacements in FliG. The diagrams show the crystal structure of residues 115 to 327 of T. maritima FliG (PDB accession no. 1lkv). The two views are related by 180° rotation, as indicated. The residue numbers are the numbers for the E. coli protein. The dashed line in FliG_C indicates the charge-bearing ridge (27, 29) that interacts with MotA (49). Double Cys replacements made for the cross-linking study are listed on the right.

Cross-linking in FliG_M. Proteins with single and double Cys replacements in FliG_M wereexpressed from plasmid pSL27 in cells of the fliG-null strainDFB225, and cross-linking was examined using the bifunctional,sulfhydryl-specific cross-linker BMH. Products of cross-linkingwere characterized by using FliG immunoblots. The majority ofthe proteins with single Cys residues in FliG_M showed littleor no cross-linking with BMH. Most of the doubly substitutedproteins also did not show significant cross-linking, but four(117/166, 117/170, 120/166, and 120/170) gave a product at aboutthe position expected for a FliG dimer and also a series oflarger products with fairly high yields. Representative resultsare shown in Fig. 3. The product yields for BMH cross-linkingof proteins with Cys residues in FliG_M were quantified by densitometryand are summarized in Fig. 4A.

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FIG. 3. Examples of BMH cross-linking of proteins with two Cys replacements in FliG_M. The positions of the Cys replacements are indicated at the bottom. DFB225 is a fliG-null strain, which is flagellated and motile when the fliG gene is provided on the plasmid. Strain RP3098 contains a deletion of the flagellar regulatory genes flhDC and does not assemble flagella even when the fliG plasmid is present. Kd, kilodaltons.

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FIG. 4. Yields of BMH-induced cross-linking in FliG proteins with one or two Cys replacements. (A) Summary of yields for Cys at positions in the middle domain (FliG_M). The shaded bars indicate the yields of cross-linked dimer, and the solid bars indicate the combined yields of all multimers larger than dimers. The error bars indicate the standard deviations of two or more determinations. (B) Summary of yields of cross-linked dimers for Cys at positions in the C-terminal domain. Larger multimers were not observed for any of the Cys replacements in FliG_C.

To verify that cross-linking occurred within flagella ratherthan by a collisional mechanism, the same experiments were conductedwith mutant strain RP3098, which does not express any chromosomalflagellar genes and does not assemble flagella. The Cys replacementproteins were expressed from the same plasmid (pSL27), and thelevels of the FliG proteins in the nonflagellated strain weresimilar to those observed in the flagellated strain. The cross-linkingyields were greatly reduced in the nonflagellated strain. Onlya small amount of dimer and none of the higher-molecular-weightproducts were observed (Fig. 3).

BMH is fairly long and might cross-link between Cys residuesthat are separated by more than 10 Å. To put tighter constraintson residue proximity, we examined disulfide cross-linking inthe 117/166, 117/170, 120/166, and 120/170 double mutants andin the corresponding single-Cys controls. Cells expressing theCys mutant proteins were treated with iodine to induce disulfidebond formation, and products of the reaction were examined asdescribed above, using anti-FliG immunoblots. The results forthe 117/166 and 120/166 double mutants are shown in Fig. 5.Iodine treatment of these double mutants yielded several cross-linkedproducts, in a pattern similar to that seen with BMH. High-yieldcross-linking was not observed in the corresponding single-Cysmutants (Fig. 4), and the dimer and higher-molecular-weightbands were essentially eliminated when samples were treatedwith DTT before electrophoresis. Thus, they were the productsof disulfide cross-linking. Experiments with the nonflagellatedflhDC strain resulted in none of the higher-molecular-weightproducts and only a small amount of dimer, indicating that thecross-linking occurred within flagella and not through collision.

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FIG. 5. (A) Iodine-induced cross-linking of proteins with double Cys replacements in FliG_M. Some preexisting disulfide cross-linking was seen in both of the mutants, and cross-linking was increased by treatment with iodine. (B) Effect of treatment with DTT. (C) Cross-linking experiment with the nonflagellated flhDC strain. Kd, kilodaltons.

