Flagella and Motility
Chapter
10
ROBERT M. MACNAB
Many, probably most, true wild-type strains of Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) are motile, moving rapidly in liquid media and undergoing abrupt directional changes. When observed by electron microscopy (Fig. 1) or (with suitable optics [125]) light microscopy (Fig. 2), the cells are seen to possess a number of thin, helical appendages. Often loosely referred to as flagella, these appendages are only the external portion of the flagella and are properly called flagellar filaments.
The flagella are varied in number (typically about 5 to 10 per cell) and originate at random points around the cell surface, a pattern called peritrichous flagellation. Other patterns of flagellation (e.g., polar monoflagellate) exist in other species, and other forms of bacterial motility also exist, such as gliding and spirochetal motility (see, e.g., reference 129).
The bacterial flagellum differs from the eukaryotic flagellum in several regards: (i) the bacterial flagellar filament, ca. 20 nm in diameter, consists of subunits of just one protein, whereas the eukaryotic flagellum has a complex architecture and is much thicker, ca. 200 nm in diameter; (ii) the filament does no chemomechanical work but is passively driven by a motor at its base; (iii) the mechanism is one of rotation (8, 173), not bending (and hence the term "flagellum," meaning "whip," is misleading); (iv) the motor can operate in either the counterclockwise (CCW) or clockwise (CW) direction (173) (i.e., it possesses a switch); and (v) the energy source is the transmembrane proton potential (proton motive force) (139), not ATP (121).
Bacterial flagella are a beautiful example of motors functioning at the molecular level. As a result of intense study in many laboratories over the last 25 years or so, they also represent one of the best-understood complex multisubunit molecular devices in terms of their genetics, structure, assembly, and function.
This chapter reviews the structure and function of the bacterial flagellum, the underlying genetic system, and the process of flagellar assembly. Only a brief description of chemotaxis is given. The molecular events surrounding chemosensory reception and signal processing, which culminate in modulation of motor function, are described in chapter 73 of this book. For other reviews of the subject of bacterial flagella and motility, see references 19, 29, 77, 89, 130, 131, 133, and 169.
The flagellar systems of E. coli and S. typhimurium are essentially identical in most aspects. Where a gene symbol, protein symbol, or other item applies to one species only, it will be subscripted by e or s for E. coli or S. typhimurium, respectively.
The nomenclature for flagellar genes has been completely changed since the first edition of this book (71), as will be described in a later section. Table 1 provides the correspondence between the old and new nomenclatures.
Table 1Operonsa and genes involved in flagellation, motility, and chemotaxis of E. coli and S. typhimurium |
The bacterial flagellum has for many years been described in terms of a long helical filament, a short curved structure called the hook, and a basal body consisting of a central rod and several rings (Fig. 3) (34). The filament and hook are external to the cell, whereas the basal body is embedded in the cell surface. The basal body is now known to be a considerably more extensive and complex structure than is shown in Fig. 3.
The filament is not a part of the chemomechanical energy-transducing apparatus (the motor), but is the component of the flagellum that performs hydrodynamic work on the medium and hence propels the cell. It represents by far the major component in terms of mass (and hence biosynthetic cost), possesses remarkable structural properties, and is significant clinically as a potent antigen.
Geometrical, Biochemical, and Mechanical Properties.
The filament is of variable length (typically 5 to 10 μm) but has a constant diameter of about 20 nm throughout its length (151). It is macroscopically helical (corkscrew-like), with helical parameters that vary slightly between the two species and even among strains; typical values are 2 to 2.5 μm for the helical wavelength (pitch) and 0.4 to 0.6 μm for the helical diameter (85). For any given filament, these values are constant throughout its length; in other words, the structure has a strictly conserved geometry and may be regarded as a unidimensional crystal.
The filament consists of an indefinite assembly of around 20,000 subunits of a single protein, flagellin (FliC), whose molecular mass is typically in the 50- to 60-kDa range but varies among strains (101) and (for S. typhimurium) between phase 1 and phase 2 flagellin gene expression (see below). S. typhimurium flagellin has the characteristic (shared by several other mechano-enzyme proteins) of containing methyl-modified residues, in this case N-methyl-lysines (3). No functional significance appears to derive from this modification, since mutants lacking the methylating enzyme (FliB) function normally.
Flagellin is a three-domain protein, with the N-terminal and C-terminal domains being responsible for the quaternary interactions between subunits (see below) and the central domain performing no obvious structural role (indeed it can be deleted without affecting assembly or function [117]). This domain projects outward and contains all of the potent antigenic epitopes; it also contains most of the lysines that are N methylated (86).
A striking characteristic of the flagellar filament is its rigidity; its coefficient of flexural rigidity is ca. 10–24 N m–2 (45), making it about two orders of magnitude stiffer than actin, for example. Thus, detached filaments in liquid show essentially no flexural Brownian motion, and even during active motility, lateral forces induce only slight elastic bending (136). Torsional rigidity is also high (ca. 1010 N m–2 [65]). Thus, the properties of the filament are well suited to its function as a propeller. The capacity of the filament to undergo inelastic conformational changes is discussed below.
Self-Assembly Characteristics.
Isolated flagellar filaments can be readily depolymerized by heat, acidic pH, or other treatments. The reverse process, polymerization into filament, occurs spontaneously under suitable conditions and yields a structure that is indistinguishable from native filament (6, 85). In vitro, the rate of elongation is constant (69), with monomer being added to the end that would be distal to the attachment point of the filament to the cell (6); the assembly of filament in vivo is discussed in a later section.
Filament Structure: Quasi-Equivalence and Polymorphism.
The flagellin subunits are arranged at points on a cylindrical or tubular lattice (193) (Fig. 4). These points can be joined in various ways to give different families of helical lines. The basic helical line passes through all lattice points and has close to (but not exactly) points per turn. As a result, there is a family of 11 helical lines oriented almost (but not exactly) parallel to the filament axis. This feature is crucial to the macroscopic helicity of the filament and hence to its function as a propeller. If all of the (identical) subunits were in identical quaternary structural environments, the filament would be straight rather than helical. The subunits on any given 1 of the 11 helical lines are in identical environments and can be thought of as a fibril. The fibrils differ from each other, however, with a shorter intersubunit distance on one side of the filament than on the other. Since the fibrils are not quite parallel to the filament axis, the length variation around the circumference introduces macroscopic helicity to the filament (6).
The situation is even more complex than this. A variety of helical forms can exist depending on physical conditions such as pH or ionic strength; in other words, flagellar filaments display polymorphism. The phenomenon can be explained in terms of the numbers of the 11 fibrils that are in the long versus the short state (27). There are two straight forms in which all 11 fibrils are in the same state, but for all other forms, n adjacent fibrils are in the extended state and the remaining 11 – n are in the compressed state. Thus theory predicts 12 forms, of which about 9 have been observed experimentally; they include the 2 straight forms and intermediate left-handed and right-handed helical forms that have been given names such as "normal," "curly," "coiled," etc. (Fig. 5). For a more detailed consideration of filament polymorphism, see references 28 and 85.
The normal form, which is left-handed, is the most stable one under physiological conditions and is the one used in propulsion. Interconversion among polymorphic forms can be caused by mechanical force; this phenomenon plays an important role in the tumbling mode of E. coli and S. typhimurium motility (136) (see below).
