FliG, FIiM, and FliN are three proteins of Salnonella typhimurium that affect the rotation and switching of direction of the flagellar motor. An analysis of mutant alleles of FliM has been described recently (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992). We have now analyzed a large number of mutations in thefliG andfliN genes that are responsible for four different types of defects: failure to assembly flagella (nonflagellate phenotype), failure to rotate flagella (paralyzed phenotype), and failure to display normal chemotaxis as a result of an abnormally high bias to clockwise (CW) or counterclockwise (CCW) rotation (CW-bias and CCW-bias phenotypes, respectively). The null phenotype for fliG, caused by nonsense or frameshift mutations, was nonflagellate. However, a considerable part of the FliG amino acid sequence was not needed for flagellation, with several substantial in-frame deletions preventing motor rotation but not flagellar assembly. Missense mutations infliG causing paralysis or abnormal switching occurred at a number of positions, almost all within the middle one-third of the gene. CW-bias and CCW-bias mutations tended to segregate into separate subclusters. The null phenotype of fliN is uncertain, since frameshift and nonsense mutations gave in some cases the nonflagellate phenotype and in other cases the paralyzed phenotype; in none of these cases was the phenotype a consequence of polar effects on downstream flagellar genes. Few positions in FliN were found to affect switching: only one gave rise to the CW mutant bias and only four gave rise to the CCW mutant bias. The different properties of the FliM, FliG, and FliN proteins with respect to the processes of assembly, rotation, and switching are discussed.The direction of rotation of the bacterial flagellum of Salmonella typhimurium is under the control of the flagellar switch, a structure that on genetic grounds is thought to consist of three proteins, FliG, FliM, and FliN, all of which are needed to permit rotation to occur and to control its direction (27). These proteins also affect the structural integrity of the flagellum, so that some mutations, especially severe ones (19), result in failure to assemble flagella at all.The default rotational state of the switch is counterclockwise (CCW), with the clockwise (CW) state being stabilized by a component of the sensory transduction system, CheY, following its phosphorylation by another component, CheA; the simplest hypothesis is that phosphorylated CheY acts by binding to the switch. Still another chemotaxis protein, CheZ, catalyzes the dephosphorylation of CheY, rendering it incapable of stabilizing the CW state. Thus, CheZ has the effect of favoring the CCW state of the switch (for a review, see reference 3).We have recently described an extensive analysis of mutations infliM of S. typhimurium (19

Previous studies have led to the conclusion that in Salmonella typhimurium (and also in Escherichia coli) there are three flagellar proteins, FliG, FliM, and FliN, which function together in enabling motor rotation and in controlling its direction of rotation (2,4,10,35,41). The part of the flagellum constructed from these proteins is called the flagellar switch. The state of the switch, counterclockwise (CCW) or clockwise (CW), is influenced by the chemotaxis sensory system, such that CCW rotation is enhanced by favorable stimuli and CW rotation is enhanced by unfavorable stimuli (14). On the basis of intergenic suppression analysis in E. coli (24) and the effect of CheY on cell envelopes lacking any of the other chemotaxis proteins (25), CheY is thought to bind to the switch and bias it to the CW state. CheZ has the opposite effect on switch bias (13), and intergenic suppression analysis (23,24) suggests that it, too, binds to the switch; however, in this case no biochemical evidence is available to support the hypothesis. CheY is activated by CheA (by phosphorylation of aspartate 57) and deactivated by CheZ (by dephosphorylation) (1,6,27,38).

The flaAlI.2, flaQ, and flaN genes of Salmonella typhimurium are important for assembly, rotation, and counterclockwise-clockwise switching of the flagellar motor. Paralyzed and nonchemotactic mutants were subjected to selection pressure for partial acquisition of motility and chemotaxis, and the suppressor mutations of the resulting pseudorevertants were mapped and isolated. Many of the intergenic suppressor mutations were in one of the other two genes. Others were in genes for cytoplasmic components of the chemotaxis system, notably cheY and cheZ; one of the mutations was found in the cheA gene and one in a motility gene, motB. (14), i.e., it contains a binary switch. The switch operates even in unstimulated cells; stimijli bias the switching events in a way that results in migration toward more favorable environments (3, 17).The motors are located at the cell surface. After digestion of the peptidoglycan layer and dissolution of the outer and cytoplasmic membranes, a structure can be isolated which is termed the flagellar basal body and consists of four rings and a rod (7,8). A variety of evidence, including a recent study of Salmonella typhimurium (1), indicates that the basal body is not the entire motor. It does not contain any of the proteins known to be involved in the two central functions of the motor, conversion of proton motive force into rotational work and switching between the two rotational senses.Five proteins are known to play important roles in rotation and switching. They are MotA and MotB (6,9,25,28,29,33) and FlaAII.2, FlaQ, and FlaN (4,5,9,11,12,19,22,23,26,[31][32][33]

SummaryFlhB, an integral membrane protein, gates the type III flagellar export pathway of Salmonella . It permits export of rod/hook-type proteins before hook completion, whereupon it switches specificity to recognize filament-type proteins. The cytoplasmic C-terminal domain of FlhB (FlhB C ) is cleaved between Asn-269 and Pro-270, defining two subdomains: FlhB CN and FlhB CC . Here, we show that subdomain interactions and cleavage within FlhB are central to substratespecificity switching. We found that deletions between residues 216 and 240 of FlhB CN permitted FlhB cleavage but abolished function, whereas a deletion spanning Asn-269 and Pro-270 abolished both. The mutation N269A prevented cleavage at the Flh-B CN -FlhB CC boundary. Cells producing FlhB(N269A) exported the same amounts of hook-capping protein as cells producing wild-type FlhB. However, they exported no flagellin, even when the fliC gene was being expressed from a foreign promoter to circumvent regulation of expression by FlgM, which is itself a filament-type substrate. Electron microscopy revealed that these cells assembled polyhook structures lacking filaments. Thus, FlhB(N269A) is locked in a conformation specific for rod/hook-type substrates. With FlhB(P270A), cleavage was reduced but not abolished, and cells producing this protein were weakly motile, exported reduced amounts of flagellin and assembled polyhook filaments.

The bacterial flagellum is a predominantly cell-external supermacromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhB C ), which is consistently and specifically cleaved into two subdomains (FlhB CN and FlhB CC ) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhB C (P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB C and FlhB C (P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.A large percentage of the bacterial flagellar structure lies outside of the cell envelope, thus requiring that the vast majority of the subunits that compose the flagellum be exported from the cytosol across both the inner and outer membranes. Salmonella employs a type III export pathway to accomplish this (1, 2). It is a Sec-independent pathway that utilizes a flagellum-specific export apparatus to transport flagellar components across the inner membrane. These exported proteins then travel the length of the developing flagellum within an interior channel prior to their incorporation at the structure's cell-distal end (3-6) (the developing structure therefore facilitates export across the outer membrane). At least nine flagellar proteins are involved in the flagellumspecific export apparatus (7). Six are integral membrane components (FlhA, FlhB, FliO, FliP, FliQ, and FliR) postulated to be located within the basal body MS ring (8, 9), and three are soluble components: an ATPase (FliI) that drives export (10), a regulator of the ATPase (FliH) (11-13), and a general chaperone (FliJ) (14, 15).One of the integral membrane proteins, FlhB, has been found to play a central role in export substrate-specificity switching; i.e. regulation of the order in which flagellar subunits are exported, such that proteins incorporated into the cell-proximal rod and hook structure...