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* Abstract The bacterial flagellum is both a motor organelle and a protein export/assembly apparatus. It extends from the cytoplasm to the cell exterior. All the protein subunits of the external elements have to be exported. Export employs a type III pathway, also utilized for secretion of virulence factors. Six of the components of the export apparatus are integral membrane proteins and are believed to be located within the flagellar basal body. Three others are soluble: the ATPase that drives export, a regulator of the ATPase, and a general chaperone. Exported substrates diffuse down a narrow channel in the growing structure and assemble at the distal end, often with the help of a capping structure.
Key Words flagellum, protein export, assembly, type HI pathway, motility
INTRODUCTION FLAGELLAR STRUCTURE The Basal Body The Flagellar Motor The Switch The Hook The Flagellar Filament Capping Proteins Junction Proteins The Export Apparatus MOTILITY Patterns of Motility The Mechanism of the Flagellar Motor CHEMOTAXIS THE MORPHOGENETIC PATHWAY AND MECHANISMS OF ASSEMBLY Assembly of the MS Ring and Membrane Components of the Export Apparatus Assembly of the Mot Proteins Assembly of the Rotor/Switch Type III Flagellar Export Assembly of FliE and the Flagellar Rod L and P Ring Assembly Hook Assembly Assembly of Junction Proteins Filament Assembly and the Role of the Filament Cap THE TYPE III EXPORT APPARATUS FlhA The Role of FlhB Cleavage and FliK in Determining Specificity of Substrate Export FliO, FliP, FliQ, and FliR THE RECOGNITION SIGNAL FOR FLAGELLAR PROTEIN EXPORT Motifs in the Substrate or in Its mRNA Local Versus Global Control of Export CONCLUDING REMARKS
Although this review is on the subject of flagellar assembly, it seems appropriate to provide a brief background on flagellar structure, motility, and chemotaxis. Emphasis is on the enteric bacteria Salmonella and Escherichia coli.
The Basal Body
The basal body consists of an integral membrane ring called the MS ring, a rod that traverses the periplasmic space, a periplasmic P ring, and an outer membrane L ring. The basal body is a passive structure, i.e., it receives torque from the motor and transmits it to the hook and then to the filament.
The Flagellar Motor
The flagellar motor, which operates by a rotary mechanism, can be subdivided into two major components: the stator and the rotor. The stator consists of multiple copies, arranged around the basal body, of an integral membrane structure made from two proteins, MotA and MotB. The stator is attached noncovalently to the peptidoglycan layer and therefore is stationary in the frame of reference of the cell. The rotor, which consists of multiple copies of a protein called FliG, is noncovalently attached to the MS ring and, together with the Mot proteins (11, 93), is responsible for torque generation (51),
Motility is under the control of environmental signals and therefore the motor needs more than one mode of operation in order to respond. The most widely studied mechanism is reversal of the direction of rotation of the motor between counterclockwise and clockwise. This requires a switch, which in Salmonella consists of subunits of three proteins, FliG, FliM, and FliN (20, 88). Their precise stoichiometry is not known, except in the case of FliG, which can form a functional fusion with the MS ring protein FliF (19), and whose stoichiometry is close to 26 (37). Best estimates for FliM and FliN are about 37 and 110, respectively (91, 92). Thus the switch is a large structure. Morphologically, FliM and FliN form a cytoplasmic cup- or ring-like structure called the C ring (20). FliG participates in switching as well as torque generation, FliM is the target for the output of the sensory transduction chain (12, 83), and the role of FliN is unknown.
The flagellar hook, a cylindrical structure constructed in a similar fashion to the filament, functions as a universal joint. In bacteria such as Salmonella, which have multiple flagella emerging from many positions on the cell, the hooks play the important role of enabling these flagella to function effectively as a bundle (52).