The various forms can also be generated by alteration of the flagellin primary sequence, even by a single amino acid substitution (86, 141), or by incorporation of an amino acid analog such as p-fluorophenylalanine (188). A number of the mutations responsible for polymorphic changes have been sequenced and found to occur in the N- and C-terminal regions. These regions are predicted to form α helices that interlock adjacent subunits; it is suggested that they make subtle perturbations in the longitudinal phasing of these α-helical bundles and so affect the intersubunit distances (86).
The filament is connected to the cell by the hook, which is structurally related to the filament but is built from a quite distinct subunit, the hook protein (FlgE) (Table 2). The same 11-fibril structure described above for the filament applies to the hook (147), as does the quasi-equivalence that produces macroscopic helicity and polymorphism (82). The helical wavelength of the hook is much shorter than that of the filament (ca. 130 nm versus 2.5 μm). Even so, the hook is so short that it only generates less than one-half of a helical turn, resulting in its characteristic curved appearance. It is much less rigid than the filament (25, 26), consistent with its presumed function as a flexible coupling that permits torque generated on an axis perpendicular to the cell surface to be applied parallel to it. The flexibility may not be elastic but could involve back-cycling of the quasi-equivalent phase of the hook so that the waveform of the rotating hook remains more or less stationary (see the discussion in reference 132).
Table 2Proteins involved in flagellation and motility of E. coli and S. typhimuriuma |
In contrast to the filament, the hook has a rather well-defined length (ca. 55 ± 6 nm [56, 80]) and consists of about 130 subunits. Between the hook and filament are two junction proteins, also known as hook-associated proteins, FlgK and FlgL (59, 60, 63) (Fig. 3B). Although the filament and hook have similar lattices (204), the mechanical properties of these structures are quite different, and it may be that the junction proteins act as adapters; consistent with this idea is the recent finding that defects in these proteins can cause structural changes in the filament (41).
The hook proteins of E. coli and S. typhimurium are rather similar to each other antigenically (83), in contrast to the range of antigenicity exhibited among flagellins. The flagellar filament constitutes a much greater antigenic challenge to the host, and presumably the bacterium has developed the potential for antigenic variability in response.
No one has yet succeeded in obtaining three-dimensional crystals of flagellin or hook protein to get a high-resolution structure, but much has been learned at lower resolution from three-dimensional reconstructions from electron micrographs (147, 151, 193, 194, 195). Analysis has thus far been confined to the two straight polymorphic forms (see the discussion on quasi-equivalence above) where all 11 fibrils are in the same state. Figure 6 shows such reconstructions, viewed in three different ways. The first, shown in the bottom panel, is the complete structure as seen from the side and reveals the three major directions referred to in Fig. 4; note that the 11-start direction is tilted slightly to the left with respect to the filament axis in one form and slightly to the right in the other. This view also emphasizes that each subunit presents a pronounced knob to the exterior. As mentioned above, this knob is important antigenically but not structurally, since there are no major knob-to-knob contacts. The second view, shown in the middle, is the structure sliced in half longitudinally; it shows the central channel and the types of intersubunit contacts that exist. The third view is a "shaved" version of the structure in which density at outer radii has been removed by computer so that intersubunit contacts can be seen from the outside. This view demonstrates vividly the substantial difference between the two straight forms; in the form to the left, the density of each subunit appears oblong and more or less along the 11-start system, whereas in the form to the right, it has been pivoted so that there is continuous density along the left-handed 5-start direction. The rotation of the subunits results in a small change in the intersubunit distance on the 11-start direction. (Recall that in the wild-type filament, this change occurs only for the fibrils on one side of the filament, and so macroscopic helicity is generated.) The resolution of the filament reconstruction has recently been improved to 9 to 11 Å (0.9 to 1.1 nm) (144, 148), resulting in a greatly refined view of the structure at inner radius, where there are inner and outer tubes of α-helical rods in the 11-start direction, with connecting spokes between the two tubes (Fig. 7); these features represent the principal intersubunit interactions that result in the rigidity of the filament. They are generated by the N- and C-terminal regions of flagellin, which have pronounced heptad repeats of hydrophobic residues (59), a characteristic of α-helical coiled coils. In solution, the terminal regions of monomeric flagellin are substantially disordered and exhibit a relatively small extent of α-helical structure, but upon polymerization into filament they order themselves into the α-helical rods seen in Fig. 7 (76, 148, 201, 202).
The hook is connected to a complex structure known as the basal body, which is embedded in the cell surface (34, 35). The filament–hook–basal-body complex (Fig. 3) can be isolated from cells after dissolution of the cell surface; depolymerization of the filament then yields the hook–basal-body complex. I first describe the basal body as it has been known until quite recently and then describe a larger and more elaborate structure, which can be called the extended basal body.
Geometry and Composition.
The basal body has approximately cylindrical symmetry, consisting of a rod and a set of four rings (34) (Fig. 8A). The outer two rings (called L and P) are spaced apart but appear to be linked by a cylindrical wall. The inner two rings (S and M) abut directly and in fact represent domains of a single protein (FliF) (196, 197); for this reason, the preferred terminology is now "MS ring." Both geometrical and biochemical considerations (34, 35) suggest that in the intact cell, the L ring is in the plane of the outer (lipopolysaccharide) membrane, the P ring is in the plane of the peptidoglycan layer, and the MS ring (membrane/supramembrane) is in the cell membrane.
There are now known to be at least eight different proteins in the limited version of the basal body shown in Fig. 8A (80, 149), four of them in the rod (FlgB, FlgC, FlgF, and FlgG), three of them in the rings (FlgH, FlgI, and FliF, forming the L, P, and MS rings, respectively), and one whose location is not known (FliE). The sequence of FliE suggests that it is not a membrane protein, a protein exported by the primary cellular pathway, or a member of the axial family of rod, hook, etc., proteins; one possibility is that it is a structural adapter between the MS ring (which has annular symmetry) and the rod (which is presumed to have helical lattice symmetry). There may be additional proteins (e.g., FliP [see below]) that remain to be identified, judging from differential radiolabeling experiments of basal bodies from conditional mutants (79).
Stoichiometry of Basal-Body Proteins.
The number of subunits of each basal-body protein has been estimated biochemically (80), and the amount of mass present in each of its morphological features has been estimated from scanning transmission electron microscopy (180). The results are in good agreement and indicate that each of the three ring proteins (FlgH, FlgI, and FliF) is present in about 26 subunits, the proximal rod proteins (FlgB, FlgC, and FlgF) are present in about 6 subunits, the distal rod protein (FlgG) is present in about 26 subunits, FliE is present in about 9 subunits, and FliP is present in about 5 subunits.
Function of Basal-Body Substructures.
On the basis of the mutant phenotypes associated with its various components, the basal body is a passive structure, not directly involved in torque generation or in switching. The best guesses for the functions of the various basal-body components are as follows. The MS ring acts as a mounting plate for components essential for motor rotation and switching (see below). The rod is a shaft that transmits torque from the motor to the external hook and filament. The LP-ring pair, together with its cylindrical wall, is a bushing through which the rod passes, protecting it against lateral shear forces. It is presumed that the MS ring, rod, hook, and filament (together with the active rotor element [see below]) constitute a single rotating unit and that the LP-ring pair is stationary.
