Vol. 175, No. 8JOURNAL OF BACTERIOLOGY, Apr. 1993, p. 2169-21740021-9193/93/082169-06$02.00/0Copyright © 1993, American Society for Microbiology

MINIREVIEW

Gene to Ultrastructure:the Case of the Flagellar Basal Body

SHAHID KHAN

Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461

INTRODUCTION

The locomotion of many bacterial species is powered bythe rotation of helical filamentous structures, the flagella.The flagellar basal body is a tiny (<0.1-1Lm) substructurecontiguous with the filament (>1 pum) that embeds eachflagellum into the cell wall. The basal body plays a key rolein flagellar assembly and in the energization and switching offlagellar rotation. For over 2 decades, the flagellar basalbody was defined as the structure which could be coisolatedwith the filament (2, 17) and subsequently copurified with thehook as the hook-basal body complex (HBB). This structurehas been analyzed extensively (27). It is now clear, primarilyon the basis of genetic and ultrastructural analyses, that anumber of additional structural components make up theflagellar basal body. Some of these structures have beenisolated biochemically. These structures are likely to playprincipal roles in the various basal body functions.Three discrete structural modules may be identified: (i)

cytoplasmic extensions contiguous with the flagellar filamentand HBB, (ii) inner membrane particle rings, and (iii) outermembrane-associated basal discs. All of these componentswere found in a number of bacterial species over the past 2decades and perhaps even earlier, yet these studies had littleimpact on ideas regarding flagellar function or assembly.Why? The answer lies in the limitations inherent in bothultrastructural analysis and biochemical isolation protocolsfor membrane-associated structures. Recent findings thattwo of these structures form part of the flagellar bases ofEscherichia coli and Salmonella typhimurium have thereforebeen significant, since they have allowed structure-functioncorrelations to be made from examination of the geneticallywell-characterized mutants available for these bacteria (36).Outer membrane-associated flagellar basal discs have notbeen found in these species, but efforts are ongoing. It is thusappropriate that a description of the methodologies and theirlimitations precedes and qualifies a description of the struc-tures.

METHODOLOGYMorphological studies. The study of the flagellar basal

body is subject to the general considerations that guideanalysis of cellular substructures. First, a self-consistentpicture of the structure needs to be obtained upon applica-tion of diverse techniques. Second, images of isolated struc-tures need to be related back to in situ images of thesestructures.

In negative-stain electron microscopy, the specimen isembedded in a metal stain. Negative staining offers the mostease of use. However, the thickness of most bacteria is suchthat the in situ structure of flagellar basal bodies cannot be

visualized by this technique, except in fortuitous cases (20).Thin-section electron microscopy is the common method forvisualizing subcellular structure in intact cells. Fixation andembedding are usual in order to prevent deformation ordisintegration of biological material, but these procedurescan cause such mishaps as frequently as prevent them. Inaddition to the fixation and embedding steps, damage mayalso occur during sectioning (1).

Physical fixation by freezing is an alternative strategy forsample preparation. Direct rapid-freeze fixation is preferableto the use of cryoprotectants, such as glycerol, which caninduce molecular rearrangements. At high freezing rates,water is frozen as an amorphous glass (vitreous ice) ratherthan as crystalline ice (4). The chemical physics of ice iscomplex, but, empirically, the damage caused by ice tobiological samples is impressive (39). With appropriatefreezing devices, freezing rates of greater than 106 K/s areobtained, and samples as thick as 10 pm can be rapidlyfrozen. Bacteria, being small, are particularly amenable torapid-freezing techniques.The rapidly frozen water in such cryofixed samples may

be subsequently substituted with an organic solvent at lowtemperature (-80'C), in the presence of a fixative, and thenprocessed and thin sectioned. There is an extensive litera-ture documenting superior preservation of animal cell archi-tecture in such freeze-substituted sections. Variations infreeze-substitution protocols are based on the solvents andstains used. Another development that seeks to minimizedamage during embedding is the use of low-temperatureresins. The antigenicity of proteins is also better preserved inlow-temperature resins (6) and, if certain fixatives areavoided, in freeze-substituted sections (39). Quantitativevalidation of the view that freeze-substitution provides bet-ter preservation has come from the analysis of periodicstructures, for which such preservation can be assessed atthe appropriate spatial resolution by comparison with X-raydata (16, 35). Advances in high-pressure freezing, section-ing, and direct imaging of cryofixed material (49) mayultimately allow objective evaluation of freeze-substitutionprotocols for complex cellular substructures.

