Mass determination and estimation of subunit stoichiometry of the bacterial hook-basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy.

Sosinsky, Gina E.; Francis, Noreen R.; DeRosier, David J.; Wall, Joseph S.; Simon, Martha N.; Hainfeld, James F.

doi:10.1073/pnas.89.11.4801

Cited by 55 publications

(43 citation statements)

References 19 publications

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“…An upper limit for the number of flagella per cell could be estimated from the FlgE content, given 130 (Ϯ 10%) FlgE subunits per hook structure (8,28). This upper limit was 7 Ϯ 1, comparable to the number measured by electron microscopy for E. coli grown under similar conditions (13).…”

Section: Vol 178 1996mentioning

confidence: 78%

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

et al. 1996

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Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ratio in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means ؎ standard errors) 41 ؎ 10 and 37 ؎ 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.The propulsion of swimming bacteria is driven by the rotation of motile organelles, the flagella. Each flagellum consists of an external, rigid, helical filament contiguous with an elaborate basal body. The flagellar basal body traverses the cell wall and cytoplasmic membrane, protruding into the cytoplasm. It harbors machinery for energization and switching of flagellar rotation. The molecular motors that drive flagellar rotation are distinct from other biological molecular motors studied thus far in that they are capable of bidirectional force generation and are energized by transmembrane ion gradients instead of ATP (26).Proteins specifically involved in motility have been identified on the basis of the analysis of paralyzed mutants (mot) in the enteric bacteria Escherichia coli and Salmonella typhimurium. Five proteins have thus been identified from a total of 50 or so proteins required for flagellation and chemotaxis (18). Two, MotA and MotB, are integral membrane proteins whose expression determines the assembly of the intramembrane particle ring structures of the flagellar basal body (13). The other three, FliG, FliM, and FliN, have been localized to recently described cytoplasmic extensions of the flagellar basal body by immunoelectron microscopy (7, 39). Mutations that give rise to nonflagellate (fla) or nonchemotactic (che) bacteria have also been isolated in each of these proteins (37). To account for these and additional suppressor mutation data, it has been postulated that these proteins form a macromolecul...

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Section: Vol 178 1996mentioning

confidence: 78%

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

et al. 1996

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“…Based on the arguments advanced in that publication and the performance of the flagella of our hinge mutants, we have concluded that, at least under some conditions, motor switching behavior cannot be entirely accounted for by a simple two-state model. We have simplified our discussion by considering only conformational changes in the 26 FliG subunits (15,20,40) in the rotor. These events can be viewed as the output of the switching event.…”

Section: Vol 186 2004 Mutations Affecting the Interdomain Region Ofmentioning

confidence: 99%

Rusty, Jammed, and Well-Oiled Hinges: Mutations Affecting the Interdomain Region of FliG, a Rotor Element of the Escherichia coli Flagellar Motor

et al. 2004

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Escherichia coli swims by spinning its four to eight flagella (1, 38), which are distributed randomly around the surface of the cell (28). When the flagella turn counterclockwise (CCW), the left-handed helical flagellar filaments coalesce into a bundle that pushes the cell in a gently curved path known as a run. When one or more flagella rotate clockwise (CW), the bundle comes apart (29, 49) and the cells undergo sudden, random changes in direction that are called tumbles. Alternation of CCW and CW flagellar rotation produces a series of runs and tumbles that generates a three-dimensional random walk. Suppression of CW rotation by signals from chemoreceptors enables a cell to migrate up an attractant gradient or down a repellent gradient (41).Most of the ϳ40 flagellar proteins are involved in the assembly of the basal body, hook, and filament. Three proteins have been shown to be directly involved in torque generation: MotA, MotB, and FliG. Null mutations in motA and motB produce paralyzed flagella (3, 4). MotA and MotB are located in the cell membrane (36) surrounding the MS ring (2 2), and they form the transmembrane channel that delivers protons to the motor (3,4,5,42,51). The large C-terminal periplasmic domain of MotB has a putative peptidoglycan-binding domain that is believed to anchor the Mot proteins to the peptidoglycan cell wall (9,11,17,51).FliG is mounted on the cytoplasmic face of the MS ring (15), which is composed of the single polypeptide FliF (50). FliG also contacts the C ring, which contains FliM and FliN (16,21,34). FliG, FliM, and FliN form the switch-motor complex (52), which is required for torque generation, flagellar assembly, and switching the direction of flagellar rotation (19,39,53).FliG from E. coli (FliG Ec contains 330 residues and can be separated into two stable, overlapping domains (25). A fliG mutant with a stop codon at position 246 has paralyzed flagella, whereas a ⌬fliG strain is not flagellated. Certain charged residues in the carboxyl-terminal region of FliG are essential for motility (26) and interact electrostatically with residues of opposite charge (55) in the cytoplasmic loop between transmembrane helices 2 and 3 of MotA (54). The picture of FliG that has emerged is that the amino-terminal region of FliG is required for flagellar assembly, whereas the carboxyl-terminal domain of FliG is required for torque generation.The C-terminal 126 residues of E. coli FliG can be expressed as a stable, soluble cytoplasmic fragment (25). The crystal structure of the C-terminal motility domain of FliG from Thermotoga maritima (FliG Tm ) has been determined (27). A more recent publication describes the structure of a larger C-terminal region that includes the putative hinge between the domains (8).The FliM protein plays an essential role in switching between CCW and CW rotation (39,44,47). FliM interfaces with the chemotactic circuit via the response regulator CheY (7,33,48). The default direction of the motor is CCW, and binding of CheY-phosphate (2) or a mutationally activated fo...