For many years, it has been thought that the proteins responsible for switching the motor between CCW and CW rotation would be located at the cytoplasmic face of the basal body, but until recently there was no direct experimental evidence. The first evidence concerned one of the three motor/switch proteins, FliG, and came from work with a spontaneous mutant in which fliG was fused in frame to the immediately upstream gene, fliF, which encodes the MS ring (43); the fusion protein permits close to normal function (indicating that FliG is part of the rotor), and electron microscopy reveals additional mass attached to the MS ring.
Since then, by using modified isolation protocols, basal bodies from wild-type strains have been obtained with a substantial additional structure (Fig. 8B and C) that consists of a thickening of the MS ring and of a cylinder projecting into the cytoplasm from the MS ring (44); in other studies, it has a more bell-like appearance (92). This structure has been called the C ring (cytoplasmic ring) or bell, and basal bodies containing this structure have been referred to as extended, to distinguish them from the simpler structures seen heretofore. Immunoblotting of simple and extended basal bodies reveals that the additional structure contains all three of the motor/switch proteins, FliG, FliM, and FliN (44; S. Khan, personal communication); FliG is much more tenaciously attached to the basal body than are the other two proteins and probably contributes more to the thickened feature of the MS ring than to the C-ring substructure. Conditional Mot– mutants (but not conditional Che– mutants) with defects in the switch proteins fail to show the C ring (88), although they frequently retain some structure, immediately attached to the cytoplasmic face of the MS ring, that can be decorated with anti-FliG antibody (Khan, personal communication). The same is true of wild-type particles that happen to have lost the C ring (44). In a few cases, conditional fliG mutants yield structures lacking FliG but retaining FliM and the C ring, indicating that FliM by itself makes some contact with the MS ring. That interaction, however, appears to be much more stable when FliG is also attached to the ring (Khan, personal communication). It is not known whether the C ring contains other proteins in addition to the switch proteins.
There are no published estimates of the stoichiometries of the FliG, FliM, and FliN proteins in the C ring. However, the fact that a FliF-FliG fusion protein sustains rotation (43) suggests a natural stoichiometry of 1:1 and therefore a FliG stoichiometry of about 26 subunits per basal body, based on the estimate for FliF (80); the fact that a FliM-FliN fusion protein sustains rotation (M. Kihara and R. M. Macnab, unpublished data) likewise suggests that the natural stoichiometric ratio of these proteins is likely to be 1:1, although the absolute stoichiometry remains unknown. (It also suggests that FliM and FliN are either both part of the rotor or both part of the stator.) In vitro experiments with purified MS-ring complexes and partially purified switch proteins indicate interaction between FliG and the MS ring protein, FliF, at a stoichiometry of about 1:1, in agreement with the above conclusion; they also indicate a lesser or weaker interaction between FliM and FliF and essentially no interaction between FliN and FliF (158). The switch proteins appear to be synthesized in considerable excess of the amounts actually incorporated into structure (D. F. Blair, personal communication).
Two proteins, MotA and MotB, are integral to the cell membrane and are necessary for motor rotation but not for motor switching or for flagellar assembly (5, 174). Freeze-fracture images of cells reveal a circlet of about 11 studs, presumed to be surrounding the MS ring, that probably consist of MotA-MotB complexes (Fig. 9), since MotA or MotB mutants show no or severely disrupted circlets (91). MotB has a single membrane-spanning segment (30, 181) and is thought to have an anchoring function as part of the stator element of the motor. In support of this is the recent finding that its periplasmic domain has a peptidoglycan binding consensus (33); binding to a covalent mesh that extends over the entire cell surface would of course create an ideal anchor. MotA is predicted to have four membrane spans with two relatively short periplasmic loops and an extensive amount of sequence in the cytoplasm (32). The functions of MotA and MotB in proton conduction are considered in a later section.
Intergenic suppression analysis of the Mot proteins (discussed further in a later section) suggests that MotB and MotA interact primarily between the periplasmic domain of the former and the periplasmic loops of the latter (47). Thus, a simple model would have MotB attached both to the peptidoglycan layer (and hence being an element of the stator) and to MotA, thereby making MotA part of the stator also.
The export of most of the external components of the flagellum is accomplished by a specialized pathway. Since the exported subunits are believed to travel down a central channel in the nascent structure (40, 68), there is presumably an apparatus somewhere in the vicinity of the flagellar base that is responsible for delivering them to that channel. Possible candidate proteins for that apparatus have been identified, but no structure has yet been detected physically. The process of export and assembly is considered in a later section.
This section describes motor function from the point of view of physiology and energetics; a later section considers molecular details of the motor and switch. A number of the quantitative characteristics of the motor that are referred to in the text are summarized in Table 3.
Table 3Geometry, energetics, and dynamics of flagellation and motility |
Swimming.
The flagellar motor operates as a reversible rotary device to cause rotation of the flagellar filament. Because the filament is normally a left-handed helix, rotation in the CCW sense causes the helical wave to travel from proximal to distal and to exert a pushing motion on the cell. Hydrodynamic and mechanical forces cause the flagellar filaments, which originate all around the cell body (Fig. 1 and 2), to sweep around the cell onto a common axis, usually the long axis of the cell body. The filaments operate in a concerted bundle (Fig. 10a and b) to propel the cell and produce swimming (4, 126). The rotation speed of the filaments within the bundle is over 100 Hz (cycles s–1) (124) and generates a swimming speed of around 25 μm s–1 (10 body lengths s–1) at room temperature in liquid medium (12, 135). Cells can also move through semisolid medium such as 0.3% agar. This is often called swarming but is in fact swimming though aqueous channels in the gel (211). True swarming entails movement in a liquid film over a solid substrate (such as hard agar). It has classically been described for hyperflagellated species such as Proteus mirabilis, but both hyperflagellation and swarming have recently been shown to be induced under appropriate conditions in many species, including E. coli and S. typhimurium (54).
Tumbling.
The other motility mode of E. coli and S. typhimurium, tumbling, occurs when the filaments are rotated in the CW direction. Under these conditions the helical wave might be expected to travel inward toward the cell body. The situation, however, is complicated by structural changes that take place in the filament (136). As described above, filaments have a polymorphic capability, including right-handed as well as left-handed examples. CW rotation of the filaments places them under right-handed torsional load and initiates, at the proximal end, a polymorphic transition to a right-handed waveform (called curly because it has about half the normal wavelength); as long as CW rotation continues, the transition proceeds outward toward the distal end. A heteromorphous filament is characterized by a curly proximal segment, a normal distal segment, and an abrupt angle between the helical axes of the two (Fig. 10c). Tumbling then occurs because the flagellar filaments are partly pulling and partly pushing and have a poorly defined orientation, as shown schematically in Fig. 10d. The cell is more or less randomly reoriented in the process, ready for the next interval of swimming. In the absence of polymorphic transitions, the probable consequence of motor reversal would be jamming of the flagellar bundle (126) and no effective reorientation.
Pausing.