In freeze-fracture and freeze-etch experiments, the cryo-fixed sample is fractured in a vacuum chamber at lowtemperatures, and exposed biological structures are metalcoated. The sample may be etched by sublimation of thefrozen water between the fracture and metal replicationsteps by covering the sample with a cold trap and raising thesample stage temperature. The metal replica is subsequentlycleaned of adherent biological material and examined.Freeze-fracture experiments provide unique views of mem-brane interiors. The technique is based on the fact that lipidbilayers separate easily upon mechanical shear at low tem-

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perature (<-100'C) and pressure (<10-4 Pa) (29). Double-replica methods allow examination of the complementarity(i.e., the match of particles on one face to complementarypits on the other) of the two membrane halves, a feature lostin knife-fractured replicas. Efforts to establish complemen-tarity (53) provide a sobering illustration of the difficultiesinherent in freeze-fracture. At normal fracture temperatures,protein particles can deform during fracture, while postfrac-ture lipid rearrangements can fill pits prior to replication. Atvery low temperatures, condensation of water vapor ontothe newly exposed lipid surface can obliterate pit-like fea-tures. Grains formed by clustering of the evaporated metalatoms upon contact with cold surfaces limit replica thicknessand hence limit resolution. The size and apparent shape ofparticles are a function of replica thickness and the shadow-ing parameters employed (31). Fine-grained tantalum-tung-sten replicas have been used to relate intramembrane parti-cle size to molecular weights of transmembrane proteins inchemically defined proteoliposome preparations (14). How-ever, the continued development of antibody labelling pro-cedures provides the only definitive guarantee for establish-ing specific intramembrane particle-protein correlations (46).

Cell biologists have produced spectacular rotary freeze-etched and freeze-dried views of detergent-extracted cellcytoskeletons (23, 25). Cross-linked cytoskeletal gels arerelatively insensitive to surface tension forces obtainedduring etching and may be particularly suited for deep-etching techniques. The sublimation rate, and thus thequality of etched replicas, is critically dependent uponprecise temperature control. Furthermore, etching requireslow-ionic-strength buffers, since otherwise biological struc-tures may be obscured by precipitation of solution salts.Damage caused to animal cell cytoskeletons during prepara-tion, etch, or replication may be assessed by inspection ofknown structural features, such as myosin-decorated actinfilaments (23). However, no such markers are available forbacteria. Preparations lacking the cytoplasm, which areneccessary for freeze-etch work, were used for electronmicroscopic studies of bacterial flagellar basal bodies sincethe 1960s, but these preparations were not characterized,and the negatively stained or resin-embedded structuresvisualized were of uncertain origin. The development ofbiochemically characterized E. coli and S. typhimunum cellenvelope preparations demonstrably containing functionalflagella has been an important advance. The preparations areheterogenous (5) but can yield valuable information (33),provided examination is restricted to clean, well-frozen cellenvelopes.

Isolated macromolecular assemblies may be studied byelectron microscopy after negative staining, cryofixation,and metal shadowing. Negatively stained samples have highcontrast, but the drying and interactions with the gridsupport can lead to denaturation and flattening (51). Sugars(e.g., glucose) have hydroxyl groups which can substitutefor water (43) and protect against dehydration. Variations instain thickness and systematic positive-staining artifacts arealso a concern. Cryofixation of macromolecular assembliesis now a routine technique (41, 43, 52). As with cryosections,the greater sensitivity to radiation damage necessitates a lowelectron dose and, thus, lowered contrast. Discrepanciesbetween negatively stained and vitreous ice images due toshrinkage and other artifacts vary depending on the biolog-ical particle (43). Metal replicas have the advantage that thereplica of the hydrated, unstained biological particle may beexamined under a normal electron dose (25). Periodic sur-face features in the specimen can be contrast enhanced by

exploitation of decoration phenomena, while different metalscan be used to distinguish decoration from true surface relief(3, 55).

