Crystallization and preliminary X-ray analysis of Salmonella FliI, the ATPase component of the type III flagellar protein-export apparatus.

Most of the structural components making up the bacterial flagellum are translocated through the central channel of the growing flagellar structure by the type III flagellar protein-export apparatus in an ATPase-driven manner and are assembled at the growing end. FliI is the ATPase that drives flagellar protein export using the energy of ATP hydrolysis. FliI forms an oligomeric ring structure in order to attain maximum ATPase activity. In this study, FliI(Delta1-18), an N-terminally truncated variant of FliI lacking the first 18 residues, was purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 8000 as a precipitant. FliI(Delta1-18) crystals grew in the monoclinic space group P2(1), with unit-cell parameters a = 48, b = 73, c = 126 A, beta = 94 degrees, and diffracted to 2.4 A resolution. Anomalous difference Patterson maps of Os-derivative and Pt-derivative crystals showed significant peaks in their Harker sections, indicating that both derivatives are suitable for structure determination.

The type III flagellar export specificity switch is dependent on FliK ruler and a molecular clock.

Salmonella flagellar hook length is controlled at the level of export substrate specificity of the FlhB component of the type III flagellar export apparatus. FliK is believed to be the hook length sensor and interacts with FlhB to change its export specificity upon hook completion. To find properties of FliK expected of such a molecular ruler, we assayed binding of FliK to the hook and found that the N-terminal domain of FliK (FliK(N)) bound to the hook-capping protein FlgD with high affinity and to the hook protein FlgE with low affinity. To investigate a possible role of FlgE in hook length control, flgE mutants with partially impaired motility were isolated and analyzed. Eight flgE mutants obtained all formed flagellar filaments. The mutants produced significantly shorter hooks while the hook-type substrates such as FlgE, FliK and FlgD were secreted in large amounts, suggesting defective hook assembly with the mutant FlgE proteins. Upon overexpression, mutant FlgEs produced hooks of normal length and wild-type FlgE produced longer hooks. These results suggest that hook length is dependent on the hook polymerization rate and that the start of hook polymerization initiates a "time countdown" for the specificity switch to occur or for significant slow down of rod/hook-type export after hook length reaches around 55 nm for later infrequent FliK(C)-FlhB(C) interaction. We propose that FliK(N) acts as a flexible tape measure, but that hook length is also dependent on the hook elongation rate and a switch timing mechanism.

FlhB regulates ordered export of flagellar components via autocleavage mechanism.

The bacterial flagellum is a predominantly cell-external super-macromolecular 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.

Crystallization and preliminary X-ray analysis of the C-terminal cytoplasmic domain of FlhA, a membrane-protein subunit of the bacterial flagellar type III protein-export apparatus.

The axial components of the bacterial flagellum and the scaffolding proteins for its assembly are exported through the flagellar-specific type III protein-export apparatus, which is believed to be located on the cytoplasmic surface of the basal body. FlhA is an essential component of the type III export apparatus of Salmonella and consists of two major portions: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhAC). FlhAC and a 38 kDa fragment of FlhAC (FlhAC38K) were purified and crystallized. The crystals were obtained by the sitting-drop vapour-diffusion technique with PEG 8000 as a precipitant. FlhAC crystals grew in the tetragonal space group I4(1)/I4(3), with unit-cell parameters a = b = 216.6, c = 65.0 A. FlhAC38K was crystallized in an orthorhombic form, with unit-cell parameters a = 53.0, b = 93.1, c = 186.5 A. X-ray diffraction data from crystals of FlhAC and the SeMet derivative of FlhAC were collected to 2.9 and 3.2 A, respectively.

Type III flagellar protein export and flagellar assembly.

Bacterial flagella, unlike eukaryotic flagella, are largely external to the cell and therefore many of their subunits have to be exported. Export is ATP-driven. In Salmonella, the bacterium on which this chapter largely focuses, the apparatus responsible for flagellar protein export consists of six membrane components, three soluble components and several substrate-specific chaperones. Other flagellated eubacteria have similar systems. The membrane components of the export apparatus are housed within the flagellar basal body and deliver their substrates into a channel or lumen in the nascent structure from which point they diffuse to the far end and assemble. Both on the basis of sequence similarities of several components and structural similarities, the flagellar protein export systems clearly belong to the type III superfamily, whose other members are responsible for secretion of virulence factors by many species of pathogenic bacteria.

