Interactions between C ring proteins and export apparatus components: a possible mechanism for facilitating type III protein export

The flagellar switch proteins of Salmonella, FliG, FliM and FliN, participate in the switching of motor rotation, torque generation and flagellar assembly/export. FliN has been implicated in the flagellar export process. To address this possibility, we constructed 10-amino-acid scanning deletions and larger truncations over the C-terminal domain of FliN. Except for the last deletion variant, all other variants were unable to complement a fliN null strain or to restore the export of flagellar proteins. Most of the deletions showed strong negative dominance effects on wild-type cells. FliN was found to associate with FliH, a flagellar export component that regulates the ATPase activity of FliI. The binding of FliM to FliN does not interfere with this FliN-FliH interaction. Furthermore, a five-protein complex consisting of FliG, His-tagged FliM, FliN, FliH and FliI was purified by nickel-affinity chromatography. FliJ, a putative general chaperone, is bound to FliM even in the absence of FliH. The importance of the C ring as a possible docking site for export substrates, chaperones and FliI through FliH for their efficient delivery to membrane components of the export apparatus is discussed.     

The FliN-FliH interaction mediates localization of flagellar export ATPase FliI to the C ring complex

FliH regulates the flagellar export ATPase FliI, preventing nonproductive ATP hydrolysis. FliH has been shown to stably associate with the C ring protein FliN. Analysis of this complex reveals that FliH is required for FliI localization to the C ring, and thus FliH not only inhibits FliI ATPase activity but also may act to target FliI to the basal body. Quantitative binding studies revealed a KD of 110 nM for FliH binding to FliN. The KD for FliH binding of a FliN variant from a temperature-sensitive nonflagellate fliN point mutant was determined to be 270 nM, suggesting a molecular explanation for its phenotype. Another variant FliN from a temperature-sensitive mutant with a different phenotype displayed binding with an intermediate affinity. Weak export activity in a fliN null mutant was greatly increased by overproduction of FliI, mimicking a previously observed FliH bypass effect and supporting the conclusion that FliN-FliH binding is important for localization of FliI to the C ring and thus the membrane-embedded export apparatus beyond. A model incorporating the present findings is presented.     

Crystal structure of the flagellar rotor protein FliN from Thermotoga maritima

FliN is a component of the bacterial flagellum that is present at levels of more than 100 copies and forms the bulk of the C ring, a drum-shaped structure at the inner end of the basal body. FliN interacts with FliG and FliM to form the rotor-mounted switch complex that controls clockwise-counterclockwise switching of the motor. In addition to its functions in motor rotation and switching, FliN is thought to have a role in the export of proteins that form the exterior structures of the flagellum (the rod, hook, and filament). Here, we describe the crystal structure of most of the FliN protein of Thermotoga maritima. FliN is a tightly intertwined dimer composed mostly of beta sheet. Several well-conserved hydrophobic residues form a nonpolar patch on the surface of the molecule. A mutation in the hydrophobic patch affected both flagellar assembly and switching, showing that this surface feature is important for FliN function. The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. T. maritima FliN is primarily a dimer in solution, and T. maritima FliN and FliM together form a stable FliM(1)-FliN(4) complex. Escherichia coli FliN forms a stable tetramer in solution. The arrangement of FliN subunits in the tetramer was modeled by reference to the crystal structure of tetrameric HrcQB(C), a related protein that functions in virulence factor secretion in Pseudomonas syringae. The modeled tetramer is elongated, with approximate dimensions of 110 by 40 by 35 Angstroms, and it has a large hydrophobic cleft formed from the hydrophobic patches on the dimers. On the basis of the present data and available electron microscopic images, we propose a model for the organization of FliN subunits in the C ring.     