Biochemical studies. A membrane-associated structure ofthe size of the flagellar basal body has not yet been purified.The task will not be easy. In spite of considerable effort, thechemical principles underlying the association of proteinassemblies with lipids and detergents are not understood(38). It is not clear, for instance, whether the methods usedfor crystallization of bacteriorhodopsin and the photosyn-thetic reaction center, both of which occur as ordered arraysin vivo, can be generalized. Flagellar basal bodies have acomplex substructure, are dispersed, and have labile com-ponents. The lability is an important consideration limitingchoice of purification protocols. Quantity is another consid-eration. Whereas the E. coli FoF, ATPase, for example, ispresent in many thousands of copies, there are only a fewflagella per cell. In particular, basal flagellar structures havea size comparable to that of the vesicular debris generatedduring cell lysis and may not be cleanly separated from thedebris by either differential centrifugation or gel filtrationchromatography.A critical shortcoming of ongoing purification attempts is

that functional assays are not available for isolation offlagellar basal body components. It is presently not evenclear whether in vitro assays for some of the more complexfunctions of the basal body, such as motor rotation andflagellar protein export, can be devised. Examination ofstructural defects in motility mutants and immunolabellingby antibodies raised against products of cloned genes pro-vide the only currently feasible course of action. Further-more, the hope in biochemical studies is that the speciesbeing purified is homogenous. This is unlikely to be the casefor the flagellar basal body, an organelle governing assemblyof the rest of the flagellum. It is possible that analogous tophage assembly, transient basal structures occur as scaffold-ing intermediates during particular assembly stages (24),while proteins that determine motor response to chemotacticsignals, such as CheY, have transient associations with theflagellar base.

Since crystallization and X-ray diffraction are not feasiblefor structures of the size and complexity of the flagellar basalbody, electron microscopy will be instrumental for analysisof the basal body. The refinement of X-ray structures isguided by stereochemical constraints (11). In contrast, it is ingeneral not possible to judge whether images of macromo-lecular structures obtained by electron microscopy arechemically reasonable. Therefore, precise evaluation of theartifacts introduced by sample preparation procedures isdifficult. Scanning transmission electron microscope mea-surements provide a notable exception (54). Molecular massinformation provided by such measurements may be com-pared with independent estimates derived from hydrody-namic and electrophoretic studies (47). Nonetheless, sampleintegrity during the freeze-drying required for mass measure-ments, and interactions with the support film, require pains-taking optimization.

In summary, biochemically isolated structures are advan-tageous in that their chemical components may be deter-mined and a structural description of higher resolution thanthat afforded by morphological studies of in situ structures ispossible. However, the relationship of the structures withthe cell wall is gone, components may be lost during purifi-cation, and numerous, albeit different, artifacts need to betaken into account in the structural analysis.

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STRUCTURAL MODULES

Genetic evidence. Motility and chemotaxis mutants havethree fundamental phenotypes; nonflagellate (fla mutants),nonmotile flagellate (mot mutants), and nonchemotactic (chemutants). In E. coli and S. typhimurium, fla genes greatlyoutnumber mot or che genes. mot mutations occur in rela-tively few loci: motA, motB, and fliG, -M, and -N. Besidesgenes coding for components of the intracellular chemotacticmachinery, some fliG, -M, and -N lesions also yield the chephenotype. mot or che mutations in genes coding for HBBstructural components have not been found. Thus, it is notclear what part, if any, the HBB plays in motor function. Theflagellar motor, which is fueled by ion gradients across thecytoplasmic membrane and must therefore be part of flagel-lar basal structure, is composed, then, of at least the MotAand -B and FliG, -M, and -N proteins. Flagellar growthproceeds by export of the structural filament, hook, and rodproteins through the hollow flagellar core, followed byassembly at the distal tip. Studies using temperature-sensi-tive mutants have implicated FliN, -I, and -H and FlhA incontrol of filament elongation (57). These proteins may makeup a basally located export control apparatus. The flagellarbase may also control the temporal expression of flagellargenes by transiently sequestering or exporting transcriptionfactors (36). Indeed, the polar flagellum of Vibrio parahae-molyticus is known to control the expression of lateralflagella by sensing changes in medium viscosity (37), al-though the molecular mechanism underlying this controlremains obscure. Finally, there are numerous genes whoseproducts have unknown functions and may play a structuralrole.Genome organization and expression, together with anal-