Analysis of the cytoplasmic domains of Salmonella FlhA and interactions with components of the flagellar export machinery.

Most flagellar proteins are exported via a type III export apparatus which, in part, consists of the membrane proteins FlhA, FlhB, FliO, FliP, FliQ, and FliR and is housed within the membrane-supramembrane ring formed by FliF subunits. Salmonella FlhA is a 692-residue integral membrane protein with eight predicted transmembrane spans. Its function is not understood, but it is necessary for flagellar export. We have created mutants in which potentially important sequences were deleted. FlhA lacking the amino-terminal sequence prior to the first transmembrane span failed to complement and was dominant negative, suggesting that the sequence is required for function. Similar effects were seen in a variant lacking a highly conserved domain (FHIPEP) within a putative cytoplasmic loop. Scanning deletion analysis of the cytoplasmic domain (FlhAc) demonstrated that substantially all of FlhAc is required for efficient function. Affinity blotting showed that FlhA interacts with several other export apparatus membrane proteins. The implications of these findings are discussed, and a model of FlhA within the export apparatus is presented.

Structural and functional analysis of the C-terminal cytoplasmic domain of FlhA, an integral membrane component of the type III flagellar protein export apparatus in Salmonella.

FlhA is an integral membrane component of the Salmonella type III flagellar protein export apparatus. It consists of 692 amino acid residues and has two domains: the N-terminal transmembrane domain consisting of the first 327 amino acid residues, and the C-terminal cytoplasmic domain (FlhAC) comprising the remainder. Here, we have investigated the structure and function of FlhAC. DNA sequence analysis revealed that temperature-sensitive flhA mutations, which abolish flagellar protein export at the restrictive temperature, lie in FlhAC, indicating that FlhAC plays an important role in the protein export process. Limited proteolysis of purified His-FlhAC by trypsin and V8 showed that only a small part of FlhAC near its N terminus (residues 328-351) is sensitive to proteolysis. FlhAC38K, the smallest fragment produced by V8 proteolysis, is monomeric and has a spherical shape as judged by analytical gel filtration chromatography and analytical ultracentrifugation. The far-UV CD spectrum of FlhAC38K showed that it contains considerable amounts of secondary structure. FlhA(Delta328-351) missing residues 328-351 failed to complement the flhA mutant, indicating that the proteolytically sensitive region of FlhA is important for its function. FlhA(Delta328-351) was inserted into the cytoplasmic membrane, and exerted a strong dominant negative effect on wild-type cells, suggesting that it retains the ability to interact with other export components within the cytoplasmic membrane. Overproduced FlhAC38K inhibited both motility and flagellar protein export of wild-type cells to some degree, suggesting that FlhAC38K is directly involved in the translocation reaction. Amino acid residues 328-351 of FlhA appear to be a relatively flexible linker between the transmembrane domain and FlhAC38K.

Domain organization and function of Salmonella FliK, a flagellar hook-length control protein.

Salmonella hook-length control protein FliK, which consists of 405 amino acid residues, switches substrate specificity of the type III flagellar protein export apparatus from rod/ hook-type to filament-type by causing a conformational change in the cytoplasmic domain of FlhB (FlhB(C)) upon completion of the hook assembly. An N-terminal region of FliK contains an export signal, and a highly conserved C-terminal region consisting of amino acid residues 265-405 (FliK((265-405))) is directly involved in the switching of FlhB(C). Here, we have investigated the structural properties of FliK. Gel filtration chromatography, multi-angle light scattering and analytical ultracentrifugation showed that FliK is monomeric in solution and has an elongated shape. Limited proteolysis showed that FliK consists of two domains, the N-terminal (FliK(N)) and C-terminal domains (FliK(C)), and that the first 203 and the last 35 amino acid residues are partially unfolded and subjected to proteolysis. Both FliK(N) and FliK(C) are more globular than full-length FliK, suggesting that these domains are connected in tandem. Overproduced His-FliK((199-405)) failed to switch export specificity of the export apparatus. Affinity blotting revealed that FlhB(C) binds to FliK and FliK((1-147)), but not to FliK((265-405)). Based on these results, we propose that FliK(N) within the central channel of the hook-basal body during the export of FliK is the sensor and transmitter of hook completion information and that the binding interaction of FliK(C) to FlhB(C) is structurally regulated by FliK(N) so as to occur only when the hook has reached a preset length. The conformational flexibility of FliK(C) may play a role in interfering with switching at an inappropriate point of flagellar assembly.

