No abstract

Three-dimensional reconstructions from electron cryomicrographs of the rotor of the flagellar motor reveal that the symmetry of individual M rings varies from 24-fold to 26-fold while that of the C rings, containing the two motor/switch proteins FliM and FliN, varies from 32-fold to 36-fold, with no apparent correlation between the symmetries of the two rings. Results from other studies provided evidence that, in addition to the transmembrane protein FliF, at least some part of the third motor/switch protein, FliG, contributes to a thickening on the face of the M ring, but there was no evidence as to whether or not any portion of FliG also contributes to the C ring. Of the four morphological features in the cross section of the C ring, the feature closest to the M ring is not present with the rotational symmetry of the rest of the C ring, but instead it has the symmetry of the M ring. We suggest that this inner feature arises from a domain of FliG. We present a hypothetical docking in which the C-terminal motor domain of FliG lies in the C ring, where it can interact intimately with FliM.The bacterial flagellum of Salmonella enterica serovar Typhimurium has a reversible rotary motor powered by the proton gradient across the cell's plasma membrane. The flagellar filament, with its corkscrew shape, converts the motor's torque to thrust. Counterclockwise (CCW) rotation of the filament by the motor pushes the cell through the liquid medium, whereas a brief intervening burst of clockwise (CW) rotation causes the cell to tumble. Following the CW burst, the motor resumes CCW rotation, and the cell swims off in a new direction. Approximately 40 proteins are involved in the regulation, assembly, and operation of the flagellum. At least 24 of them are components of the completed flagellum. Of the 24, only 5, i.e., MotA, MotB, FliG, FliM, and FliN, appear to be involved in torque generation (for reviews, see references 1, 2, and 25). MotA and MotB (8, 37) form a proton channel through the plasma membrane (3, 38); they are assumed to form the stator (7, 18). The remaining three are cytoplasmic proteins that form the switch complex (49) and are assumed to be part of the rotor. FliG appears to be most directly involved in torque generation (14,22), and the C-terminal domain of FliG contains key charged residues that interact with charged residues in MotA (53). The N-terminal portion of FliG interacts with FliF, the transmembrane component of the rotor (20, 31), which functions as a mechanical mount for the rotor and couples the motor to the rod or drive shaft. FliM is involved in switching between CCW and CW rotation (34) and binds phospho-CheY (48), tipping the motor's bias toward CW rotation. FliN appears to play a smaller role in rotation and switching (14, 22) but has an important role in flagellar export (46).The gross structural features of the flagellum are a filamentous axial structure and a set of coaxial rings (Fig. 1). MotA and MotB, the putative stator, form a ring of about 10 studs in the plasma membrane (17, 18)...

Image analysis of isolated F-actin filaments shows that the actin helix can be described by a constant rise per subunit but a considerably variable and randomized twist (number of units per turn). The ability of actin subunits to rotate through angles of the order of 10 degrees from their helically ideal positions helps to explain actin's capacity to form many different polymorphic structures.

The direction of rotation of the bacterial flagellum is determined by the flagellar switch. We have localized FliG, one of the switch proteins of Sabnonella typhimurium, to the cytoplasmic face of the M ring of the llar basal body. This lozation was made possible by the discovery of two spontaneous mutants in which thefliF (M ring) and JUG (switch) genes were fused in-frame. In the first mutant, a deletion of 7 base pairs at the 3' end of fliF resulted in an essentially full-length fusion protein. In the second mutant, a larger deletion resulted in a fusion in which 56 amino acids from the carboxyl terminus ofFliF and 94 amino acids from the amino terminus of FliG were lost. Both strains were motile and underwent switching; the first strain had a clockwise bias, and the second strain had a counterclockwise bias. Gel electrophoresis and immunoblotting of isolated hook-basal-body complexes verified that they contained the fusion proteins. Electron microscopy revealed additional mass at the cytoplasmic face of the M ring, which could be decorated with anti-FliG antibody. We conclude that the natural location for FIUG is at the cytoplasmic face of the M ring and that the stoichiometric ratio between FliF and FIG in wild-type cells is probably 1:1.The bacterial flagellum contains a switch that determines whether its motor is in counterclockwise or clockwise rotation. Genetic evidence has indicated that in Salmonella typhimurium the switch is a complex containing subunits of three proteins-FliG, FliM, and FliN (1, 2). Based on the fact that some mutants with defects in the fliG, fliM, and fliN genes are paralyzed (2), the switch is also presumed to be needed for the actual process of rotation, along with two other proteins, MotA and MotB (3).A long-standing question has been the physical location of the switch. A membrane-associated structure called the hook-basal-body (HBB) complex (Fig. 1) has been described, but this complex was not found to contain any of the three switch proteins (4, 9); however, the protocol for isolating this structure is rather harsh and could dissociate the labile components.If the switch is not part of the basal body, where is it likely to be located? The facts that (i) the direction of motor rotation is modulated by the cytoplasmic protein CheY (10, 11) and (ii) che Y mutations can be suppressed by switch mutations and vice versa (1,(12)(13)(14) suggest that the switch should be near the cytoplasmic face of the basal body. The most proximal feature of the basal body is the M ring, which in the intact cell is integral to the cell membrane (15) (Fig. 1)

SummaryMost of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum-specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are FliP (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS-ring protein FliF and immunoblotting isolated hook basal body complexes with anti-FLAG monoclonal antibody, we estimate (using the FLAG-tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti-FLAG antibody and gold-protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains FliP and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.