While the high-yield cross-linking was observed only in cellsthat assembled flagella, this cross-linking could neverthelesshave involved a protein that was not in the flagellum, suchas a protein nearby in the membrane. To rule out cross-linkingto nearby proteins in the membrane, we solubilized the flagellawith the detergent Triton X-100 (final concentration, 1%) beforecross-linking with iodine. Triton X-100 releases the basal bodiesfrom the membrane but leaves the C rings intact (14). For boththe 117C/166C and 120C/166C proteins, the yields and distributionof cross-linked products were similar to those seen in the experimentswith whole cells (Fig. 6).

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FIG. 6. Cross-linking in samples solubilized in Triton X-100. Cys replacements are indicated at the top. Kd, kilodaltons.

FliM contains a single native Cys residue and is thought tobe near FliG in the flagellum (30, 42, 45). To see if any ofthe cross-linked products contained FliM, we cross-linked the117C/166C and 120C/166C double-mutant proteins with BMH andused anti-FliM immunoblots to examine the products. The blotsshowed a single strong band corresponding to the FliM monomerbut no bands at higher molecular weight (data not shown). FliMis therefore not a component of the cross-linked products.

Because the high-yield cross-linking required assembled flagellaand two Cys residues in FliG_M, still occurred in flagella solubilizedin detergent, and did not involve FliM, we conclude that theproducts are dimers and larger multimers of FliG formed by cross-linkingbetween adjacent subunits within the flagellum. The positionsin FliG_M that gave the highest yields of FliG dimers and multimers(positions 117, 120, 166, and 170) are at opposite ends of theFliG_M domain (cf. Fig. 8).

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FIG. 8. Model for the arrangement of FliG subunits. Three copies of the FliG_MC protein from T. maritima are shown. The Gly-Gly motif and the positions giving high-yield cross-linking are indicated by space-filled alpha carbons and E. coli residue numbers. The FliG_M domain is oriented with residues 117 and 120 of one subunit near residues 166 and 170 of the adjacent subunit. The FliG_C domain is positioned with the active site ridge (indicated by the dashed line) toward the viewer and oriented in an approximately radial direction relative to the axis of the flagellum. The relative orientation of FliG_M and FliG_C domains is somewhat different from that in the crystal structure and was adjusted by making sterically allowed changes in torsion angles of the Gly-Gly linker. The three positions in FliG_C that gave a cross-linked dimer with a moderate yield are also indicated. One of the residues in FliG_C that gave a cross-linked dimer (residue 297 in E. coli; residue 299 in T. maritima) is at the end of the charge-bearing ridge and is among the charged residues important for function.

Cross-linking in FliG_C. We next examined cross-linking of proteins with single or doubleCys replacements in the C-terminal domain. The product yieldsfor BMH cross-linking experiments with FliG_C are summarizedin Fig. 4B. Most of the Cys-containing proteins did not showmeasurable cross-linking with BMH. A moderate level of cross-linkingwas observed for the proteins with single Cys replacements atposition 297, 298, or 299 and also for double mutants containingCys at any of these positions. Residues 297, 298, and 299 aregrouped at one end of the active site ridge. Cross-linking gavea single product, at the position expected for a FliG dimer(Fig. 7), and was again much stronger in the strain that assemblesflagella than in the nonflagellated control strain. We concludethat the product is a FliG dimer formed by cross-linking betweenadjacent FliG_C domains in the flagellum.

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FIG. 7. BMH-induced cross-linking of proteins with single Cys residues in FliG_C. The positions of the Cys replacements are indicated at the bottom; double Cys mutants containing any of these replacements showed similar yields of cross-linking (see Fig. 4). The 297C, 298C, and 299C proteins gave similar dimer yields when cross-linking was induced by iodine (not shown). Kd, kilodaltons.

The results were similar when cross-linking was induced by iodine.Upon oxidation, the proteins with Cys residues at position 297,298, or 299 cross-linked to form a FliG dimer with a moderateyield (data not shown). Other positions in FliG_C gave littleor no cross-linking. A small number of experiments were doneusing the bifunctional cross-linker DPDPB, which is longer thanBMH. Even under conditions expected to maximize cross-linking(high concentration, long incubation time, room temperature)we observed only dimer formation, and only with Cys at position297, 298, or 299 (data not shown).

Function of the proteins showing strong cross-linking. To confirm that the proteins that showed high-yield cross-linkingwere not altered by the Cys replacements, we measured the swarmingrates of fliG-null cells expressing the mutant proteins. Allof the mutant proteins that exhibited cross-linking (117/166,117/170, 120/166, 120/170, 297, 298, and 299) supported swarmingat normal rates (data not shown).