The Flagellar Filament
The filament is a long, thin cylindrical structure that is helical in shape and therefore when rotated functions like an Archimedes screw or propeller. The basis of its helicity is subtle. The structure can be thought of as consisting of 11 fibrils that form the cylinder and are at a slight tilt angle to the cylindrical axis. The quaternary interactions between subunits break symmetry (53), so that the intersubunit distance in the fibrils varies around the circumference. Several helical forms are possible, depending on how many of the fibrils are in the short versus the long state (14). The two extreme forms (11 short and 11 long, respectively) are straight, while several of the others participate in motility. The straight forms have been examined by both cryo-electron microscopy (59, 67) at ~10 [Angstrom] resolution and, more recently, by X-ray crystallography at 2 [Angstrom] resolution (80). The subtle structural differences between the two fibril states are beginning to emerge. An essential feature of the structures that employ the flagellum-specific export pathway is a ~3-nm channel down their center that extends all the way from the periplasmic face of the MS ring to the tip of the filament [(59, 66, 67); D.G. Morgan & D.J. DeRosier, personal communication].
At the tip of the growing filament is a capping structure, the filament cap (31). At an earlier stage of assembly the same is true of the hook, and at an even earlier stage the same is probably true of the rod. Their role in flagellar assembly is discussed below.
Between the hook and the filament are two short zones of proteins that are called junction proteins (33). A reasonable supposition for their function is that they are structural adaptors, since the hook and the filament have different mechanical properties, the former as a universal joint and the latter as a rigid propeller (discussed in Reference 37). Recent crystallographic studies support this supposition (K. Imada, personal communication).
The Export Apparatus
Consideration of the export apparatus is postponed until we consider the process of flagellar assembly.
Patterns of Motility
Bacterial flagella rotate (82), rather than flex, as eukaryotic flagella do. The consequences depend on the direction of rotation and the handedness of the helical filament. In Salmonella the handedness in its normal form is left-handed, and so counterclockwise rotation causes the helical waveform to travel outward from the cell, generating a pushing motion. The multiple filaments of a bacterium such as Salmonella then form into a bundle at one or the other pole of the cell, propelling the cell forward. Bundle formation is simply a consequence of hydrodynamic and mechanical interactions (52).
The other motility mode, tumbling, is a consequence of clockwise rotation. It is a complex phenomenon, involving polymorphic structural changes of the filaments generated by hydrodynamic torsional load. The changes entail conversion to forms such as curly or semicoiled, which are of opposite handedness to the normal form (55, 85). The bundle unravels; the pattern of thrust and torque becomes chaotic; and the cell tumbles before reverting to counterclockwise rotation, bundle formation, and swimming.
The Mechanism of the Flagellar Motor
The rotary flagellar motor utilizes the transmembrane potential of protons, both electrical ([DELTA][PSI]) and chemical ([DELTA]pH) (57, 58). In some marine and alkalophilic bacteria (for example, Vibrio and Bacillus species) sodium ions are used instead (35). Detailed mutational analyses by Blair and coworkers (42) suggest that when the protons cross the membrane, they bind to a specific aspartate residue within MotB, causing a conformational change in the stator that drives the rotor through an elementary rotational step. This is followed by depretonation of the aspartate residue with release of the proton into the cytoplasm, and restoration of the stator to its original conformation.
Bacteria sense environmental signals via chemoreceptors located in their cytoplasmic membrane. The ligand binding site is on the periplasmic domain, and binding causes a conformational change that is transmitted across the transmembrane domain to the cytoplasmic domain. This domain includes binding sites for the histidine autokinase CheA and its regulator CheW (involved in sensory excitation) and sites for methylation and demethylation of receptor glutamyl side chains (involved in sensory adaptation). Decreasing ligand occupancy results in autophosphorylation of CheA and transfer of its phosphoryl group to a specific aspartate residue on either of two small proteins, CheY and CheB. Phosphorylated CheY (CheY-P) binds to the motor switch and biases it toward clockwise rotation. CheB-P is the activated form of the receptor-demethylating enzyme. CheY-P gradually becomes dephosphorylated by the phosphatase CheZ. [For recent reviews on chemotaxis see (9, 13).]
In spatial gradients of attractants, such as aspartate, cells swimming upgradient sense a temporal gradient (54) as an increasing occupancy of their receptors by ligand, and this results in decreased probability of tumbling. The result is a random three-dimensional zigzag trajectory that is biased by the spatial gradient information, and so the cell displays chemotaxis (7).
THE MORPHOGENETIC PATHWAY AND MECHANISMS OF ASSEMBLY
It is …