A variety of experiments indicate that flagella intermittently undergo brief pauses, during which the rotation velocity goes to zero (39, 120). The frequency with which pauses occur correlates with the reversal frequency of the cells under the conditions examined (i.e., smooth swimming mutants pause rarely, whereas tumbly mutants pause often), suggesting that the two events are linked; a plausible hypothesis is that a pause is a failed reversal. There is no evidence for a distinct signal for pausing or for a physiologically important role for pausing in chemotaxis.
Much of our knowledge regarding flagellar motor energetics and dynamics comes from observations made (for technical reasons) on species other than E. coli and S. typhimurium, but there is no reason to believe that the characteristics of flagellar motors will be fundamentally different in different species. A major source of knowledge has been a motile Streptococcus strain that, conveniently, lacks endogenous energy sources (cited in reference 139). For a recent summary of motor energetics, see reference 11.
The energy source for rotation is the transmembrane proton potential, or proton motive force (51, 94, 121, 139, 140, 142, 165, 170); motor rotation has been demonstrated in the laboratory with an artificially imposed proton motive force, as well as with naturally energized cells. Both chemical potential (ΔpH) and electrical potential (Δψ) can be used, with comparable efficiencies.
The source of the proton motive force varies. Under respiratory conditions, it is the electron transport chain, in which case the proton motive force is above the saturating value for motility, so that the swimming speed is insensitive to the actual value (94). Under conditions of anaerobic glycolysis, ATP hydrolysis via the proton-linked membrane ATPase is responsible; at least on a short time scale after oxygen removal, the ATPase apparently generates a subsaturating proton motive force, and therefore swimming speed is appreciably diminished (R. M. Macnab, unpublished data).
From the amount of hydrodynamic work performed, it can be estimated that at least several hundred protons must pass through the motor per revolution (8). Although there are likely to be quantized elementary steps in the motion, they have thus far escaped detection (10), perhaps because of elastic damping.
There are several reasons for suspecting that the motor can be a highly efficient device, operating close to equilibrium: it has constant torque characteristics over a wide range of loads (16) all the way to a stalling load; its torque is proportional to the proton motive force; it lacks either a temperature dependence or a deuterium isotope dependence (90); it can operate equally with an artificially imposed proton motive force of either polarity (14); CCW and CW rotation (achieved via the motor switch mechanism) are equivalent in terms of torque and other characteristics (8).
Most of the above statements refer to the relatively high-load conditions of tethered cells. When loads are low and therefore rotation speeds are high, as with free-swimming cells (93, 124, 170) or the rotation of a filament on a cell attached by its body to glass (105), the torque (and therefore the efficiency) is considerably reduced, with internal events now becoming rate limiting. In the extreme case, speed becomes independent of either load or proton motive force; i.e., the motor is saturated. Under these conditions, the motors are operating far from equilibrium.
Recent experiments in which external torque is applied to a tethered cell have allowed measurements to be made over a much wider range, so that both the low-speed, constant-torque domain and the high-speed, variable-torque domain can be measured on the same cell and with the same geometry (17, 205). The results are generally consistent with earlier measurements of free-swimming and tethered cells. When an external torque of opposite sign to the motor torque is applied, so that the cell is slowed rather than speeded, the data conform to the expected pattern until the speed has been reduced to zero. At that point, the applied torque has to be increased to about twice the stall value before the cell will rotate backward, and there is considerable likelihood of irreversible damage to the motor (17).
The above discussion has focused on the issue of thermodynamic efficiency. A related issue is whether the stoichiometric coupling between proton flux and motor rotation is tight or loose; i.e., is the number of protons per revolution fixed, or does it depend on speed or load? Close-to-equilibrium operation of course implies tight coupling, but far-from-equilibrium operation can occur with either tight stoichiometric coupling (as with an enzymatic reaction with a large free energy change) or loose coupling (as has been postulated in certain transport processes [37]). Advocates of both conditions for the flagellar motor exist (13, 127, 157).
To answer the question experimentally, one needs to know the proton flux through the motor. The rotation-dependent proton flux in Streptococcus cells has been estimated to have a constant value (about 1,200 per revolution) regardless of motor speed (143), strongly arguing for tight coupling. Also, estimates of the amount of hydrodynamic work performed (at high load) versus the electrochemical energy dissipated, although subject to considerable uncertainty, indicate a high thermodynamic efficiency, further supporting the hypothesis of tight coupling (143). Finally, the extreme difficulty of driving the motor backward (17) is consistent with tight coupling, with protons having to bind at sites where their free energy is low and be released at sites where their free energy is high.
Whereas flagellar rotation in E. coli and S. typhimurium is driven by protons, in certain alkaliphilic species it can be driven by sodium ions (57, 58). If we assume a broadly similar mechanism regardless of the ion used, mechanisms that entail hydrogen bonding arrays seem unlikely. There is no evidence that clearly suggests or favors any specific mechanism for the flagellar motor, but there are various experimental observations (described above) with which a successful model must be compatible.
In one detailed model (13, 90), the proton potential is used to gate and therefore bias a diffusional random walk of force-generating units within the motor. The energy is proposed to be stored via the elastic tethering of these units, with the stored elastic potential almost equal to the driving proton potential, so that the device is operating close to equilibrium. Conceptually similar is a model in which rotor and stator surfaces each contribute half of the proton-binding site; arrays of such half-sites have a mutually tilted geometry, such that they come into register only if the two surfaces are rotated (97). Other classes of models (51, 146, 157) involve the conversion of the proton potential via specific binding sites into an electrical potential between components of the motor so that the motor operates as an electrostatically driven device. In at least one such proposed mechanism (157), the transfer of protons is not obligatorily coupled to rotation; i.e., the mechanism is a loosely coupled one. Yet another proposal (203) is for a proton-generated conformational change (axial twist of the central rod) with cyclic attachment at the proximal and distal ends.
For a recent summary and evaluation of published models, see reference 17.
The flagellar motors of well-energized cells of E. coli and S. typhimurium rotate and switch direction of rotation incessantly, whether or not there is gradient information in the environment of the cell. Provided that they are not forced to interact with each other mechanically or hydrodynamically, the switching of one motor is not synchronized with that of any other (75, 134); in other words, each motor is an autonomous unit.
In a constant environment, the switching probability per unit time is constant. For a tethered wild-type cell, mean intervals are typically around 1 to 2 s for both the CCW and CW directions (15). However, with free cells, there is considerable asymmetry in the mean intervals for swimming and tumbling (ca. 1 and 0.1 s, respectively [12]). A quantitative correlation between motor switching rotation probabilities and free-swimming behavior has not been achieved as yet. Part of the discrepancy between the measurements on free and tethered cells probably derives from the presence of mechanical interactions among flagellar filaments in a free-swimming cell, so that switching probabilities of individual motors may no longer be independent (compare discussions in references 75, 93, and 131).
Mutants can be conveniently grouped into just a few phenotypic classes, which are described below.
Nonflagellate or Partially Flagellate (Fla–).
Fla– is by far the most common mutant phenotype, as illustrated by the fact that many "wild-type" laboratory strains of E. coli and S. typhimurium are in fact nonflagellate. Indeed, spontaneous nonflagellate mutants rapidly (in ca. 10 days) overtake a motile population in stirred culture because of the metabolic cost of synthesizing the flagellar apparatus. The growth disadvantage of this synthesis is estimated to be about 2% (Macnab, unpublished). Another illustration is the fact that mutants in which flagellin synthesis is constitutive because of defective regulation of late genes (see below) grow much more slowly than wild-type cells (110).
