Mechanisms of type III protein export for bacterial flagellar assembly

Flagellar type III protein export is highly organized and well controlled in a timely manner by dynamic, specific and cooperative interactions among components of the export apparatus, allowing the huge and complex macromolecular assembly to be built efficiently. The bacterial flagellum, which is required for motility, consists of a rotary motor, a universal joint and a helical propeller. Most of the flagellar components are translocated to the distal, growing end of the flagellum for assembly through the central channel of the flagellum itself by the flagellar type III protein export apparatus, which is postulated to be located on the cytoplasmic side of the flagellar basal body. The export specificity switching machinery, which consists of at least two proteins that function as a molecular ruler and an export switch, respectively, monitors the state of hook-basal body assembly in the cell exterior and switches export specificity, thereby coupling sequential flagellar gene expression with the flagellar assembly process. The export ATPase complex composed of an ATPase and its regulator acts as a pilot to deliver its export substrate to the export gate and helps initial entry of the substrate N-terminal chain into a narrow pore of the export gate. The energy of ATP hydrolysis appears to be used to disassemble and release the ATPase complex from the protein about to be exported, and the rest of the successive unfolding/translocation process of the long polypeptide chain is driven solely by proton motive force (PMF), perhaps through biased one-dimensional Brownian diffusion. Interestingly, the subunits of the ATPase complex have significant sequence similarities to subunits of F(0)F(1)-ATP synthase, a rotary motor that drives the chemical reaction of ATP synthesis using PMF, and the entire crystal structure of the export ATPase is extremely similar to the alpha/beta subunits of F(0)F(1)-ATP synthase, suggesting that the flagellar export apparatus and F(0)F(1)-ATP synthase share the mechanism for their two distinct functions.     

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.     

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.     

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.     

Role of FliJ in flagellar protein export in Salmonella

We isolated and characterized spontaneous mutants with defects in the 147-amino-acid Salmonella protein FliJ, which is a cytoplasmic component of the type III flagellar export apparatus. These mutants, including ones with null mutations, have the ability to form swarms on motility agar plates after prolonged incubation at 30 degrees C; i.e., they display a leaky motile phenotype. One mutant, SJW277, which formed significantly bigger swarms than the others, encoded only the N-terminal 73 amino acids of FliJ, one-half of the protein. At 30 degrees C, overproduction of this mutant protein improved, to wild-type levels, both motility and the ability to export both rod/hook-type (FlgD; hook capping protein) and filament-type (FliC; flagellin) substrates. At 42 degrees C, however, export was inhibited, indicating that the mutant FliJ protein was temperature sensitive. Taking advantage of this, we performed temperature upshift experiments, which demonstrated that FliJ is directly required for the export of FliC. Co-overproduction of FliJ and either of two export substrates, FliE or FlgG, hindered their aggregation in the cytoplasm. We conclude that FliJ is a general component of the flagellar export apparatus and has a chaperone-like activity for both rod/hook-type and filament-type substrates.     

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.     

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.     

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.     

Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa

Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (DeltafliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (DeltamotAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.     

Similar modes of polypeptide recognition by export chaperones in flagellar biosynthesis and type III secretion

Assembly of the bacterial flagellum and type III secretion in pathogenic bacteria require cytosolic export chaperones that interact with mobile components to facilitate their secretion. Although their amino acid sequences are not conserved, the structures of several type III secretion chaperones revealed striking similarities between their folds and modes of substrate recognition. Here, we report the first crystallographic structure of a flagellar export chaperone, Aquifex aeolicus FliS. FliS adopts a novel fold that is clearly distinct from those of the type III secretion chaperones, indicating that they do not share a common evolutionary origin. However, the structure of FliS in complex with a fragment of FliC (flagellin) reveals that, like the type III secretion chaperones, flagellar export chaperones bind their target proteins in extended conformation and suggests that this mode of recognition may be widely used in bacteria.     

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.     

Mutational analysis of the flagellar rotor protein FliN: identification of surfaces important for flagellar assembly and switching

FliN is a component of the flagellar switch complex in many bacterial species. The crystal structure is known for most of FliN, and a targeted cross-linking study (K. Paul and D. F. Blair, J. Bacteriol. 188:2502-2511, 2006) showed that it is organized in ring-shaped tetramers at the bottom of the basal body C ring. FliN is essential for flagellar assembly and direction switching, but its precise functions have not been defined. Here, we identify functionally important regions on FliN by systematic mutagenesis. Nonconservative mutations were made at positions sampling the surface of the protein, and the effects on flagellar assembly and function were measured. Flagellar assembly was disrupted by mutations in a conserved hydrophobic patch centered on the dimer twofold axis or by mutations on the surface that forms the dimer-dimer interface in the tetramer. The assembly defect in hydrophobic-patch mutants was partially rescued by overexpression of the flagellar export proteins FliH and FliI, and coprecipitation assays demonstrated a binding interaction between FliN and FliH that was weakened by mutations in the hydrophobic patch. Thus, FliN might contribute to export by providing binding sites for FliH or FliH-containing complexes. The region around the hydrophobic patch is also important for switching; certain mutations in or near the patch caused a smooth-swimming chemotaxis defect that in most cases could be partially rescued by overexpression of the clockwise-signaling protein CheY. The results indicate that FliN is more closely involved in switching than has been supposed, possibly contributing to the binding site for CheY on the switch.     