Analysis of an engineered Salmonella flagellar fusion protein, FliR-FlhB.

Salmonella FliR and FlhB are membrane proteins necessary for flagellar export. In Clostridium a fliR-flhB fusion gene exists. We constructed a similar Salmonella fusion gene which is able to complement fliR, flhB, and fliR flhB null strains. Western blotting revealed that the FliR-FlhB fusion protein retains the FlhB protein's cleavage properties. We conclude that the FliR and FlhB proteins are physically associated in the wild-type Salmonella basal body, probably in a 1:1 ratio.

Interactions of FliJ with the Salmonella type III flagellar export apparatus.

FliJ, a 17-kDa protein, is a soluble component of the Salmonella type III flagellar protein export system that has antiaggregation properties and several other characteristics that suggest it may have a chaperone-like function. We have now examined this protein in detail. Ten-amino-acid scanning deletions covering the entire 147-amino-acid sequence were tested for complementation of a fliJ null strain; only the first and last deletions complemented. A few of the deletions, especially towards the C terminus, exerted a dominant negative effect on wild-type cells, indicating that they were actively interfering with function. Two truncated versions of FliJ, representing its N- and C-terminal halves, failed to complement and were not dominant. We tested for FliJ self-association by several techniques. Size-exclusion chromatography (Superdex 200) indicated an apparent molecular mass of around 50 kDa, which could reflect either multimerization or an elongated shape or both. Multiangle light scattering gave a peak value of 20 kDa, close to the molecular mass of the monomer. Analytical ultracentrifugation gave evidence for weak self-association as a trimer or tetramer. It was known from previous studies that FliJ interacts with the N-terminal region of FliH, a negative regulator of the ATPase FliI. Using both truncation and deletion versions of FliJ, we now show that it is its C-terminal region that is responsible for this interaction. We also show that FliJ interacts with the soluble cytoplasmic domain of the largest membrane component of the export apparatus, FlhA; although small deletions in FliJ did not interfere with the association, both truncated versions failed to associate, indicating that a substantial amount of the central region of the FliJ sequence participates in the association. We present a model summarizing these multiple interactions.

The ATPase FliI can interact with the type III flagellar protein export apparatus in the absence of its regulator, FliH.

Salmonella FliI is the ATPase that drives flagellar protein export. It normally exists as a complex together with the regulatory protein FliH. A fliH null mutant was slightly motile, with overproduction of FliI resulting in substantial improvement of its motility. Mutations in the cytoplasmic domains of FlhA and FlhB, which are integral membrane components of the type III flagellar export apparatus, also resulted in substantially improved motility, even at normal FliI levels. Thus, FliH, though undoubtedly important, is not essential.

Substrate specificity of type III flagellar protein export in Salmonella is controlled by subdomain interactions in FlhB.

FlhB, 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 (FlhBC) is cleaved between Asn-269 and Pro-270, defining two subdomains: FlhBCN and FlhBCC. Here, we show that subdomain interactions and cleavage within FlhB are central to substrate-specificity switching. We found that deletions between residues 216 and 240 of FlhBCN permitted FlhB cleavage but abolished function, whereas a deletion spanning Asn-269 and Pro-270 abolished both. The mutation N269A prevented cleavage at the FlhBCN-FlhBCC 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.

How bacteria assemble flagella.

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.