The Fla– phenotype is defined as any substantial diminution of the flagellar apparatus. The defects can range all the way from total lack of any flagellar structure to partial basal structures to defective external structures lacking, for example, the filament.
Paralyzed (Mot–).
In Mot– mutants, no morphological defects are manifest in the flagellar apparatus, yet it does not rotate. In some cases (motA and motB mutants), the gene product can be totally lacking; in other cases (mutants with mutations in the motor/switch genes fliG, fliM, and fliN), the paralyzed phenotype occurs with certain missense mutations or in-frame deletions, but complete absence of the gene product prevents flagellar assembly.
Switch Defective (Che–[CW] or Che–[CCW]).
Che– mutants possess flagella that can rotate but have abnormal switching properties, with either a high CCW or a high CW rotational bias compared with the wild type. Although many such mutants are actually defective in components of the sensory apparatus (see chapter 73 of this volume), the three motor/switch genes described above can generate a switch-defective phenotype also, depending on the particular allele.
Overview.
The flagellar gene system is a large one (Table 1), consisting of over 40 genes for flagellar assembly, structure, and function in addition to the genes involved in sensory reception and transduction (130). In view of the extensive mutant analysis and molecular genetics that have been performed, it does not seem likely that many more genes remain to be discovered. All of the known genes have at this point been cloned and sequenced in one or both species; for the relevant references, see Table 1. Homologs to many of these genes have now been found in other species such as Caulobacter crescentus (182) and Bacillus subtilis (159).
Nomenclature.
Genes whose null mutant phenotype is Fla– used to carry the basic symbol fla plus an extension (spilling over to flb when the end of the alphabet, flaZ, was reached); this nomenclature, which exists in papers published through 1988 (including the first edition of this book), became increasingly baroque (e.g., flaAII.3) and disparate between E. coli and S. typhimurium, even though the actual gene systems are extremely similar (e.g., flaK E stood for a positive regulatory gene while flaK S stood for the structural gene for the hook protein).
A new simplified and unified nomenclature was adopted in 1988 (71). Gene symbols all begin with fl, have a third letter that is either g, h, or i, depending on the chromosomal region in which the gene is located (Fig. 11), and an extension that follows chromosomal order within the region: flgA, flgB...., flhA, flhB...., etc. A few genes have been discovered since the nomenclature was instituted and have been assigned the next available alphabetical extension; they are flgM and flgN in region I (50, 116), flhE in region II (145; M. C. Kim, Ph.D. thesis, University of Illinois, Chicago, 1989), and fliS and fliT in region IIIb (87). Genes whose null phenotype is Mot– are given the symbol mot plus an extension; there are only two, motA and motB. Genes whose null phenotype is Che– are given the symbol che plus an extension, unless they are receptor genes, in which case they carry special symbols like tar (taxis for aspartate and repellents). (The nomenclature for the mot, che, and receptor genes was not affected by the change in flagellar gene nomenclature.) Table 1 lists the genes in chromosomal order.
Chromosomal Organization of Flagellar Genes.
Flagellar, motility and chemotaxis genes are highly clustered (171) (Fig. 11). Region I (at 24E/23S min) contains many of the structural genes. Region II (41E/40S min) contains some flagellar genes involved in regulation and assembly, the mot genes, and the che genes. Region III, originally thought to be a single contiguous region, was shown recently to consist of two regions, IIIa and IIIb (43E/40S min), with a disruption consisting of DNA unrelated to the flagellar gene system (87). The disruption is an ancient one, having taken place prior to the divergence of E. coli and S. typhimurium. Although regions II, IIIa, and IIIb are indicated as being at roughly the same map position, they are in fact appreciably separated. Regions II and IIIa are separated by about 23 kb of sequence that contains unrelated genes such as uvrC. Regions IIIa and IIIb are separated by a smaller distance, about 7E/5S kb, that contains a previously unknown gene, amyA, encoding a cytoplasmic α-amylase (163), as well as several other putative genes (87). Interestingly, it also contains the longest contiguous stretch (2.7 kb) of apparently noncoding DNA yet described for E. coli (164). Although the limits of the flagellar regions are known qualitatively, the only definitive examples are the amylase/noncoding region between regions IIIa and IIIb and the rightmost boundary of region IIIb, where fliR is followed by a short noncoding region (137; Kihara and Macnab, unpublished) and then a gene unrelated to flagellation, rcsA (regulation of capsule synthesis) (187).
Flagellar Regulon.
The large number of flagellar and related genes are organized into 15 (E. coli) or 17 (S. typhimurium) operons (99, 115). There are also separate operons for various chemoreceptors such as the serine receptor, Tsr. The flagellar operons are organized in a hierarchical fashion as a regulon, with expression of operons at a given level affecting expression of operons at lower levels (70, 110, 130) (Fig. 12). The coding regions of adjacent genes within an operon closely abut each other and in many cases overlap (e.g., ATGA). A notable exception is the existence of a 54-bp noncoding region containing a stem-loop sequence between the rod and ring genes in the flgB operon (78), possibly a regulatory feature.
Master (flhDC) Operon.
The flhDC operon lies at the top of the hierarchy as the sole class 1 operon, with both of its gene products being absolutely required for the expression of all other genes in the flagellar regulon, i.e., those belonging to the class 2 and class 3 operons. It was originally thought that either FlhC or FlhD or both might be transcription initiation (σ) factors (55). The idea fell into disfavor with the experimental proof that a distinct protein, FliA, was a flagellum-specific σ factor (see below). It is not clear how expression of the master operon itself is achieved (i.e., what it employs for a σ factor), although it is known to be activated by cyclic AMP via binding of catabolite activator protein to a site upstream of the promoter (100, 172).
Class 2 and Class 3 Operons.
Class 2 and class 3 operons both have a flagellum-specific consensus –10 sequence (GCCGATAA) that is distinct from the general one (7, 114). Class 3 operons have additionally a flagellum-specific –35 sequence (TAAA), whereas class 2 operons have no obvious consensus at that position. This suggests that the expression of the two classes must be related yet distinct.
FliA is required for expression of class 3 operons, with some exceptions; in Fig. 12, the class 3 operons are broken down into classes 3a and 3b to distinguish those that have some FliA-independent expression from those that do not. The exceptions are as follows. (i) flgM and flgN can be expressed independently of FliA by readthrough of the transcript from the class 2 flgA promoter (50, 107). (ii) fliA mutants assemble all three of the hook-associated proteins (64), indicating that there must be low-level FliA-independent expression of the flgK and fliD operons. In the case of the flgK operon, this is a result of readthrough from the upstream operon (flgB), which is class 2 (109). In the case of the fliD operon, it is because the operon itself possesses a class 2 promoter in addition to the class 3 one (109).