A trans-envelope protein complex needed for filamentous phage assembly and export

Assembly and export of filamentous phage requires four non-capsid proteins: the outer membrane protein, pIV; the inner membrane proteins, pI and pXI; and a cytoplasmic host factor, thioredoxin. Chemical cross-linking of intact cells demonstrates a trans-membrane complex containing pI and pIV. Formation of the complex protects pI from proteolytic cleavage by an endogenous protease. This protection also requires pXI, which is identical to the C-terminal portion of pI. This indicates that pXI, which is required for phage assembly in its own right, is also part of the complex. This complex forms in the absence of any other phage proteins or the DNA substrate; hence, it represents the first preinitiation step of phage morphogenesis. On the basis of protease protection data, we propose that the preinitiation complex is converted to an initiation complex by binding phage DNA, thioredoxin and the initiating minor coat protein(s).     

Retroviral mRNA nuclear export elements regulate protein function and virion assembly

Rodent cells are notable for their inability to support normal assembly of HIV particles. In this report, we address possible causes for this defect by considering the hypothesis that mRNA-associated events occurring in the nucleus can regulate the activity of their encoded proteins in the cytoplasm. We show that altering the RNA nuclear export element used by HIV gag-pol mRNA from the Rev response element to the constitutive transport element restores both the trafficking of Gag to cellular membranes and efficient HIV assembly in murine cells. These results suggest that two phases of the HIV life cycle, RNA export and capsid assembly, that have hitherto been regarded as distinct are, in fact, linked. Thus, protein function and fate may depend upon the full and precise history of its encoding mRNA.     

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.     

A structural feature in the central channel of the bacterial flagellar FliF ring complex is implicated in type III protein export

The FliF ring complex, which consists of the M-S ring and a proximal portion of the rod of the flagellar basal body, is the base structure for the bacterial flagellar assembly. The FliF ring is also thought to be part of the export apparatus for flagellar proteins from its amino acid sequence homology to proteins involved in type III protein export systems. We established a new purification procedure for the FliF ring particles and carried out electron microscopic image analyses in their two distinct forms: well-dispersed single particles in the presence of salt and ordered monolayer arrays of hexagonal packing formed in the absence of salt. In both cases, the axial projection maps showed a common feature, a pair of concentric rings: the inner ring corresponds to the proximal rod; the outer ring represents the thick, edge portion of the M-S ring. However, the central channel of the FliF ring, the putative pathway for the flagellar protein export, appeared to show distinct structural features in the two forms. This suggests that a domain of FliF partially occupies the central channel to be involved in the export and gate mechanism, and the domain changes its conformation depending on the ionic strength.     

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.     

β-lactamase as a probe of membrane protein assembly and protein export

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.     

ODA16p, a Chlamydomonas flagellar protein needed for dynein assembly

Dynein motors of cilia and flagella function in the context of the axoneme, a very large network of microtubules and associated proteins. To understand how dyneins assemble and attach to this network, we characterized two Chlamydomonas outer arm dynein assembly (oda) mutants at a new locus, ODA16. Both oda16 mutants display a reduced beat frequency and altered swimming behavior, similar to previously characterized oda mutants, but only a partial loss of axonemal dyneins as shown by both electron microscopy and immunoblots. Motility studies suggest that the remaining outer arm dyneins on oda16 axonemes are functional. The ODA16 locus encodes a 49-kDa WD-repeat domain protein. Homologues were found in mammalian and fly databases, but not in yeast or nematode databases, implying that this protein is only needed in organisms with motile cilia or flagella. The Chlamydomonas ODA16 protein shares 62% identity with its human homologue. Western blot analysis localizes more than 90% of ODA16p to the flagellar matrix. Because wild-type axonemes retain little ODA16p but can be reactivated to a normal beat in vitro, we hypothesize that ODA16p is not an essential dynein subunit, but a protein necessary for dynein transport into the flagellar compartment or assembly onto the axoneme.     

Regulated assembly of the transenvelope protein complex required for lipopolysaccharide export

Gram-negative bacteria are impervious to many drugs and environmental stresses because they possess an outer membrane (OM) containing lipopolysaccharide (LPS). LPS is biosynthesized at the cytoplasmic (inner) membrane and is transported to the OM by an unknown mechanism involving the LPS transport proteins, LptA-G. These proteins have been proposed to form a bridge between the two membranes; however, it is not known how this bridge is assembled to prevent mistargeting of LPS. We use in vivo photo-cross-linking to reveal the specific protein-protein interaction sites that give rise to the Lpt bridge. We also show that the formation of this transenvelope bridge cannot proceed before the correct assembly of the LPS translocon in the OM. This ordered sequence of events may ensure that LPS is never transported to the OM if it cannot be translocated across it to the cell surface.