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.     

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.     

Assembly and stoichiometry of FliF and FlhA in Salmonella flagellar basal body

The bacterial flagellar export apparatus is required for the construction of the bacterial flagella beyond the cytoplasmic membrane. The membrane‐embedded part of the export apparatus, which consists of FlhA, FlhB, FliO, FliP, FliQ and FliR, is located in the central pore of the MS ring formed by 26 copies of FliF. The C‐terminal cytoplasmic domain of FlhA is located in the centre of the cavity within the C ring made of FliG, FliM and FliN. FlhA interacts with FliF, but its assembly mechanism remains unclear. Here, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the C‐termini of FliF and FlhA and investigated their subcellular localization by fluorescence microscopy. The punctate pattern of FliF–YFP localization required FliG but neither FliM, FliN, FlhA, FlhB, FliO, FliP, FliQ nor FliR. In contrast, FlhA–CFP localization required FliF, FliG, FliO, FliP, FliQ and FliR. The number of FlhA–YFP molecules associated with the MS ring was estimated to be about nine. We suggest that FlhA assembles into the export gate along with other membrane components during the MS ring complex formation in a co‐ordinated manner.     

Function of the conserved FHIPEP domain of the flagellar type III export apparatus,protein FlhA

The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly‐conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68‐amino acid FHIPEP region. Fifty‐two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short‐stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un‐polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook‐cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook‐filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook‐length control protein FliK and facilitated growth of full‐length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore.     

Genetic characterization of conserved charged residues in the bacterial flagellar type III export protein FlhA

For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΔfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate.     

Interaction of the Extreme N-Terminal Region of FliH with FlhA Is Required for Efficient Bacterial Flagellar Protein Export

The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH(EN)) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH(EN) with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.     

The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H⁺–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na⁺ increased it dramatically. Phenamil, a blocker of Na⁺ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na⁺ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na⁺ channel. In wild-type cells, however, neither Na⁺ nor phenamil affected protein export, indicating that the Na⁺ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na⁺ concentration.     

Cross-Complementation Study of the Flagellar Type III Export Apparatus Membrane Protein FlhB

The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram-negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of-function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, which are required for ubiquinone biosynthesis. The mutations were shown by reversed-phase high performance liquid chromatography to reduce the quinone pool of the cytoplasmic membrane. Ubiquinone biosynthesis could be restored for the strain bearing a mutated gene for 4-hydroxybenzoate octaprenyltransferase by the addition of excess exogenous 4-hydroxybenzoate. Restoring the level of ubiquinone reduced flagella biogenesis with the AquSalFlhB chimera demonstrating that the respiratory chain quinone pool is responsible for this phenomenon.     

Molecular characterization of the Salmonella typhimurium flhB operon and its protein products.

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, may constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.     

Mechanism of type‐III protein secretion: Regulation of FlhA conformation by a functionally critical charged‐residue cluster

The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram‐negative pathogens to export virulence factors into host cells. This mode of protein export is termed type‐III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton‐binding groups that might function in transport. Conserved proton‐binding residues in all the membrane components were tested. The results identify residues R147, R154 and D158 of FlhA as most critical. These lie in a small, well‐conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5. Two‐hybrid experiments demonstrate self‐interaction of the domain, and targeted cross‐linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton‐actuated movements in the cytoplasmic domains of FlhA.     

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.     

Protein export through the bacterial flagellar type III export pathway

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly–disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

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.     

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.     

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.     

Interactions of bacterial flagellar chaperone–substrate complexes with FlhA contribute to co‐ordinating assembly of the flagellar filament

Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C‐terminal cytoplasmic domain of FlhA (FlhA_C), a membrane component of the export apparatus, provides a binding‐site for these chaperone–substrate complexes but it remains unknown how it co‐ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhA_C is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhA_C for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14‐fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhA_C for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.     

Interaction of a bacterial flagellar chaperone FlgN with FlhA is required for efficient export of its cognate substrates

FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteolysis in the cytoplasm. Docking of the FlgN-FlgK complex with the FliI ATPase of the flagellar type III export apparatus is key to the protein export process. However, a ΔfliH-fliI flhB(P28T) mutant forms some flagella even in the absence of FliH and FliI, raising the question of how FlgN promotes the export of its cognate substrates. Here, we report that the interaction of FlgN with an integral membrane export protein, FlhA, is directly involved in efficient protein export. A ΔfliH-fliI flhB(P28T) ΔflgN mutant caused extragenic suppressor mutations in the C-terminal domain of FlhA (FlhA(C) ). Pull-down assays using GST affinity chromatography showed an interaction between FlgN and FlhA(C) . The FlgN-FlgK complex bound to FlhA(C) and FliJ to form the FlgN-FlgK-FliJ-FlhA(C) complex. The FlgN-FlhA(C) interaction was enhanced by FlgK but not by FliJ. FlgN120 missing the last 20 residues still bound to FlgK and FliJ but not to FlhA(C) . A highly conserved Tyr-122 residue was required for the interaction with FlhA(C) . These results suggest that FlgN efficiently transfers FlgK/L subunits to FlhA(C) to promote their export.     

Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export

FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N‐terminal transmembrane domain (FlhA_TM) and a C‐terminal cytoplasmic domain (FlhA_C). FlhA_C is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhA_C at 2.8 Å resolution. FlhA_C consists of four subdomains (A_CD1, A_CD2, A_CD3 and A_CD4) and a linker connecting FlhA_C to FlhA_TM. The sites of temperature‐sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation suggested that FlhA_C changes its conformation for its function. Molecular dynamics simulation demonstrated an open‐close motion with a 5–10 ns oscillation in the distance between A_CD2 and A_CD4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhA_C is essential for protein export.     

The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer

YscV (FlhA in the flagellum) is an essential component of the inner membrane (IM) export apparatus of the type III secretion injectisome. It contains eight transmembrane helices and a large C-terminal cytosolic domain. YscV was expressed at a significantly higher level than the other export apparatus components YscR, YscS, YscT, and YscU, and YscV-EGFP formed bright fluorescent spots at the bacterial periphery, colocalizing in most cases with YscC-mCherry. This suggested that YscV is the only protein of the export apparatus that oligomerizes. Oligomerization required the cytosolic domain of YscV, as well as YscR, -S, -T, but no other Ysc protein, indicating that an IM platform can assemble independently from the membrane-ring forming proteins YscC, -D, -J. However, in the absence of YscC, -D, -J, this IM platform moved laterally at the bacterial surface. YscJ, but not YscD could be recruited to the IM platform in the absence of the secretin YscC. As YscJ was shown earlier to be also recruited by the outer membrane (OM) platform made of YscC and YscD, we infer that assembly of the injectisome proceeds through the independent assembly of an IM and an OM platform that merge through YscJ.