The requirement for FlhC and FlhD for expression of class 3 operons appears to be an indirect one, namely, for the synthesis of FliA (a class 2 product). If fliA is expressed from a foreign promoter, class 3 genes are no longer FlhC-FlhD dependent. Class 2 operons, in contrast, are absolutely dependent on FlhC/FlhD (110). However, expression is enhanced by FliA (with the enhancement being negatively regulated by FlgM, just as with class 3 operons). What then does this say about the relative roles of these proteins? The existence of the shared –10 consensus suggests that FliA might be acting as a σ factor for both classes of operons. However, if this is the case, what are the roles of FlhC and FlhD? Do they modify the recognition specificity so that the –35 sequence is no longer necessary? There is also the question of what functions as a σ factor in the absence of FliA. FlhC/FlhD might be that factor, or there may be another flagellum-specific factor. Alternatively, some other σ factor (such as the housekeeping factor, σ 70) might acquire the ability to function with flagellar promoters as a result of FlhC-FlhD action.
Flagellum-Specific s Factor (FliA) and Its Antagonist (FlgM).
A few years ago, the gene flgM (the symbols flgR and rflB have also been used), with a repressor-like activity, was discovered (48, 49, 154). Mutants defective in flgM had elevated levels of expression of class 3 operons. With the characterization of the products of the fliA and flgM genes, it emerged that FlgM acted not as a repressor (in the sense of a DNA-binding protein) but as a protein that bound the σ factor FliA and thereby prevented it from binding to class 3 promoters (111, 154). Thus, FlgM may best be described as an anti-σ factor. This raises the question about how this anti-σ activity is itself controlled, so that class 3 operons can be expressed under the appropriate conditions.
Control of FlgM Activity by Its Export from the Cell.
A long-standing and puzzling observation has been that expression of all of the large number of class 2 genes, many of them structural, is needed for expression of class 3 operons (114). Even before it was known that FlgM was an anti-σ factor rather than a DNA-binding protein, it was difficult to imagine how all these structural proteins could be operating in a direct fashion to control gene expression. The answer is a truly remarkable one, intimately related to the process of flagellar assembly itself (66, 107, 123) (Fig. 12). Most of the flagellar components that lie beyond the cell membrane are exported by a specialized pathway through the nascent organelle itself (see below). Although the pathway functions primarily for proteins that are going to be assembled into structure, it has been co-opted for another, quite distinct function, namely, the removal of FlgM from the cell at the point when expression of class 3 operons is needed. FliA, which remains behind in the cell, is no longer inactivated and is capable of binding to class 3 promoters. An important feature of the export pathway is that there is control of which proteins can be exported at what stage of assembly: the relevant stage for FlgM is upon completion of the hook, just prior to addition of hook-associated proteins and assembly of filament—in other words, at precisely the boundary defined genetically by operon class.
Negative Regulation by the fliD Operon.
As well as the repressor-like activity now known to be associated with FlgM, there is a repressor-like activity associated with the fliD operon (114), which encodes the filament-capping protein; this, again, is a case of a structural gene apparently playing a genetic regulatory role. The subsequent discovery of two small genes of unknown function (fliS and fliT) in the fliD operon (87) raised the possibility that these were regulatory. This may still be true, but a regulatory mechanism for FliD itself can now be envisaged, since fliD mutants were found to excrete more FlgM than wild-type cells (107) (presumably because of the absence of the long filament channel) and hence to demonstrate a strongly derepressed phenotype. Thus, the repressor activity may be a simple consequence of the capping function of FliD rather than a physiologically important regulatory activity.
Flagellin Phase Variation.
For reasons that are not really understood but may relate to survival against host defense systems, Salmonella species (but not E. coli) have two genes, at different locations on the chromosome, that code for the filament protein flagellin (Table 1). The two versions of the gene are highly similar but not identical (81, 118, 156); the divergence is most pronounced in the central part of the sequence and results in distinct antigenicities. In wild-type cells, either product is perfectly functional in assembling into a helical flagellar filament that can propel the cell.
At any given time, only one of the two genes is being expressed, and therefore a cell possesses homogeneous filaments. In a stochastic fashion, on a timescale on the order of 103 to 105 generations, the genes change roles so that the nonexpressed one becomes expressed and vice versa.
The mechanism of this unusual phenomenon has been shown to be as follows (Fig. 13) (113, 175, 176): The fliC S gene is subject to repression by the product of the fljA S gene, which is contained within the fljB S operon. Expression of the fljB S operon therefore precludes expression of the fliC S gene. Alternating expression and nonexpression of the fljB S operon is caused by the inversion of a 970-bp region of DNA upstream of the fljB S gene that contains the fljB S promoter; in one orientation the promoter is in the correct position and orientation for transcription initiation, while in the other it is not. The inversion region is bounded by an inverted repeat (hix) that permits homologous recombination. It also contains the promoter and coding sequence of a gene, hin S, whose product is the site-specific recombinase responsible for mediating the inversion event; hin is expressed in either orientation, possibly under negative autoregulation to ensure that inversion is a relatively infrequent event.
Interestingly, the Hin protein shows extensive sequence homology with the TnpR protein, which performs a related recombinational function, resolution of the cointegrate structure of Tn3 and the host chromosome. It is suggested that the flagellin phase variation mechanism may have evolved from a host-associated transposon (178, 192). A variety of other site-specific rearrangements related to hin are also known (see chapter 125 of this volume).
As described above, the flagellar gene system consists of clusters of operons. Table 1 lists the genes and operons in chromosomal order, indicates the class to which operons belong, and provides brief comments. Table 2 emphasizes the gene products by functional category (control of gene expression, flagellar structure, etc.). Many of the genes and products are discussed at greater length in other sections of this chapter.
For a detailed recent review of the flagellar switch, see reference 131. Motor/switch components should be identifiable genetically by looking for mutants that either are paralyzed or have abnormal switching characteristics. Aside from chemotaxis genes such as cheA and cheY, only three genes (fliG, fliM, and fliN) have been found to cause switching defects (160, 213); since the searches have been extensive, it seems unlikely that other switch genes exist. Intergenic suppression analysis had suggested that the three proteins existed as a multisubunit complex (212), and recent studies employing the di-hybrid reporter assay indicate that FliG and FliM at least do interact strongly, with positions in FliG important for the interaction lying mostly within the middle one-third of the protein (D. Marykwas, personal communication). The switch proteins have now been located as a large structure attached to the cytoplasmic face of the basal-body MS ring (44, 88) (see above).
All three genes give rise to four distinct mutant phenotypes depending on the mutation involved: (i) nonflagellate (Fla–), (ii) paralyzed (Mot–), (iii) smooth-swimming (Che–[CCW]), and (iv) tumbly (Che–[CW]). Thus, these proteins are important for flagellar structure as well as function, and they are important for the mechanism of rotation as well as for controlling its direction. Note that the switch does not consist of separate CCW-generating and CW-generating subunits; all three proteins contribute to both states of the switch.
A large number of mutations in these three genes, most generated as suppressors of cheY or cheZ mutations, have been sequenced (74, 179). FliM has many more amino acid positions that are important in switching than does either FliG or FliN, but it has relatively few that affect rotation. Positions important for one switch state (say, CCW) tend to exist in clusters that are separate from clusters for the other switch state; these clusters may contribute to alternate interfaces between subunits in the two switch states. Many of the mutations that affect switching represent a shift in charge, from more positive to more negative, indicating that electrostatic interactions may be important in switching.
The fact that FliM has so many residues important for switching suggests that it is the most central component of the switch and therefore the most likely target for binding of the switch regulator, CheY-P; in vitro binding assays indicate this is in fact the case (207).
