Soluble components of the flagellar export apparatus,FliI, FliJ,and FliH,do not deliver flagellin,the major filament protein,from the cytosol to the export gate

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook–filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear.     

ATP-induced FliI hexamerization facilitates bacterial flagellar protein export

FliI ATPase forms a homo-hexamer to fully exert its ATPase activity, facilitating bacterial flagellar protein export. However, it remains unknown how FliI hexamerization is linked to protein export. Here, we analyzed the capability of ring formation by FliI and its catalytic mutant variants. Compared to ATP a non-hydrolysable ATP analog increased the probability of FliI hexamerization. In contrast, FliI(E221Q), which retained the affinity for ATP but has lost ATPase activity, efficiently formed the hexamer even in the presence of ATP. The mutations, which reduced the binding affinity for ATP, significantly abolished the ring formation. These results indicate that ATP-binding induces FliI hexamerization and that the release of ADP and Pi destabilizes the ring structure. FliI(E221Q) facilitated flagellar protein export in the absence of the FliH regulator of the export apparatus although not at the wild-type FliI level while the other did not. We propose that FliI couples ATP binding and hydrolysis to its assembly-disassembly cycle to efficiently initiate the flagellar protein export cycle.     

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.     

Role of the N-terminal domain of FliI ATPase in bacterial flagellar protein export

FliI, the ATPase involved in bacterial flagellar protein export, forms a complex with its regulator FliH in the cytoplasm and hexamerizes upon docking to the export gate composed of integral membrane proteins. The extreme N-terminal region of FliI is involved not only in its interaction with FliH but also in its oligomerization, but the regulatory mechanism of oligomerization remains unclear. Using in-frame 10-residue deletions within the 100 residues of the N-terminal domain, we demonstrate that the first 20 residues are required for FliH binding and that the conformation of the N-terminal domain is sensitive to the export function, even though the oligomerization and FliH-binding ability are retained and the ATPase activity is maintained in most of the deletion variants.     

Interaction of FliS flagellar chaperone with flagellin

Premature polymerization of flagellin (FliC), the main component of flagellar filaments, is prevented by the FliS chaperone in the cytosol. Interaction of FliS with flagellin was characterized by isothermal titration calorimetry producing an association constant of 1.9x10(7) M-1 and a binding stoichiometry of 1:1. Experiments with truncated FliC fragments demonstrated that the C-terminal disordered region of flagellin is essential for FliS binding. As revealed by thermal unfolding experiments, FliS does not function as an antifolding factor keeping flagellin in a secretion-competent conformation. Instead, FliS binding facilitates the formation of alpha-helical secondary structure in the chaperone binding region of flagellin.     

Intergenic suppression between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane component of its export apparatus

The MS ring of the flagellar basal body of Salmonella is an integral membrane structure consisting of about 26 subunits of a 61-kDa protein, FliF. Out of many nonflagellate fliF mutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of export function and flagellar assembly. The three parental mutants were all found to carry the same mutation, a six-base deletion corresponding to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of the FliF protein. The 19 intergenic suppressors identified all lay in flhA, and they consisted of 10 independent examples at the nucleotide level or 9 at the amino acid level. Since two of the nine corresponded to different substitutions at the same amino acid position, only eight positions in the FlhA protein have given rise to suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare event. FlhA is a component of the flagellar protein export apparatus, with an integral membrane domain encompassing the N-terminal half of the sequence and a cytoplasmic C-terminal domain. All of the suppressing mutations lay within the integral membrane domain. These mutations, when placed in a wild-type fliF background, had no mutant phenotype. In the fliF mutant background, mutant FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA did not exert significant negative dominance in the pseudorevertant background, indicating that it does not compete effectively with mutant FlhA for interaction with mutant FliF. Mutant FliF was partially dominant over wild-type FliF in both the wild-type and second-site FlhA backgrounds. Membrane fractionation experiments indicated that the fliF mutation, though preventing export, was mild enough to permit assembly of the MS ring itself, and also assembly of the cytoplasmic C ring onto the MS ring. The data from this study provide genetic support for a model in which at least the FlhA component of the export apparatus physically interacts with the MS ring within which it is housed.     

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.     

Components of the Salmonella flagellar export apparatus and classification of export substrates

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.     

Functional defect and restoration of temperature-sensitive mutants of FlhA, a subunit of the flagellar protein export apparatus

The flagellar axial component proteins are exported to the distal end of the growing flagellum for self-assembly by the flagellar type III export apparatus. FlhA is a key membrane protein of the export apparatus, and its C-terminal cytoplasmic domain (FlhA_C) is a part of an assembly platform for the three soluble export components, FliH, FliI, and FliJ, as well as export substrates and chaperone–substrate complexes. FlhA_C is composed of a flexible linker region and four compact domains (A_CD1–A_CD4). At 42 °C, a temperature-sensitive (TS) G368C mutation in FlhA_C blocks the export process after the FliH–FliI–FliJ–substrate complex binds to the assembly platform, but it remains unknown how it does so. In this study, we analyzed a TS mutant variant, FlhA_C(G368C), and its pseudorevertant variants FlhA_C(G368C/L359F), FlhA_C(G368C/G364R), FlhA_C(G368C/R370S), and FlhA_C(G368C/P550S) using far-ultraviolet circular dichroism. Whereas the denaturation of the wild-type FlhA_C occurs in a single step, FlhA_C(G368C) and its pseudorevertant variants showed thermal transitions, at least, in two steps. The first transition of FlhA_C(G368C) can further be divided into reversible and following irreversible transitions, which correspond to the denaturation of A_CD2 and A_CD1, respectively. We show the relation between the reversible transition and the TS defect in the exporting function of FlhA_C(G368C) and that the loss of function is caused by denaturation of A_CD2. We suggest that A_CD2 is directly involved in the translocation of export substrates.     