Roles of the extreme N‐terminal region of FliH for efficient localization of the FliH–FliI complex to the bacterial flagellar type III export apparatus

Most bacterial flagellar proteins are exported by the flagellar type III protein export apparatus for their self‐assembly. FliI ATPase forms a complex with its regulator FliH and facilitates initial entry of export substrates to the export gate composed of six integral membrane proteins. The FliH–FliI complex also binds to the C ring of the basal body through a FliH–FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. Here, we analysed subcellular localization of FliI–YFP by fluorescence microscopy. FliI–YFP was localized to the flagellar base, and its localization required both FliH and the C ring. The ATPase activity of FliI was not required for its localization. FliI–YFP formed a complex with FliHΔ1 (missing residues 2–10) but the complex did not show any localization. FliHΔ1 did not interact with FliN, and alanine‐scanning mutagenesis revealed that only Trp‐7 and Trp‐10 of FliH are essential for the interaction with FliN. Overproduction of the FliH–FliI complex improved the export activity of the fliN mutant whereas neither of the FliH(W7A)‐FliI nor FliH(W10A)‐FliI complexes did, suggesting that Trp‐7 and Trp‐10 of FliH are also required for efficient localization of the FliH–FliI complex to the export gate.     

A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations.

CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression of fliG mutations. Interestingly, these cheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch.     

Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium.

The flagellar switch of Salmonella typhimurium and Escherichia coli is composed of three proteins, FliG, FliM, and FliN. The switch complex modulates the direction of flagellar motor rotation in response to information about the environment received through the chemotaxis signal transduction pathway. In particular, chemotaxis protein CheY is believed to bind to switch protein FliM, inducing clockwise filament rotation and tumbling. To investigate the function of FliM and its interactions with FliG and FliN, we engineered a series of 34 FliM deletion mutant proteins, each lacking a different 10-amino-acid segment. We have determined the phenotype associated with each mutant protein, the ability of each mutant protein to interfere with the motility of wild-type cells, and the effect of additional FliG and FliN on the function of selected FliM mutant proteins. Overall, deletions at the N terminus produced a counterclockwise switch bias, deletions in the central region of the protein produced poorly motile or nonflagellate cells, and deletions near the C terminus produced only nonflagellate cells. On the basis of this evidence and the results of a previous study of spontaneous FliM mutants (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992), we propose a division of the FliM protein into four functional regions: an N-terminal region primarily involved in switching, an extended N-terminal region involved in switching and assembly, a middle region involved in switching and motor rotation, and a C-terminal region primarily involved in flagellar assembly.     

Mutational analysis of the flagellar protein FliG: sites of interaction with FliM and implications for organization of the switch complex

The switch complex at the base of the bacterial flagellum is essential for flagellar assembly, rotation, and switching. In Escherichia coli and Salmonella, the complex contains about 26 copies of FliG, 34 copies of FliM, and more then 100 copies of FliN, together forming the basal body C ring. FliG is involved most directly in motor rotation and is located in the upper (membrane-proximal) part of the C ring. A crystal structure of the middle and C-terminal parts of FliG shows two globular domains connected by an alpha-helix and a short extended segment. The middle domain of FliG has a conserved surface patch formed by the residues EHPQ(125-128) and R(160) (the EHPQR motif), and the C-terminal domain has a conserved surface hydrophobic patch. To examine the functional importance of these and other surface features of FliG, we made mutations in residues distributed over the protein surface and measured the effects on flagellar assembly and function. Mutations preventing flagellar assembly occurred mainly in the vicinity of the EHPQR motif and the hydrophobic patch. Mutations causing aberrant clockwise or counterclockwise motor bias occurred in these same regions and in the waist between the upper and lower parts of the C-terminal domain. Pull-down assays with glutathione S-transferase-FliM showed that FliG interacts with FliM through both the EHPQR motif and the hydrophobic patch. We propose a model for the organization of FliG and FliM subunits that accounts for the FliG-FliM interactions identified here and for the different copy numbers of FliG and FliM in the flagellum.     

Assembly of the switch complex onto the MS ring complex of Salmonella typhimurium does not require any other flagellar proteins.

The cytoplasmic portion of the bacterial flagellum is thought to consist of at least two structural components: a switch complex and an export apparatus. These components seem to assemble around the MS ring complex, which is the first flagellar basal body substructure and is located in the cytoplasmic membrane. In order to elucidate the process of assembly of cytoplasmic substructures, the membrane localization of each component of the switch complex (FliG, FliM, and FliN) in various nonflagellated mutants was examined by immunoblotting. It was found that all these switch proteins require the MS ring protein FliF to associate with the cell membrane. FliG does not require FliM and FliN for this association, but FliM and FliN associate cooperatively with the membrane only through FliG. Furthermore, all three switch proteins were detected in membranes isolated from fliE, fliH, fliI, fliJ, fliO, fliP, fliQ, fliR, flhA, flhB, and flgJ mutants, indicating that the switch complex assembles on the MS ring complex without any other flagellar proteins involved in the early stage of flagellar assembly. The relationship between the switch complex and the export apparatus is discussed.     