Substrate specificity classes and the recognition signal for Salmonella type III flagellar export.

Most flagellar proteins of Salmonella are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ). In this study, we have measured the amounts of these proteins exported before and after hook completion. Their amounts in the culture supernatant from a flgE mutant (which is still at the hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export.

Structural properties of FliH, an ATPase regulatory component of the Salmonella type III flagellar export apparatus.

FliH is a regulatory component for FliI, the ATPase that is responsible for driving flagellar protein export in Salmonella. FliH consists of 235 amino acid residues, has a quite elongated shape, exists as a homodimer and together with FliI forms a heterotrimer. Here, we have investigated the structural properties of the FliH homodimer in further detail. Like intact His-tagged FliH homodimer, fragment His-FliH(N2) (consisting of the first 102 amino acid residues of FliH), exhibited anomalous elution behavior in gel filtration chromatography; the same was true of His-FliH(C1) (consisting of amino acid residues 119-235), but to a much lesser degree. Thus the elongated shape of FliH appears to derive primarily from its N-terminal region. A deletion version of N-His-FliH, lacking amino acid residues 101-140, does not dimerize and so we were able to establish the gel filtration properties of an almost full-size monomeric form; it also exhibited anomalous elution behavior. We performed trypsin proteolysis of the FliH homodimer and subjected the cleavage products to gel filtration chromatography. FliH was degraded by trypsin and a contaminating protease into two stable fragments: FliH(Prt1) (missing both the first ten and the last 12 amino acid residues), and FliH(Prt2) (missing both the first ten and the last 63 amino acid residues); however, substantial amounts remained undigested even after 24 hours. Under native conditions, both FliH(Prt1) and FliH(Prt2) co-eluted with undigested His-FliH from the gel filtration column, indicating that the fragments exist as a hybrid dimer with intact FliH. These results suggest that the two subunits within the dimer differ in their proteolytic susceptibility. No heterotrimer was observed by gel filtration chromatography when His-FliI was mixed with either His-FliH/FliH(Prt1) or His-FliH/FliH(Prt2) hybrid dimers. A hybrid dimer of FliH and His-FliHDelta1 (lacking the first ten amino acid residues) retained the ability to form a complex with His-FliI. In contrast, hybrid dimers consisting of FliH and either His-FliH(W223ochre) or His-FliH(V172ochre) failed to complex to His-FliI, demonstrating that the C-terminal region of both FliH monomers within the FliH dimer are required for heterotrimer formation.

Molecular dissection of Salmonella FliH, a regulator of the ATPase FliI and the type III flagellar protein export pathway.

FliH is a soluble component of the flagellar export apparatus that binds to the ATPase FliI, and negatively regulates its activity. The 235-amino-acid FliH dimerizes and interacts with FliI to form a hetero-trimeric (FliH)2FliI complex. In the present work, the importance of different regions of FliH was examined. A set of 24 scanning deletions of 10 amino acids was constructed over the entire FliH sequence, along with several combined deletions of 40 amino acids and truncations of both N- and C-termini. The mutant proteins were examined with respect to (i) complementation; (ii) dominance and multicopy effects; (iii) interaction with wild-type FliH; (iv) interaction with FliI; (v) inhibition of the ATPase activity of FliI; and (vi) interaction with the putative general chaperone FliJ. Analysis of the deletion mutants revealed a clear functional demarcation between the FliH N- and C-terminal regions. The 10-amino-acid deletions throughout most of the N-terminal half of the sequence complemented and were not dominant, whereas those throughout most of the C-terminal half did not complement and were dominant. FliI binding was disrupted by C-terminal deletions from residue 101 onwards, indicating that the C-terminal domain of FliH is essential for interaction with FliI. FliH dimerization was abolished by deletion of residues 101-140 in the centre of the sequence, as were complementation, dominance and interaction with FliI and FliJ. The importance of this region was confirmed by the fact that fragment FliHC2 (residues 99-235) interacted with FliH and FliI, whereas fragment FliHC1 (residues 119-235) did not. FliHC2 formed a relatively unstable complex with FliI and showed biphasic regulation of ATPase activity, suggesting that the FliH N-terminus stabilizes the (FliH)2FliI complex. Several of the N-terminal deletions tested permitted close to normal ATPase activity of FliI. Deletion of the last five residues of FliH caused a fivefold activation of ATPase activity, suggesting that this region of FliH governs a switch between repression and activation of FliI. Deletion of the first 10 residues of FliH abolished complementation, severely reduced its interaction with FliJ and uncoupled its role as a FliI repressor from its other export functions. Based on these data, a model is presented for the domain construction and function of FliH in complex with FliI and FliJ.