DISCUSSION

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Discussion

Zhao and coworkers measured levels of FliG in purified flagellarbasal bodies of Salmonella and estimated that the basal bodycontains around 40 molecules of FliG and a similar number ofFliM molecules (47). En face electron micrographs of C ringsshowed a subunit structure with rotational symmetry that wasmost often 34-fold but varied somewhat (±2) between specimens(41, 46). Because FliM is thought to be in the part of the structurewith $~$ 34-fold symmetry, the number of FliM molecules is probably34 per basal body, which is close to the estimate of Zhao etal. (47). The MS ring/proximal rod is formed from the proteinFliF (18, 43). The number of FliF subunits was estimated tobe approximately 26, using both scanning transmission electronmicroscopy (35) and ³⁵S labeling of basal body proteins (20).A recent electron microscopic reconstruction of the MS ringshowed 26-fold symmetry (38). FliG is believed to be presentat the same number of copies as FliF, because when FliF andFliG are fused genetically into a single protein, flagella arestill assembled and can function well (13). The 26-fold symmetryof MS rings is retained and becomes somewhat more pronouncedwhen the rings become associated with FliG (38). Thus, whilethe subunit number might vary slightly among specimens (46),a typical motor should have close to 26 copies of FliG.

Because the C ring has the appearance of an axially symmetricstructure, we might expect its subunits to be arranged withaxial symmetry. More complex arrangements involving local symmetryaxes can be imagined, however, and are probably relevant inthe case of FliN, which can form a stable tetramer in solution(9). The cross-linking results obtained with FliG_M are mostsimply interpreted in terms of an axially symmetric array ofsubunits, oriented so that residues 117 and 120 of one subunitare near residues 166 and 170 of the adjacent subunit (Fig.8). Within the FliG_M domain, the residue 117/120 pair is separatedfrom the 166/170 pair by about 2.8 nm (average of the four C-Cdistances). The alpha carbons of Cys residues in a disulfidebond are separated by about 0.5 nm, and so if disulfide cross-linkingoccurred with no movement of the subunits, the intersubunitspacing would be approximately 3.3 nm. For 26 subunits and aspacing of 3.3 nm, the subunits would fall on a ring with adiameter of 27 nm (3.3 x 26/ ${pi}$ ). This represents a lower boundaryfor the subunit spacing and ring diameter, because the subunitsmight have some freedom to move and cross-linking could trapthem in positions away from equilibrium.

It has been difficult to locate FliG precisely in electron micrographsof flagellar basal bodies. The MS rings appear to be thickerin samples that contain FliG than in samples depleted of FliG(14, 40), which might indicate that part of the protein lieson the underside of the MS ring. The increased thickness wouldaccount for only a small part of the mass of FliG, however,so the bulk of FliG might be located somewhat apart from theMS ring. FliF has two transmembrane segments, a large periplasmicdomain that forms much of the MS ring and the proximal rod,and a C-terminal domain consisting of about 90 residues locatedin the cytoplasm. FliG attaches to a fairly well conserved segmentof about 15 residues near the C terminus of FliF (16). Otherparts of the FliF C-terminal domain are less conserved and arepredicted to be largely helical and possibly nonglobular (i.e.,extended in conformation) in E. coli and its close relatives.This segment of FliF could form a linker that reaches out fromthe ordered part of the MS ring that is visible in micrographsto contact FliG at a greater radius. If the linker were relativelyflexible, then FliG would remain largely disordered and invisiblein micrographs until FliM and FliN joined the assembly and completedthe C ring.

In an effort to locate FliG, Thomas et al. (40) examined thestructures of basal bodies containing FliF-FliG fusion proteins,including a fusion protein that lacks a substantial portionof the FliG amino-terminal domain. Careful comparison of wild-typeand fusion mutant basal bodies indicated that the amino-terminaldomain of FliG might lie at a radius of 16.5 nm. This wouldrequire an intersubunit spacing of 4 nm for the FliG_M domainsif the motor contains 26 FliG molecules and the FliG_M domainsare evenly spaced. This is somewhat greater than the estimategiven above based on immobile subunits, but it is within therange of the cross-linking results if we allow for some movement.