Binding of the chemotaxis response regulator CheY to the isolated,intact switch complex of the bacterial flagellar motor: lack of cooperativity

In bacteria, the chemotactic signal is greatly amplified between the chemotaxis receptors and the flagellar motor. In Escherichia coli, part of this amplification occurs at the flagellar switch. However, it is not known whether the amplification results from cooperativity of CheY binding to the switch or from a post-binding step. To address this question, we purified the intact switch complex (constituting the switch proteins FliG, FliM, and FliN and the scaffolding protein FliF) in quantities sufficient for biochemical work and used it to investigate whether the binding of CheY to the switch complex is cooperative. As a negative control, we used complexes of switchless basal bodies, formed from the proteins FliF and FliG and similarly isolated. Using double-labeling centrifugation assays for binding, we found that CheY binds to the isolated, intact switch complex in a phosphorylation-dependent manner. We observed no significant phosphorylation-dependent binding to the negative control of the switchless basal body. The dissociation constant for the binding between the switch complex and phosphorylated CheY (CheY approximately P) was 4.0 +/- 1.1 microm, well in line with the published range of CheY approximately P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient approximately 1). This lack of CheY approximately P-switch complex binding cooperativity, taken together with earlier in vivo studies suggesting that the dependence of the rotational state of the motor on the fraction of occupied sites at the switch is sigmoidal and very steep (Bren, A., and Eisenbach, M. (2001) J. Mol. Biol. 312, 699-709), indicates that the chemotactic signal is amplified within the switch, subsequent to the CheY approximately P binding.     

Organization of FliN subunits in the flagellar motor of Escherichia coli

FliN is a major constituent of the C ring in the flagellar basal body of many bacteria. It is present in >100 copies per flagellum and together with FliM and FliG forms the switch complex that functions in flagellar assembly, rotation, and clockwise-counterclockwise switching. FliN is essential for flagellar assembly and switching, but its precise functions are unknown. The C-terminal part of the protein is best conserved and most important for function; a crystal structure of this C-terminal domain of FliN from Thermotoga maritima revealed a saddle-shaped dimer formed mainly from beta strands (P. N. Brown, M. A. A. Mathews, L. A. Joss, C. P. Hill, and D. F. Blair, J. Bacteriol. 187:2890-2902, 2005). Equilibrium sedimentation studies showed that FliN can form stable tetramers and that a FliM1FliN4 complex is also stable. Here, we have examined the organization of FliN subunits by using targeted cross-linking. Cys residues were introduced at various positions in FliN, singly or in pairs, and disulfide cross-linking was induced by oxidation. Efficient cross-linking was observed for certain positions near the ends of the dimer and for some positions in the structurally uncharacterized N-terminal domain. Certain combinations of two Cys replacements gave a high yield of cross-linked tetramer. The results support a model in which FliN is organized in doughnut-shaped tetramers, stabilized in part by contacts involving the N-terminal domain. Electron microscopic reconstructions show a bulge at the bottom of the C-ring whose size and shape are a close match for the hypothesized FliN tetramer.     

Domain Analysis of the FliM Protein of Escherichia coli

The FliM protein of Escherichia coli is required for the assembly and function of flagella. Genetic analyses and binding studies have shown that FliM interacts with several other flagellar proteins, including FliN, FliG, phosphorylated CheY, other copies of FliM, and possibly MotA and FliF. Here, we examine the effects of a set of linker insertions and partial deletions in FliM on its binding to FliN, FliG, CheY, and phospho-CheY and on its functions in flagellar assembly and rotation. The results suggest that FliM is organized into multiple domains. A C-terminal domain of about 90 residues binds to FliN in coprecipitation experiments, is most stable when coexpressed with FliN, and has some sequence similarity to FliN. This C-terminal domain is joined to the rest of FliM by a segment (residues 237 to 247) that is poorly conserved, tolerates linker insertion, and may be an interdomain linker. Binding to FliG occurs through multiple segments of FliM, some in the C-terminal domain and others in an N-terminal domain of 144 residues. Binding of FliM to CheY and phospho-CheY was complex. In coprecipitation experiments using purified FliM, the protein bound weakly to unphosphorylated CheY and more strongly to phospho-CheY, in agreement with previous reports. By contrast, in experiments using FliM in fresh cell lysates, the protein bound to unphosphorylated CheY about as well as to phospho-CheY. Determinants for binding CheY occur both near the N terminus of FliM, which appears most important for binding to the phosphorylated protein, and in the C-terminal domain, which binds more strongly to unphosphorylated CheY. Several different deletions and linker insertions in FliM enhanced its binding to phospho-CheY in coprecipitation experiments with protein from cell lysates. This suggests that determinants for binding phospho-CheY may be partly masked in the FliM protein as it exists in the cytoplasm. A model is proposed for the arrangement and function of FliM domains in the flagellar motor.     

