Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly

During flagellum assembly by motile enterobacteria, flagellar axial proteins destined for polymerization into the cell surface structure are thought to be exported through the 25–30 Å flagellum central channel as partially unfolded monomers. How are premature folding and oligomerization in the cytosol prevented? We have shown previously using hyperflagellated Proteus mirabilis and a motile but non-swarming flgN transposon mutant that the apparently cytosolic 16.5 kDa flagellar protein FlgN facilitates efficient flagellum filament assembly. Here, we investigate further whether FlgN, predicted to contain a C-terminal amphipathic helix typical of type III export chaperones, acts as a chaperone for axial proteins. Incubation of soluble radiolabelled FlgN from Salmonella typhimurium with nitrocellulose-immobilized cell lysates of wild-type S. typhimurium and a non-flagellate class 1 flhDC mutant indicated that FlgN binds to flagellar proteins. Identical affinity blot analysis of culture supernatants from the wild-type and flhDC, flgI, flgK, flgL, fliC or fliD flagellar mutants showed that FlgN binds to the flagellar hook-associated proteins (HAPs) FlgK and FlgL. This was confirmed by blotting artificially expressed individual HAPs in Escherichia coli. Analysis of axial proteins secreted into the culture medium by the original P. mirabilis flgN mutant demonstrated that export of FlgK and FlgL was specifically reduced, with concomitant increased release of unpolymerized flagellin (FliC), the immediately distal component of the flagellum. These data suggest that FlgN functions as an export chaperone for FlgK and FlgL. Parallel experiments showed that FliT, a similarly small (14 kDa), potentially helical flagellar protein, binds specifically to the flagellar filament cap protein, FliD (HAP2), indicating that it too might be an export chaperone. Flagellar axial proteins all contain amphipathic helices at their termini. Removal of the HAP C-terminal helical domains abolished binding by FlgN and FliT in each case, and polypeptides comprising each of the HAP C-termini were specifically bound by FlgN and FliT. We suggest that FlgN and FliT are substrate-specific flagellar chaperones that prevent oligomerization of the HAPs by binding to their helical domains before export.     

Substrate complexes and domain organization of the Salmonella flagellar export chaperones FlgN and FliT

The flagellar proteins FlgN and FliT have been proposed to act as substrate-specific export chaperones, facilitating incorporation of the enterobacterial hook-associated axial proteins (HAPs) FlgK/FlgL and FliD into the growing flagellum. In Salmonella typhimurium flgN and fliT mutants, the export of target HAPs was reduced, concomitant with loss of unincorporated flagellin into the surrounding medium. Gel filtration chromatography of wild-type S. typhimurium cell extracts identified stable pools of FlgN and FliT homodimers in the cytosol, but no chaperone-substrate complexes were evident. Nevertheless, stable unique complexes were assembled efficiently in vitro by co-incubation of FlgN and FliT with target HAPs purified from recombinant Escherichia coli. The sizes of the chaperone-substrate complexes indicated that, in each case, a chaperone homodimer binds to a substrate monomer. FlgN prevented in vitro aggregation of FlgK monomers, generating a soluble form of the HAP. Recombinant polypeptides spanning the potentially amphipathic C-terminal regions of FlgN or FliT could not complement in trans the chaperone deficiency of the respective flgN and fliT mutants, but efficient flagellar assembly was restored by homodimeric translational fusions of these domains to glutathione S-transferase, which bound FlgK and FlgL like the wild-type FlgN. These data provide further evidence for the substrate-specific chaperone function of FlgN and FliT and indicate that these chaperones comprise common N- and C-terminal domains mediating homodimerization and HAP substrate binding respectively. In support of this view, the flgN mutation was specifically complemented by a hybrid chaperone comprising the N-terminal half of FliT and the C-terminal half of FlgN.     

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.     

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.     

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

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

The type III secretion chaperone FlgN regulates flagellar assembly via a negative feedback loop containing its chaperone substrates FlgK and FlgL

The type III secretion (TTS) chaperones are small proteins that act either as cytoplasmic bodyguards, protecting their secretion substrates from degradation and aggregation, facilitators of their cognate substrate secretion or both. FlgN has been previously shown to be a TTS chaperone for the hook-associated proteins FlgK and FlgL (FlgKL), and a translational regulator of the anti-sigma28 factor FlgM. Protein stability assays indicate that a flgN mutation leads to a dramatic decrease in the half-life of intracellular FlgK. However, using gene reporter fusions to flgK we show that a flgN mutation does not affect the translation of a flgK-lacZ fusion. Quantification of FlgM protein levels showed that FlgKL inhibit the positive regulation on flgM translation by FlgN when secretion of FlgKL is inhibited. Suppressors of the motility-defective phenotype of a flgN mutant were isolated and mapped to the clpXP and fliDST loci. Overexpression of flgKL on a plasmid also suppressed the motility defect of a flgN null mutant. These results suggest that FlgN is not required for secretion of FlgKL and that FlgN typifies a class of TTS chaperones that allows for the minimal amount of their substrates expression required in the assembly process by protecting the substrate from proteolysis. Our data leads us to propose a model in which the interaction between FlgN and FlgK or FlgL is a sensing mechanism to determine the stage of flagellar assembly. Furthermore, the interaction between FlgN and FlgK or FlgL inhibits the translational regulation of flgM via FlgN in response to the stage of flagellar assembly.     