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.     

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.     

Molecular basis of the interaction between the flagellar export proteins FliI and FliH from Helicobacter pylori

Bacterial flagellar protein export requires an ATPase, FliI, and presumptive inhibitor, FliH. We have explored the molecular basis for FliI/FliH interaction in the human gastric pathogen Helicobacter pylori. By using bioinformatic and biochemical analyses, we showed that residues 1-18 of FliI very likely form an amphipathic alpha-helix upon interaction with FliH, and that residues 21-91 of FliI resemble the N-terminal oligomerization domain of the F1-ATPase catalytic subunits. A truncated FliI-(2-91) protein was shown to be folded, although the N-terminal 18 residues were likely unstructured. Deletion and scanning mutagenesis showed that residues 1-18 of FliI were essential for the FliI/FliH interaction. Scanning mutation of amino acids in the N-terminal 10 residues of FliI indicated that a cluster of hydrophobic residues in this segment was critical for the interaction with FliH. The interaction between FliI and FliH has similarities to the interaction between the N-terminal alpha-helix of the F1-ATPase alpha-subunit and the globular domain of the F1-ATPase delta-subunit, respectively. This similarity suggests that FliH may function as a molecular stator.     

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.     

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.     

Biochemical and antigenic properties of the Campylobacter flagellar hook protein.

The flagellar filament-hook complex was removed from Campylobacter cells by shearing and was purified by differential solubilization and ultracentrifugation at pH 11 followed by cesium chloride buoyant density ultracentrifugation. Flagellar filaments were then dissociated in 0.2 M glycine-HCl (pH 2.2), and purified hooks were collected by ultracentrifugation. The hooks (105 by 24 nm) each displayed a conical protrusion at the proximal end, a concave cavity at the distal end, and helically arranged subunits. The apparent subunit molecular weight of the hook protein of seven of the eight Campylobacter strains studied was 92,500, while that of the other was 94,000. N-terminal amino acid analysis of the hook protein of two strains of Campylobacter coli and one strain of Campylobacter jejuni demonstrated that the first 15 residues were identical. Amino acid composition analysis showed that the Campylobacter hook protein contained 35.7% hydrophobic and 9.5% basic residues. Isoelectric focusing determined that the hook protein was acidic, with a pI of 4.9. Comparisons with the Salmonella and Caulobacter hook protein compositions and N-terminal amino acid sequences indicated that the Campylobacter protein was related, but more distantly than these two proteins were to each other. Immunochemical analysis with four different antisera and a panel of eight strains showed that serospecific epitopes were immunodominant. The Campylobacter hook proteins carried both cross-reactive and specific non-surface-exposed epitopes, as well as serospecific epitopes which were exposed on the surface of the assembled hook. One class of these surface-exposed hook epitopes was shared with serospecific flagellin epitopes and may involve posttranslational modification, while the second class of epitopes was hook specific and not shared with flagellin.     

Characterization of the type III export signal of the flagellar hook scaffolding protein FlgD of Escherichia coli

Transport of flagellar structural proteins beyond the cytoplasmic membrane is accomplished by a type III secretory pathway [flagellar type III secretion system (fTTSS)]. The mechanism of substrate recognition by the fTTSS is still enigmatic. Using the hook scaffolding protein FlgD of Escherichia coli as a model substrate, it is demonstrated that the export signal is contained within the N-terminal 71 amino acids of FlgD. Analysis of frame-shift mutations and alterations of the nucleotide sequence suggest a proteinaceous nature of the signal. Furthermore, the physicochemical properties of the first about eight amino acids are crucial for export.     

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.     

SecG is an auxiliary component of the protein export apparatus of Escherichia coli

SecY, SecE and SecG form the membrane-embedded core complex of the Escherichia coli protein export apparatus. These three proteins co-purify and can be co-immunoprecipitated, demonstrating that they are closely associated. While SecE and SecY are generally accepted as essential components of translocase, the role of SecG is more ambiguous. It is commonly believed that deletion of secG causes a cold-sensitive phenotype and a severe defect in export, even though some reports have indicated otherwise. However, we demonstrate that deletion of secG does not produce a cold-sensitive phenotype or a strong export defect in most genetic backgrounds. The more common result is that deletion of secG causes only a mild export defect and does not result in conditional lethality. We propose that the role of SecG is not fundamental to the export process, but is merely auxiliary – as suggested previously by biochemical data – and is physiologically important only when cells are otherwise compromised. Received: 22 July 1999 / Accepted: 11 November 1999     

Interaction of the disordered terminal regions of flagellin upon flagellar filament formation

Helical filaments of bacterial flagella are built up by a self-assembly process from thousands of flagellin subunits. To clarify how the disordered terminal regions of flagellin interact upon filament formation, polymerization ability of various terminally truncated fragments was investigated. Fragments deprived of 19 N-terminal residues were able to bind to the end of filaments, however, only a single layer was formed. Removal of C-terminal segments or truncation at both ends resulted in the complete loss of binding ability. Our observations are consistent with the coiled-coil model of filament formation, which suggests that the alpha-helical N- and C-terminal regions of axially adjacent subunits form an interlocking pattern of helical bundles upon polymerization.     

FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly

In wild-type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at approximately 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild-type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook-filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N-terminal region of FliK, but not by C-terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook-length control and the switching of export substrate specificity.