Regulated underexpression and overexpression of the FliN protein of Escherichia coli and evidence for an interaction between FliN and FliM in the flagellar motor.

The FliN protein of Escherichia coli is essential for the assembly and function of flagella. Here, we report the effects of regulated underexpression and overexpression of FliN in a fliN null strain. Cells that lack the FliN protein do not make flagella. When FliN is underexpressed, cells produce relatively few flagella and those made are defective, rotating at subnormal, rapidly varying speeds. These results are similar to what was seen previously when the flagellar protein FliM was underexpressed and unlike what was seen when the motility proteins MotA and MotB were underexpressed. Overexpression of FliN impairs motility and flagellation, as has been reported previously for FliM, but when FliN and FliM are co-overexpressed, motility is much less impaired. This and additional evidence presented indicate that FliM and FliN are associated in the flagellar motor, in a structure distinct from the MotA/MotB torque generators. A recent study showed that FliN might be involved in the export of flagellar components during assembly (A. P. Vogler, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). We show here that approximately 50 amino acid residues from the amino terminus of FliN are dispensable for function and that the remaining, essential part of FliN has sequence similarity to a part of Spa33, a protein that functions in transmembrane export in Shigella flexneri. Thus, FliN might function primarily in flagellar export, rather than in torque generation, as has sometimes been supposed.     

Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor.

FliG, FliM, and FliN are three proteins of Salmonella 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 the fliG and fliN 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 in fliG 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.     

Interaction between FliI ATPase and a flagellar chaperone FliT during bacterial flagellar protein export

FliT is a flagellar type III export chaperone specific for the filament-capping protein FliD. The FliT/FliD complex binds to the FliI ATPase of the flagellar export apparatus. The C-terminal α4 helix of FliT controls its interaction with FliI but it remains unknown how it does so. Here, we analysed the FliI-FliT interaction by pull-down assays using GST affinity chromatography. FliT94, missing the C-terminal α4 helix, bound to the extreme N-terminal region of FliI (FliI(EN)) with high affinity and to the C-terminal ATPase domain (FliI(CAT)) with low affinity. The C-terminal α4 helix of FliT suppressed the interaction with FliI(EN). FliH and FliT94 bound to a common binding site on FliI(EN) and hence FliH induced the release of FliI from FliT94 in an ATP-independent manner. FliD increased the binding affinity of FliI(CAT) for FliT. These results raise a possible hypothesis that the FliH/FliI complex binds to the FliT/FliD complex through FliI(CAT) to escort it from the cytoplasm to the export gate made up of six integral membrane proteins and that, upon dissociation of FliD from FliT, FliT94 may bind to FliI(EN) and then FliI may transfer from FliT94 to FliH by the direct competition of FliT94 and FliH for FliI(EN).     

Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH

The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.     

Interactions among components of the Salmonella flagellar export apparatus and its substrates