ysis of suppressor mutations, provide clues to related func-tion and/or structural interaction. In E. coli and S. typhimu-num, the motility-chemotaxis genes are clustered in fourregions (36). fliG, -M, and -N are expressed early relative tomotA and -B. motA and -B are adjacent in one operon. fliMand -N are adjacent in another operon, the remainder ofwhich encodes flagellar proteins with unknown functions.fliG is adjacent to fliF (the HBB M-S ring protein) in yetanother operon, but in the same operon cluster as fliM and-N. In Bacillus subtilis (8) and Caulobacter crescentus (18),the genome organization of the motility-chemotaxis genes isdifferent, although the gene order is similar in some cases.There is evidence for structural interactions between theMot proteins (48) and, on the basis of suppressor mutationalanalysis, between FliG, -M, and -N (26, 36). Mutations inCheY and CheZ suppress mutations in FliG, FliM, and FliNand vice versa. CheY suppressor mutations for FliG andFliM are clustered on one face of the CheY protein, consis-tent with physical protein-protein interaction (42) and evi-dence that CheY switches motor rotation sense (5).

Cytoplasmic structures. Evidence for flagellar cytoplasmitstructure was obtained from thin sections of intact, polarlyflagellated bacteria more than a decade ago (reviewed inreferences 33 and 50). Freeze-substituted thin sections,freeze-etch replicas, and preparations of isolated flagellahave provided evidence that similar structures exist in E.coli and S. typhimurium (19, 22, 30, 33, 45). These studies,together with the earlier work, indicate that a morphologi-cally conserved cytoplasmic structure is a general feature ofbasal flagellar structure and make possible the analysis ofstructure-function relationships based on examination ofmutant strains carrying precisely determined genetic lesions.The S. typhimurium basal flagellar structure, as revealed in

FIG. 1. S. typhimurium cytoplasmic flagellar structure in afreeze-substituted cell envelope section. Bar = 50 nm. Taken fromreference 33 with permission.

freeze-substituted cell envelope sections, comprises a bell-shaped module with an axial rod-like feature (Fig. 1). Thismodule is larger than the HBB complex. Smaller flagellarcytoplasmic structures were also observed in cell envelopepreparations. These may be partial structures derived fromthe large bell structure.

S. typhimurium HBB-associated structures may be iso-lated and purified if extremes of pH and ionic strength areavoided (30). These structures are probably a routine feature(from their frequency of occurence) and cytoplasmic (fromtheir relationship with the HBB). The most common struc-ture isolated had a bell-like morphology when negativelystained, with axial disc- and rod-like features. However, inspite of similar morphology, its dimensions were smallerthan those of the largest class of structures found in cellenvelope preparations. Extended, ring-like S. typhimuriumHBB-associated structures have also been visualized bycryoelectron microscopy (52).Examination of S. typhimurium mutants has implicated

FliG, -M, and -N as components of the isolated structures.Mutants containing fliF-fliG gene fusions assemble basalbodies comprising the FliF-FliG fusion protein and aremotile (22). It is possible that the fusion protein can substi-tute for FliF in the HBB and perform an unidentified FliGfunction, without blocking assembly or rotation. However,the simplest interpretation is that FliG is also a structuralprotein located adjacent to FliF. The bell-like structure is notisolated, or rarely so, in mutant strains carrying mot lesionsin either fliG, -M or -N (30). The simplest interpretation ofthis result is that FliG, -M, and -N are components of theisolatable bell and that mot lesions accentuate the lability ofthis structure, preventing its isolation. In physiological stud-ies, mutant strains carrying temperature-sensitivefla lesionsin fliG and -M lost motility irreversibly when shifted to therestrictive temperature (57). The motility of strains carryingcorresponding lesions in genes coding for HBB componentswas not affected in such experiments, consistent with thenotion that FliG and -M comprise part of a structure that islabile relative to the more stable HBB. The larger bellstructures visualized in cell envelope preparations mayinclude additional modules such as the postulated exportapparatus, transient structures found only at a specific stageof flagellar morphogenesis, or artifactual aggregations. Inany case, it is clear that the cytoplasmic basal flagellarstructure is an extensive and complex multimodular entity.