When expression of the fliM gene is held to artificially low levels, the number of flagella is subnormal, and those that do assemble operate in an erratic fashion, with rapid speed fluctuations around a low mean value (Blair, personal communication). (This is in contrast with similar experiments with the Mot proteins [see below].) Thus, a full complement of FliM appears to be necessary, perhaps because only then can the switching process be a fully cooperative phenomenon; the role of FliM in torque generation may be an indirect one.
Recall that the Mot proteins, MotA and MotB, give rise to the Mot– phenotype only, indicating that they play no role in switching or in assembly of the rest of the flagellum. In experiments in which expression is repressed and then progressively induced, a succession of relatively stable submaximal speed values are seen, indicating that individual subunits of the Mot proteins contribute as independent entities to the generation of torque (20, 24). Their membrane location strongly suggests a role in transmembrane proton conductance. Wild-type MotA incorporated into vesicles in fact causes proton conductance, whereas mutant versions do not. Overexpression of MotA was found to impair growth (21, 208), presumably by making the membranes leaky to protons, but co-overexpression with MotB reversed the effect (186, 209). Interestingly, growth impairment is not caused by the absence of MotB, as first thought, but by the presence of an artifactual fusion protein consisting of N-terminal MotB sequence and plasmid-encoded sequence; total elimination of MotB sequence or introduction of mutations into the N-terminal sequence results in close to normal growth rates (186). The available evidence thus suggests that MotA and MotB jointly contribute to the proton conductance pathway.
A number of mutant versions of the Mot proteins have been sequenced, using dominant mutants to avoid the trivial cases in which the protein cannot even assemble (22). In the case of MotA, most of the mutations abolishing or severely impairing function occurred in the transmembrane regions. However, several mutations gave rise to the relatively mild defect of slow-swimming phenotype and reduced proton conductance; at the high load of tethered cells, the torque was normal. These mutations were mostly in the cytoplasmic domain and may represent impaired kinetics at sites of delivery of protons to the motor/switch complex.
In the case of MotB, most of the mutations were in the periplasmic domain and resulted either in complete paralysis or subnormal torque at all load conditions (23). A possible explanation of the latter category would be impaired but not abolished interaction with the peptidoglycan layer or with MotA, resulting in defective stator function.
Interestingly, some mutational sites in the periplasmic domain of MotB can be suppressed by mutations in the switch protein FliG (47, 74). Given what is known about the structure of the motor/switch (Fig. 8B), this may be an indirect effect, possibly via FliM.
Formation of flagella is coupled with the cell cycle and is dependent on the process of cell division itself; it is not directly dependent on the process of DNA replication (152). Control is at the transcriptional level, operating on the flagellar master operon, but the mechanism is unknown. Flagellar assembly is also dependent on growth phase: cells taken from stationary phase and placed in fresh medium have their existing flagella diluted out by cell division, and new flagella do not appear for several generations. Maximal flagellation and motility are not achieved until late log phase (S.-I. Aizawa, personal communication).
The pathway of flagellar morphogenesis is illustrated in Fig. 14 (72, 79, 104, 190, 191). The earliest known structure is the MS ring. The next stages involve peripheral structures such as the switch-containing C ring. A number of genes are needed for the next stage, rod assembly, including the rod structural genes (flgB, flgC, flgF, and flgG) and another structural gene (fliE), whose product location in the basal body is not known. Also needed are several gene products that are thought to function in export and assembly.
Subunits of the P and L rings are exported to the periplasm and outer membrane, respectively, in a process that involves signal peptide cleavage (61, 78) and presumably the primary (Sec) export pathway. These subunits assemble into rings around the basal-body rod. P-ring assembly requires FlgA (104, 190), a protein which has not been found in the flagellar structure and might be acting as a chaperone, for example; assembly also requires formation of an internal disulfide bond in FlgI, mediated enzymatically by the Dsb system (31).
The transition from rod assembly to hook assembly requires FlgD (104, 190, 191), which assembles as a cap at the rod tip (155), enabling hook subunits to assemble by insertion at the distal end of the growing structure. If the outer rings of the basal body are lacking, the nascent hook cannot penetrate the outer membrane and assembly terminates prematurely (104).
When the hook is complete, FlgM is exported, and expression of the late (class 3) operons commences (66, 107). FlgK (hook-associated protein 1 [HAP1]) is also exported at this stage and displaces the hook cap to form the first hook-filament junction zone; in other words, FlgD is used as a scaffolding protein (155). The FlgK zone is followed by distal assembly of the second hook-filament junction zone, made out of FlgL (HAP3) subunits. Finally, a third zone, made out of FliD (HAP2) subunits, is added distally to form a cap (73).
This cap enables flagellin subunits to insert into the distal end of the nascent filament rather than being lost to the external medium. In contrast to the hook cap, however, the filament cap is retained indefinitely, presumably because filament assembly is the final stage of flagellar morphogenesis and proceeds indefinitely.
MotA and MotB are probably assembled into the cytoplasmic membrane surrounding the MS ring as soon as they are expressed as products of a class 3 operon, i.e., at the point when FlgM has been exported from the cell (66, 107). However, setting aside the question of expression, they can probably assemble as soon as the MS ring and C ring are in place; they can also be assembled extremely late, after the rest of flagellar assembly is complete, as experiments with the motA and motB genes under inducible control have shown (20, 24).
With the P- and L-ring subunits as the only known exceptions (61, 78), flagellar proteins that lie beyond the cell membrane are exported by a specialized flagellum-specific pathway, distinct from the primary protein export (Sec) pathway. The proteins exported by the flagellum-specific pathway are those for the rod, hook, hook cap, hook-filament junction, filament, and filament cap; collectively, they are called the axial family of proteins, since they form a continuous axial structure that extends from the proximal rod to the filament cap (59, 62). The evidence for a flagellum-specific pathway is as follows: (i) The exported proteins do not have a cleaved signal peptide sequence (59, 62, 81, 118, 155), whereas all known Sec-mediated proteins do. (ii) Individual protein subunits insert at the distal end of the nascent structure (40, 68), a target that would be exceedingly improbable by bulk diffusion through the medium, especially in the case of the filament, which can be over 10 μm long. (iii) A channel exists in the filament (144, 148), the hook (147), and presumably also the rod.
The channel should be thought of as a conduit for proteins once they have been exported, rather than as the export apparatus itself. It seems likely that the export process is an active one, requiring the supply of energy, given the presumably substantial frictional coefficient as the subunits travel the enormous distance down the channel in a single-file process like peas in a peashooter. FliI, a flagellum-specific ATPase (see below), might be involved in the process.
Substrate Specificity.
The flagellar proteins that are exported must be recognizable as different from all other proteins (and small molecules) in the cell, but the basis for this recognition is not known. Although their amino acid sequences do show sequence similarities at their termini (59, 62), these probably reflect their similarities as axial structural proteins rather than signals for export. It has been shown that the N-terminal one-third of the flagellin sequence suffices for its own export (119), and in the case of the hook protein of Caulobacter crescentus, the relevant sequence has been further localized to a stretch of 21 amino acids 38 amino acids in from the N terminus (102). However, this sequence bears no resemblance to that in the corresponding position in the S. typhimurium hook protein.