We have examined the cytoplasmic components (FliH, FliI and FliJ) of the type III flagellar protein export apparatus, plus the cytoplasmic domains (FlhAC and FlhBC) of two of its six membrane components. FliH, FlhAC and FliJ, when overproduced, caused inhibition of motility of wild-type cells and inhibition of the export of substrates such as the hook protein FlgE. Co-overproduction of FliH and FliI substantially relieved the inhibition caused by FliH, suggesting that it is excess free FliH that is inhibitory and that FliH and FliI form a complex. We purified His-FLAG-tagged versions of: (i) export components FliH, FliI, FliJ, FlhAC and FlhBC; (ii) rod/hook-type export substrates FlgB (rod protein), FlgE (hook protein), FlgD (hook capping protein) and FliE (basal body protein); and (iii) filament-type export substrates FlgK and FlgL (hook-filament junction proteins) and FliC (flagellin). We tested for protein-protein interactions by affinity blotting. In many cases, a given protein interacted with more than one other component, indicating that there are likely to be multiple dynamic interactions or interactions that involve more than two components. Interactions of FlhBC with rod/hook-type substrates were strong, whereas those with filament-type substrates were very weak; this may reflect the role of FlhB in substrate specificity switching. We propose a model for the flagellar export apparatus in which FlhA and FlhB and the other four integral membrane proteins of the apparatus form a complex at the base of the flagellar motor. A soluble complex of at least three proteins (FliH, FliI and FliJ) bind the protein to be exported and then interact with the complex at the motor to deliver the protein, which is then exported in an ATP-dependent process mediated by FliI.     

Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium.

The flaAII.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. Suppression among the three fla genes was allele specific, and many of the pseudorevertants were either cold sensitive or heat sensitive. We conclude that the FlaAII.2, FlaQ, and FlaN proteins form a complex which determines the rotational sense, either counterclockwise or clockwise, of the motor and also participates in the conversion of proton energy into mechanical work of rotation. This switch complex is probably mounted to the base of the flagellar basal body and, via binding of the CheY and CheZ proteins, receives sensory information and uses it to control flagellar operation.     

Interactions of the chemotaxis signal protein CheY with bacterial flagellar motors visualized by evanescent wave microscopy

The chemotaxis signal protein CheY of enteric bacteria shuttles between transmembrane methyl-accepting chemotaxis protein (MCP) receptor complexes and flagellar basal bodies [1]. The basal body C-rings, composed of the FliM, FliG and FliN proteins, form the rotor of the flagellar motor [2]. Phosphorylated CheY binds to isolated FliM [3] and may also interact with FliG [4], but its binding to basal bodies has not been measured. Using the chemorepellent acetate to phosphorylate and acetylate CheY [5], we have measured the covalent-modification-dependent binding of a green fluorescent protein-CheY fusion (GFP-CheY) to motor assemblies in bacteria lacking MCP complexes by evanescent wave microscopy [6]. At acetate concentrations that cause solely clockwise rotation, GFP-CheY molecules bound to native basal bodies or to overproduced rotor complexes with a stoichiometry comparable to the number of C-ring subunits. GFP-CheY did not bind to rotors lacking FIiM/FliN, showing that these subunits are essential for the association. This assay provides a new means of monitoring protein-protein interactions in signal transduction pathways in living cells.     

Flipping the switch: bringing order to flagellar assembly

The bacterial flagellum is a complex self-assembling nanomachine that contains its own type III protein export apparatus. Upon completion of early flagellar structure, this apparatus switches substrate specificity to export late structural subunits, thereby coupling sequential flagellar gene expression with flagellar assembly. The switch is achieved by a conformational change of the export apparatus component FlhB driven by the flagellar hook-length control protein FliK. Two basic models of FliK- and FlhB-based switching are currently being pursued, together with the investigation of another factor, Flk, which prevents premature export of late substrates. Here, we review in detail each of these three export switch components and present the current understanding of how they work in concert in the making of a flagellum.     

Bacillus subtilis hydrolyzes CheY-P at the location of its action,the flagellar switch

In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P, also causes hydrolysis of CheY-P to form CheY and Pi. This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch. In particular we have identified the phosphatase as FliY, which resembles E. coli switch protein FliN only in its C-terminal part, while an additional N-terminal domain is homologous to another switch protein FliM and to CheC, a protein found in the archaea and many bacteria but not in E. coli. Previous E. coli studies have localized the CheY-P binding site of the switch to FliM residues 6-15. These residues are almost identical to the residues 6-15 in both B. subtilis FliM and FliY. We were able to show that both of these proteins are capable of binding CheY-P in vitro. Deletion of this binding region in B. subtilis mutant fliM caused the same phenotype as a cheY mutant (clockwise flagellar rotation), whereas deletion of it in fliY caused the opposite. We showed that FliY increases the rate of CheY-P hydrolysis in vitro. Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.