Inner membrane particle rings. Rings of intramembraneparticles are a general ultrastructural motif in biologicalmembranes. Diverse functions have been ascribed to suchstructures in plant and animal cells (see, e.g., reference 44).

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FIG. 2. Thin platinum freeze-fracture replica of B. finnus OF4intramembrane particle ring. Bar = 25 nm. Taken from reference 32with permission.

However, the absence of well-characterized genetic markershas prevented structure-function relationships from beingestablished. In bacteria, distinctive intramembrane particlerings have been found in a variety of species, includingspirilla, streptococci, spirochetes, bacilli, and eubacteria(15, 31, 32). These structures consist of a large, centralparticle, with dimensions comparable to the HBB rod, whichis separated by a space, with dimensions comparable to theHBB M-S ring, from an outer ring of smaller, uniformlysized particles (Fig. 2). The explicit postulate that thesestructures are involved in energization of flagellar rotation(7) was based on freeze-fracture images of polar membraneregions of the polarly multiflagellated species Aquaspirillumserpens, which revealed clusters of such structures (15).Subsequently, it was shown that analogous particle rings inE. coli were flagellar structures, since they were present inmotile bacteria but absent in nonflagellate mutants (31).Presumably the central particle corresponds to the basal rodsegment, since it is known that the flagellum breaks readilyat the basal-distal rod junction (40). The ring particles werehighlighted by high-angle rotary shadowing, a procedure thatcontrast enhances tall particles, consistent with the particlesprotruding into the periplasmic space. Nevertheless, the factthat the ring particles partition to the P face of freeze-fractured cytoplasmic membranes indicates that they havestronger contacts with cytoplasm-based, rather thanperiplasm-based, structures (29). In lightly etched replicas,the space between the central and outer particles wasdepressed relative to the rest of the membrane P face. Thisphenomenon was consistent with the space being proteinrich relative to the rest of the membrane, as would be thecase if it were occupied by the HBB M-S ring, thus allowingmore rapid sublimation of the frozen intracellular waterbeneath.By light-etch procedures, it was shown that both MotA

and MotB were required for the assembly of the outerparticle rings (31). In E. coli motA motB double-deletionmutants, partial structures comprising the central particleand surrounding depression, but lacking the outer particles,were obtained. Both MotA and MotB are integral membraneproteins. MotB has a large periplasmic domain (13). MotAexpressed together with a MotB fragment conducts protons,while MotA expressed alone does not; this implies MotA-MotB interaction (10, 48). These data, together with themorphology, are consistent with the postulate that the ringparticles are composed of MotA-MotB complexes and func-tion to conduct protons and/or contact the rigid cell wall,thus enabling energization and force generation (7). Thisperipheral location would also allow unrestricted assembly-disassembly of the Mot proteins from the flagellar base,

FIG. 3. Negatively stained W. succinogenes outer membranebasal disc containing a centrally located HBB L ring. Bar = 100 nm.Taken from reference 45 with permission.

consistent with physiological data (9). Nevertheless, a num-ber offla gene products of unknown function have predictedtransmembrane-spanning hydrophobic stretches (36) andmay also be integral membrane proteins. Estimates of wild-type copy levels of MotA and MotB (56) indicate that a poolof excess protein is present in the membrane after allowingfor incorporation into flagellar intramembrane ring particles.Although this is not necessarily evidence against the postu-late noted above, a functional rationale for an excess-proteinpool is unclear. Thus, proof of the postulate will requirechemical characterization of the ring structures by eitherbiochemical isolation and characterization or immunolabel-ling.An unresolved issue regarding particle ring assembly is