Because FliG_C interacts with MotA (49) and MotA is located inthe membrane surrounding the MS ring (21), we think that FliG_Cis likely to lie at a greater radius than FliG_M. Electron microscopicreconstructions revealed two concentrations of electron densityin the upper (membrane-proximal) part of the C ring, at radiiof about 17 nm and 22 nm (14, 40). These two features mightcorrespond to the FliG_M and FliG_C domains, respectively. Alternatively,FliG_C might correspond to the inner feature (at a radius of17 nm), with FliG_M attached to the MS ring at an even smallerradius. These alternative arrangements for FliG are illustratedin Fig. 9C and D.

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FIG. 9. Model for the arrangement of FliG subunits in the flagellum. (A) Overall arrangement of 25 FliG subunits, viewed along the axis of rotation. The FliG_M domains are at an inner position to allow interaction with FliF, while the FliG_C domains are at a larger radius to allow interaction with the stator complexes. The ridge bearing the conserved charged residues is oriented radially. The positions in FliG_M that yielded cross-linked dimers and multimers are indicated in the enlarged view by black circles, and the region in FliG_C where Cys residues gave cross-linked dimers is indicated by gray circles. (B) Hypothesized small rotations of FliG_C that could bring together positions 297, 298, and 299 in adjacent subunits. (C) Image of the flagellar basal body obtained from electron microscopic reconstructions (40). The outlines indicate the features sketched in panel D. (D) Two possibilities for the location of FliG relative to features seen in the electron microscopic reconstruction.

Previously, we suggested that the ridge on FliG_C that bearsthe functionally important charged residues might be orientedparallel to the edge of the rotor, so that charged residueson the ridge could interact successively with charged groupson the stator as the rotor turns. If the ridge were orientedin this way, then adjacent subunits would approach each othermost closely through positions at opposite ends of the ridge,and we would expect the highest yield of cross-linking in mutantswith Cys residues at both ends of the ridge. This was not observed.Instead, cross-linking occurred in fair yield for proteins withsingle Cys replacements at one end of the ridge and was notsignificantly greater in proteins with Cys at the other endalso. Cross-linking between the single-Cys proteins impliesthat equivalent positions in adjacent subunits can approachclose enough to form a disulfide cross-link. This would requiresome movement of the domains or a more drastic rearrangementsuch as local unfolding. The required movement would be especiallylarge if the ridge were oriented parallel to the edge of therotor, as it would then involve the rotation of one or bothdomains by more than 90°. An approximately radial orientationof the ridge appears more likely, because small rotations ofthe domain would then be sufficient to bring together the positionsthat showed cross-linking, particularly if the residue 297/298/299end of the ridge were pointed inward (toward the axis of theflagellum [Fig. 9]).

If the charged ridge is oriented radially, then the shorterdimension of the domain would be directed toward the adjacentsubunit, and we would predict a sizable gap between the domains.The gap between adjacent FliG_C domains could be anywhere inthe range from 2 nm to 3.3 nm, depending upon whether FliG isin the outer or inner location discussed above and what symmetryFliG_C adopts (even if 26 copies of FliG_C are present, it mightlocally adopt the 34-fold symmetry of the C ring). This relativelylarge spacing could account for the absence of any strong cross-linkingbetween proteins with Cys replacements on the sides of the domain,even with the long reagents BMH and DPDPB. Thus, while the FliG_Cdomains appear to be able to undergo rotational movements thatbring the 297/298/299 ends of adjacent domains into proximity,they are constrained from bringing their sides together. Motorswitching seems likely to involve movements of FliG_C, and weare presently examining switch mutants to determine whethercross-linking yields are affected by mutations that alter motorbias or rates of switching.

ACKNOWLEDGMENTS

This work was supported by grant 8R01-EB2041 from the NationalInstitutes of Biomedical Imaging and Bioengineering. B.J.L.received partial support from NIH training grant 5T32-GM08537.M.D. was supported by a grant from the Biosciences UndergraduateResearch Program at the University of Utah. The Protein-DNAcore facility at the University of Utah receives support fromthe National Cancer Institute (grant 5P30 CA42014).

We thank Matthew Borrowman, Karen Jordan, Monica Longtin, KateSadler, Akiko Tsunoda, and Christina M. Yong for assistancewith cross-linking experiments and swarming assays.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biology, University of Utah, Salt Lake City, UT 84112. Phone: (801) 585-3709. Fax: (801) 581-4668. E-mail: blair{at}bioscience.utah.edu.

REFERENCES

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