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“…It is the first substructure to be assembled and anchors the flagellum in the cytoplasmic membrane (17). Twenty-six FliF monomers assemble into this ring structure (9,40,43), forming a central pore where the integral membrane components of flagellar export are located (4,16,27). The MS ring also interacts with the axial extension, the rod, on the periplasmic side and with the C ring in the cytoplasm ( Fig.…”

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confidence: 99%

Role of the Cytoplasmic C Terminus of the FliF Motor Protein in Flagellar Assembly and Rotation

2003

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Bacteria swim by virtue of a complex organelle, the flagellum. Motility is conferred by rotating a long filament that acts as a propeller and is connected via a flexible hook and a rod to a membrane-embedded motor (37) that uses the proton motive force as an energy source (19,24). At least 20 proteins are assembled into the highly complex flagellar structure, and another 30 are used for its construction (23). Correct assembly and function of this macromolecular structure require complex regulation on the levels of both gene expression and protein interaction (36).A key component of the flagellar structure is the MS ring (Fig. 1). It is the first substructure to be assembled and anchors the flagellum in the cytoplasmic membrane (17). Twenty-six FliF monomers assemble into this ring structure (9, 40, 43), forming a central pore where the integral membrane components of flagellar export are located (4, 16, 27). The MS ring also interacts with the axial extension, the rod, on the periplasmic side and with the C ring in the cytoplasm (Fig. 1) (6). The latter is required for torque generation and for the switching of flagellar rotation in response to signals from the environment (20,42). The C ring is composed of three proteins, FliG, FliM, and FliN (6,25,41), and is mounted onto the membraneintegral MS ring through a direct interaction between FliG and FliF in a 1:1 stoichiometry (12,18,21,25,30). It has been shown that this interaction occurs between the C terminus of FliF and the N terminus of FliG (5,15,25), and deletion analysis of FliG has revealed that only the N-terminal 46 amino acids of FliG are required for interaction with FliF (15). However, for FliF, the specific requirements for FliG interaction have not been analyzed. The data presented here provided a first indication of the precise position of the FliG interaction site in the FliF C terminus.Along with enteric bacteria, Caulobacter crescentus has been used as a model system by which to understand the assembly process of the flagellar structure and its regulation by the cell cycle. Both gain and loss of motility are integral parts of the C. crescentus developmental cycle, which is, in turn, intimately connected with cell proliferation (10). A flagellum is assembled in the predivisional cell at the pole opposite the stalk. As a consequence, cell division generates two distinct daughter cells, a motile, flagellated swarmer cell and a nonflagellated stalked cell. After a defined period of chemotactic activity, the swarmer cell differentiates into a stalked cell and restarts the division cycle. During this process, the flagellum is ejected into the medium. The FliF protein presumably plays a key role in both cell cycle-dependent assembly and ejection of the flagellum. FliF is expressed very early during the C. crescentus cell cycle and is specifically localized to the swarmer pole of the predivisional cell, where the flagellum is assembled (12). Since the MS ring forms in the absence of any other flagellar component (17), FliF is a good candidate by whic...

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Mass determination and estimation of subunit stoichiometry of the bacterial hook-basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy.

Cited by 55 publications

References 19 publications

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

Rusty, Jammed, and Well-Oiled Hinges: Mutations Affecting the Interdomain Region of FliG, a Rotor Element of the Escherichia coli Flagellar Motor

Role of the Cytoplasmic C Terminus of the FliF Motor Protein in Flagellar Assembly and Rotation

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