Interactions among membrane and soluble components of the flagellar export apparatus of Salmonella.

Interactions among several components of the flagellar export apparatus of Salmonella were studied using affinity chromatography, affinity blotting, and fluorescence resonance energy transfer (FRET). The components examined were two integral membrane proteins, FlhA and FlhB, and two soluble components, FliH and the ATPase FliI. Affinity chromatography and affinity blotting demonstrated a heterologous interaction between FlhA and FlhB but not homologous FlhA-FlhA or FlhB-FlhB interactions. Both FlhA and FlhB consist of N-terminal transmembrane domains and C-terminal cytoplasmic domains (FlhA(C) and FlhB(C)). To study the interactions among the cytoplasmic components (FlhA(C), FlhB(C), FliH, and FliI), FRET measurements were carried out using fluorescein-5-isothiocyanate (FITC) as donor and tetramethylrhodamine-5- (and 6-) isothiocyanate (TRITC) as acceptor. To reveal the nature of the binding interactions, measurements were carried out in physiological buffer, at high salt (0.5 M NaCl) and in 30% 2-propanol. The results indicated that FlhA(C) could bind to FlhB(C) and both FlhA(C) and FlhB(C) could bind to themselves. Both FlhA(C) and FlhB(C) bound to FliH and FliI. Several in-frame deletion mutants of FliH were examined and found to have only minor effects of decreased binding to FlhA(C) and FlhB(C); deletions in the interior of the FliH sequence had a greater effect than those at the N terminus. The FliI mutants examined bound FlhA(C) and FlhB(C) about the same as or slightly more weakly than wild-type FliI. FliH bound more weakly to FliI carrying the N-terminal double mutation R7C/L12P than it did to wild-type FliI, confirming the importance of the N terminus of FliI for its interaction with FliH.

Effects of lipoprotein biogenesis mutations on flagellar assembly in Salmonella.

Flagellar assembly requires the expression of a large number of flagellum-specific genes. However, mutations in a number of other genes in Salmonella and Escherichia coli have been shown to have pleiotropic effects that affect flagellar assembly. FlgH (the L-ring subunit of the flagellar basal body) is a lipoprotein whose modification is important for L-ring assembly. We therefore tested whether the lack of motility of Salmonella mutants defective in lipoprotein biogenesis is a result of inability to modify FlgH. Our results show that temperature-sensitive apolipoprotein N-acyltransferase [lnt(Ts)] mutants are nonflagellate at 42 degrees C. However, the flagellar assembly defect occurs at a much earlier step in the pathway than L-ring assembly. These mutants failed to assemble even an MS ring, presumably because of the observed decrease in transcription of fliF. In contrast, temperature-sensitive diacylglycerol transferase [lgt(Ts)] mutants were motile at 42 degrees C, provided the strains carried an lpp (Braun lipoprotein) mutation to permit growth. We have isolated second-site mutants from an lgt(Ts) lpp(+) strain that grow but are nonflagellate at 42 degrees C. Thus, lipoprotein biogenesis is a factor that is important for flagellar assembly.

FliL is a membrane-associated component of the flagellar basal body of Salmonella.

FliL is one of the least understood proteins in the flagellar systems of Salmonella and Escherichia coli. There is no apparent mutant phenotype associated with it, even when virtually the entire coding sequence has been eliminated. In this study it has been shown that FliL is a cytoplasmic membrane protein associated with the basal body. Although it has a sequence that conforms to the consensus cleavage site for lipoproteins, FliL does not undergo cleavage or modification under physiological conditions.