whether discrete sites exist for attachment of the ringparticles to the flagellar base. Alternatively, the particlesmight simply pack closely around the flagellum. The ringstructures identified in the various species may be dividedinto two classes based on the number of outer ring particles:rings with 10 to 12 particles (i.e., E. coli, S. typhimurium,Bacillus firmus, and B. subtilis) and those with 14 to 16particles (i.e., A. serpens and Streptococcus spp.). Thereplicas used thus far are too coarse to allow determinationof a periodic spacing between the ring particles. Possibledisplacements of intramembrane particles during fracturealso complicate the endeavor. The study of changes inparticle number brought about by controlled mot geneexpression and characterization of particle ring interactionswith other flagellar components, rather than morphologyalone, are likely to be decisive for resolution of this issue.Outer membrane-associated basal discs. Outer membrane-

associated basal discs, which have been found only inpolarly flagellated bacteria thus far, were also described overa decade ago (reviewed in references 34 and 45). In A.serpens and Wolinella succinogenes, these structures havebeen coisolated together with the HBB. In the isolatedstructures, the discs contact the HBB periplasmic face,consistent with their location next to the outer membrane asrevealed in whole-cell thin sections. The discs are of large,variable diameter and possess a central hole, which in W.succinogenes is sometimes partially filled with the HBB Lring, indicating that the HBB L ring is coplanar with the discstructures (Fig. 3).The size of the discs has prompted speculation that they

may mechanically stabilize the basal body and perhapsprovide anchorage for force-generating complexes (45). Ifso, it is not clear why similar structures have not been foundin nonpolarly flagellated bacteria. There is evidence frombacteria such as Bdellovibrio bacteriovorus that these struc-tures may be quite labile (34). Perhaps the discs of nonpolar

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flagella are lost during detergent extraction. Consistent withthis possibility, basal cytoplasmic and intramembrane ringstructures of nonpolar flagella seem to be more labile thantheir polar counterparts, since visualization in situ of thenonpolar basal flagellar structures has required rapid-freezemethods of sample preparation (31, 33). Alternatively, thediscs may function to position polar structures, includingflagella. In stalks of C. crescentus, "crossband" structuresmorphologically almost identical to the flagellar basal discsare found (20), consistent with this view. Even if this is so,the nature of the intracellular position-specific signals gen-erated by such a periplasm-based structure is tantalizinglyobscure.

STRUCTURE, FUNCTION, AND MORPHOGENESIS

The outstanding issue for future structural investigation isthe nature of the contacts between the described structuralmodules, the HBB, and the bacterial cell wall (see references33, 45, and 50 for models). In E. coli and S. typhimurium,differences in the number of structural and functional unitsfurther confound the issue. There are 10 to 12 ring particles,compared with the eight force generators measured in phys-iological experiments or the ca. 6 or 27 subunits comprisingthe rod or M-S ring HBB substructures, respectively (9, 28,31, 47). Chemical labelling methods need to be developed forstudy of the in situ distribution, as well as localization withinthe basal body, of the component proteins. Scanning probemicroscopies (21) may prove potent for obtaining topograph-ical information, such as the height of the intramembraneparticles seen in freeze-fracture replicas. However, thestudy of biological material by these technologies is in itsinfancy. Their application to the flagellar basal structuremight prove more distracting than fruitful at the presenttime.At the resolution at which it has been examined thus far,

the flagellar basal structure has morphologically conservedfeatures. For the flagellar filament and HBB, this conserva-tion reflects homologies between the proteins (18). An im-plicit assumption made in this article is that morphologicallysimilar structural modules found in different species havesimilar functions. If so, this implies that there is a commonplan underlying the construction of the rotary motor offlagellated bacteria, which may consist of all or part of therecently described modules. However, identification of therotor and stator components is not presently possible, andthe structures responsible for ion transport and CheY-basedswitching of rotation sense have not been unambiguouslydetermined.Fundamental issues regarding biological supramolecular

structure assembly and its control are posed by study offlagellar basal substructures. The morphology of the flagellarcytoplasmic bell, for example, is unprecedented. Geometri-cal considerations relevant for understanding its assembly,such as those that guided studies of the self-assembly of viralcapsids (12), are not available. Possible control by thisstructure of the assembly of more-distal flagellar structuresremains entirely mysterious. Understanding will probablyneed to be painstakingly pieced together by structural andphysicochemical analyses of the protein-protein interactionsof selected components, in addition to analysis of the ultra-structural modules. It has become apparent that proteinchemistry is the critical missing link between gene andultrastructure.

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