Components of the Export Apparatus.
For flagellar proteins to be transferred into the axial channel, they must cross the plane of the cell membrane, presumably via the MS ring. What is the apparatus that is responsible for recognizing them and carrying out the transfer? There is no clear answer to this yet. In one set of experiments, temperature-sensitive mutants were grown at the permissive temperature, subjected to hydrodynamic shear to remove their flagellar filaments, and then placed at the restrictive temperature (200). For mutants with mutations in most of the genes tested, filaments regrew and motility was restored. For mutants with mutations in three genes, flhA, fliH, and fliI, however, regrowth did not occur, suggesting that the export process was not functioning. Both flhA and fliI belong to the set of flagellar genes that have equivalents in virulence systems (see below). There are a number of other proteins, several of them integral membrane proteins (FlhB, FliO, FliP, FliQ, FliR), that may also be involved in the export process.
The location of the export apparatus has not been demonstrated, but it is likely to be at the cytoplasmic face of the basal body, in the same general location as the switch. The MS ring appears to have a central pore of about 10 nm (Aizawa, personal communication), and so the export apparatus may occupy that pore, spanning the membrane and perhaps projecting into the cytoplasm. FliP, an integral membrane protein implicated in the export process, has been shown to be associated with the basal body (K. Ohnishi and R. M. Macnab, unpublished data), but it is not yet known whether its location is at the center of the MS ring.
Control of the Order and Extent of Export.
For assembly to proceed correctly, there has to be control of the sequential order of export of the various proteins and (at least in the case of the hook) of the number of subunits exported. There are probably at least three phases of this process: export of the rod proteins, then export of the hook-capping protein and hook protein, and finally export of the anti-σ factor FlgM, the hook-filament junction proteins, the filament-capping protein, and flagellin. To some extent, control is at the level of gene expression, but this cannot be the complete answer, since (i) the rod genes and the hook gene both belong to the same operon and so are subject to the same transcriptional control and (ii) complementation usually occurs even when flagellar genes are not being expressed under control of the flagellar regulon. There must therefore be something about the nascent structure itself that determines which of the flagellar proteins can be exported. The most puzzling aspect of this process is how the export apparatus can know the extent of assembly of an external structure.
Hook length control and the roles of FliK and FlhB.
A protein which is key to control of hook assembly is FliK. In wild-type cells, the hook assembles to a length of ca. 55 ± 6 nm (56, 80). fliK mutants are characterized by a double failure: (i) they fail to terminate hook assembly at the proper length but instead generate very long structures called polyhooks, and (ii) they fail to initiate filament assembly (161, 189). These two aspects of FliK function are separable, since suppressor mutants are found that have recovered the second aspect of function, filament assembly. Such mutants are called polyhook-filament mutants. They are of two types, intragenic and extragenic (56, 112). Suppressible first-site fliK mutations are nonsense or frameshift mutations, while the intragenic suppressor mutations are compensating mutations (such as a further deletion) that result in restoring frame (A. W. Williams and R. M. Macnab, unpublished data). Both the N and C termini appear to be important for terminating hook assembly correctly, while the C terminus alone appears to be responsible for initiating filament assembly. fliK mutations capable of being suppressed extragenically tend to be near the 3' end of the fliK gene; the suppressing mutations have all been found to map to flhB (56, 112) (one of the genes that has a homolog in virulence systems [see below]). The sequence of FlhB (145; Kim, Ph.D. thesis) indicates that it is an integral membrane protein and that the suppressor mutations all lie in a presumed cytoplasmic domain toward the C terminus (112; Williams and Macnab, unpublished). The evidence argues against a direct interaction between FliK and FlhB, since the suppression is not allele specific and the damage to FliK that is being suppressed is severe. A plausible general model is that FlhB exerts a negative control on filament initiation and FliK counters this control (112). Loss of both controls, in the extragenic pseudorevertant, results in filament addition but poor control of hook termination.
The deduced amino acid sequence of FliI (200) clearly indicates that it is homologous to the catalytic β subunit of the proton-translocating F0F1 ATPase, with moderate identity levels (ca. 30%) overall and much higher levels in those regions of the sequence that in the β subunit correspond to the nucleotide- binding regions. Site-directed mutation of FliI residues corresponding to ones known to be essential for activity in the β subunit results in a nonflagellate phenotype (36). Purified FliI binds ATP (36); preliminary evidence indicates that it can also hydrolyze it (G. Dreyfus, personal communication; Williams and Macnab, unpublished). No other flagellar proteins resemble any other components of the F0F1 ATPase, and attempts to identify proteins interacting with FliI have been unsuccessful. Intergenic suppression analysis of fliI mutants has thus far yielded examples involving control of gene expression only; for example, one class of suppressors lies in fliA, the gene encoding the flagellum-specific σ factor (S. Yamaguchi, personal communication).
The role of FliI in export is not known. Possibilities include acting as a chaperone to deliver the proteins to be exported to the export apparatus or as the energy-supplying component of the export apparatus itself.
Shortly after the similarity of FliI to the F0F1 β subunit was discovered, an even more pronounced set of similarities was found between several flagellar proteins and proteins that are involved in either the expression of virulence genes or the export of virulence proteins or both. The examples for which similarities have been seen are listed in Table 4; see chapter 151. The flagellar proteins include FliI and FlhA (two of those that affected filament growth), FlhB (which can affect hook versus filament assembly), and a basal-body protein, FliP. It should be emphasized that the proteins that are related are ones implicated in the export process, not the actual proteins that are exported.
Table 4Homologs between proteins involved in flagellar assembly and proteins involved in export of virulence factors |
Flagellar motility is an extremely ancient system, pre-dating the divergence of archaebacteria and prokaryotes. This suggests that the export systems for virulence factors (which would presumably be of value only when potential hosts existed) may have evolved from those for flagellar proteins.
There have been major advances in our knowledge of bacterial flagellation and motility in the 8 years since the first edition of this book appeared. Some of the molecular mechanisms responsible for controlling expression of the flagellar regulon have been clarified, including the discovery of a flagellum-specific σ factor, an anti-σ factor, and an inactivation process for the latter involving its export through the flagellar structure itself. All of the known flagellar and motility genes have been cloned and sequenced, and in many instances the roles of their products have been substantially clarified; it is unlikely that any significant number of genes remain to be discovered. The definition of the basal body has been extended to include a large cytoplasmic ring structure containing the motor/switch proteins. The Mot and motor/switch proteins have been subjected to analysis at the level of individual amino acid changes and their consequences for function. Analysis of the dynamics of the motor has been greatly refined and extended over a large speed range by the application of external torque. The process of flagellar assembly, including the control of hook length and the use of a scaffolding process during its assembly, is better (although still not well) understood. Two remarkable connections have been made to other systems, namely, the F0F1 ATPase and export of virulence factors.
Probably the biggest gaps in our current knowledge concern (i) the molecular mechanisms for conversion of proton motive force into torque and for determining whether it is applied to CCW or CW rotation and (ii) the process of flagellar protein export and how it determines the identity, quantity, and order of its substrates.
I gratefully acknowledge the many colleagues who have provided information prior to publication. Work in my laboratory is supported by USPHS grants AI12202 and GM40335.
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