Identification of the flagellar chaperone FlgN in the phytopathogen <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar <Emphasis Type="Italic">citri</Emphasis> by its interaction with hook-associated FlgK

Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.     

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.     

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti‐σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far‐UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.     

Subdivision of flagellar region III of the Escherichia coli and Salmonella typhimurium chromosomes and identification of two additional flagellar genes.

The many genes involved in flagellar structure and function in Escherichia coli and Salmonella typhimurium are located in three major clusters on the chromosome: flagellar regions I, II and III. We have found that region III does not consist of a contiguous set of flagellar genes, as was thought, but that in E. coli there is almost 7 kb of DNA between the filament cap gene, fliD, and the next known flagellar gene, fliE; a similar situation occurs in S. typhimurium. Most of this DNA is unrelated to flagellar function, since a mutant in which 5.4 kb of it had been deleted remained fully motile and chemotactic as judged by swarming on semi-solid agar. We have therefore subdivided flagellar region III into two regions, IIIa and IIIb. The known genes in region IIIa are fliABCD, all of which are involved in filament structure and assembly, while region IIIb contains genes fliEFGHIJKLMNOPQR, all of which are related to formation of the hook (basal-body)-complex or to even earlier assembly events. We have found that fliD, the last known gene in region IIIa, is immediately followed by two additional genes, both necessary for flagellation, which we have designated fliS and fliT. They encode small proteins with deduced molecular masses of about 15 kDa and 14 kDa, respectively. The functions of FliS and FliT remain to be determined, but they do not appear to be members of the axial family of structural proteins to which FliD belongs.     

FlgN Is Required for Flagellum-Based Motility by Bacillus subtilis

The assembly of the bacterial flagellum is exquisitely controlled. Flagellar biosynthesis is underpinned by a specialized type III secretion system that allows export of proteins from the cytoplasm to the nascent structure. Bacillus subtilis regulates flagellar assembly using both conserved and species-specific mechanisms. Here, we show that YvyG is essential for flagellar filament assembly. We define YvyG as an orthologue of the Salmonella enterica serovar Typhimurium type III secretion system chaperone, FlgN, which is required for the export of the hook-filament junction proteins, FlgK and FlgL. Deletion of flgN (yvyG) results in a nonmotile phenotype that is attributable to a decrease in hag translation and a complete lack of filament polymerization. Analyses indicate that a flgK-flgL double mutant strain phenocopies deletion of flgN and that overexpression of flgK-flgL cannot complement the motility defect of a ΔflgN strain. Furthermore, in contrast to previous work suggesting that phosphorylation of FlgN alters its subcellular localization, we show that mutation of the identified tyrosine and arginine FlgN phosphorylation sites has no effect on motility. These data emphasize that flagellar biosynthesis is differentially regulated in B. subtilis from classically studied Gram-negative flagellar systems and questions the biological relevance of some posttranslational modifications identified by global proteomic approaches.     

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.     

FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium.

FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum. We have overproduced and purified FlgD and raised an antibody against it. By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies from flgA, flgE, flgH, flgI, flgK, and fliK mutants, in much smaller amounts in those from the wild type and flgL, fliA, fliC, fliD, and fliE mutants, and not at all in those from flgB, flgD, flgG, and flgJ mutants. In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip. Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook. Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament. However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein. When FlgD was added to the culture medium of a flgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously. When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant. Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export.     

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.     

Chaperone Recycling in Late-Stage Flagellar Assembly

The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.     

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.     

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.     

The HP0256 gene product is involved in motility and cell envelope architecture of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Background  

Helicobacter pylori is the causative agent for gastritis, and peptic and duodenal ulcers. The bacterium displays 5-6 polar sheathed flagella that are essential for colonisation and persistence in the gastric mucosa. The biochemistry and genetics of flagellar biogenesis in H. pylori has not been fully elucidated. Bioinformatics analysis suggested that the gene HP0256, annotated as hypothetical, was a FliJ homologue. In Salmonella, FliJ is a chaperone escort protein for FlgN and FliT, two proteins that themselves display chaperone activity for components of the hook, the rod and the filament.     

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.     

The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation

Assembly of each Salmonella typhimurium flagellum filament requires export and polymerisation of ca. 30000 flagellin (FliC) subunits. This is facilitated by the cytosolic chaperone FliS, which binds to the 494 residue FliC and inhibits its polymerisation. Yeast two-hybrid assays, co-purification and affinity blotting showed that FliS binds specifically to the C-terminal 40 amino acid component of the disordered D0 domain central to polymerisation. Without FliS binding, the C-terminus is degraded. Our data provide further support for the view that FliS is a domain-specific bodyguard preventing premature monomer interaction.