Flha Provides the Adaptor for Coordinated Delivery of Late Flagella Building Blocks to the Type III Secretion System

FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system

Gert Bange1, Nico Kümmerer1, Christoph Engel, Gunes Bozkurt, Klemens Wild, and Irmgard Sinning2

Biochemie-Zentrum der Universität Heidelberg, INF328, 69120 Heidelberg, Germany

Edited* by Peter Walter, University of California, San Francisco School of Medicine, San Francisco, CA, and approved May 7, 2010 (received for review February 4, 2010)

Flagella are the bacterial organelles of motility and can play impor- by six integral membrane proteins (FlhA, FlhB, FliP, FliQ, FliR, tant roles in pathogenesis. Flagella biosynthesis requires the coor- FliO) and is thought to reside within the MS-ring (19, 20). In the dinated export of huge protein amounts from the cytosol to the cytosol three proteins (FliI, FliH, and FliJ) are functionally asso- nascent flagellar structure at the cell surface and employs a type ciated to the T3SS. The FliI ATPase (21) together with its nega- III secretion system (T3SS). Here we show that the integral mem- tive regulator FliH (22) seems to regulate initial entry and sorting brane protein FlhA from the gram-positive bacterium Bacillus of export substrates into the T3SS (14, 23). The membrane-asso- subtilis acts as an adaptor for late export substrates at the T3SS. ciated protein FliJ is essential for the export of flagella building The major filament protein (flagellin) and the filament-cap protein blocks (20). It binds to empty chaperones of the hook-filament (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex junction and of filament-cap substructures but not of the filament with their cognate chaperones (FliS and FliT). To understand (24). This led to the proposal that FliJ selectively recycles empty the molecular details of these interactions we determined the chaperones during flagella assembly (24, 25). FlhA-C crystal structure at 2.3 Å resolution. FlhA-C consists of an The integral transmembrane protein FlhA (InvA, YscV in N-terminal linker region, three subdomains with a novel fold, pathogen related T3SSs) is the largest component of the T3SS and a disordered region essential for the adaptor function. We (26) and belongs to the “flagellum/hypersensitive response/inva- show that the export protein FliJ associates with the linker region sion” (FHIPEP) family of proteins (27). It consists of an and modulates the binding properties of FlhA-C. While the interac- N-terminal transmembrane (TM) domain with eight predicted tion of FliD/FliT is enhanced, flagellin/FliS is not affected. FliJ also TM α-helices and a large cytosolic domain at its C terminus keeps FliTassociated with FlhA-C and excess of FliT inhibits binding (FlhA-C) (Fig. 1A) (28). Disruption of the FlhA gene in gram- of FliD/FliT, suggesting that empty FliT chaperones stay associated positive or gram-negative bacteria leads to nonmotile cells, which with FliJ after export of FliD. Taken together, these results allow to lack flagella and are unable to export flagellar proteins (20, 29). propose a model that explains how the T3SS may switch from the Although FlhA has been implicated in substrate translocation stoichiometric export of FliD to the high-throughput secretion of (30, 31), and even though interactions of its cytoplasmic domain flagellin. with all components of the T3SS were reported (28, 30, 32–34), its precise function is still unknown. Our biochemical and structural chaperone ∣ flagella biosynthesis ∣ motility ∣ protein transport analyses establish FlhA-C as an adaptor that receives the late flagella building blocks FliD and Flagellin only when bound to lagella represent one of nature’s largest molecular machines their cognate chaperones FliT and FliS, respectively. Fand are the bacterial organelles of locomotion (reviewed in refs. 1–3). In many pathogenic bacteria, they also act as virulence Results and Discussion factors, because motility is needed to reach the primary site of FlhA-C Interacts with Late Flagella Substrate-Chaperone Complexes. infection, and thereby establish the first step of a bacterial infec- To specify the role of FlhA-C in the T3SS of flagella, we tion (4, 5). A flagellum consists of the cytosolic C-ring, the mem- performed Glutathione-S-transferase (GST) pulldown assays in brane-embedded basal body and the exterior hook, hook- Bacillus subtilis cell lysates. FlhA-C interacts with the major fila- filament junction, and filament structures. The flagellar filament ment protein flagellin (Fig. 1B). Addition of the flagellin-specific is a long, tubular structure and consists of about 20,000 subunits chaperone FliS (11) to the cell lysates enhanced the interaction of of the protein flagellin. The assembly of the filament is strictly FlhA-C with flagellin. This indicates that the flagellin/FliS complex binds to FlhA-C and that FliS was probably limiting in the

sequential: At first the pentameric FliD cap must be formed BIOCHEMISTRY to promote flagellin self-assembly at the distal end of the growing cell lysate. Next we performed the same assay with recombinantly filament (6). Flagellar proteins (e.g., flagellin and FliD) are expressed and purified flagellin and FliS. The same results as synthesized in the cytosol, transferred into the central channel before were obtained, indicating that no other cytosolic factors of the growing flagellar structure, and travel as monomers to

the nascent end of the flagellum where they assemble (7). Trans- Author contributions: G. Bange and I.S. designed research; G. Bange, N.K., C.E., and location into the central channel is achieved by a flagellum- G. Bozkurt performed research; G. Bange, N.K., C.E., G. Bozkurt, K.W., and I.S. analyzed specific type 3 secretion system (T3SS) in the cytoplasmic mem- data; and G. Bange and I.S. wrote the paper. brane and driven by the proton-motive force (8–10). To prevent The authors declare no conflict of interest. futile self-assembly/aggregation within the cytosol, specialized *This Direct Submission article had a prearranged editor. chaperones (FliS for flagellin and FliT for FliD) prevent prema- Freely available online through the PNAS open access option. ture interactions of flagellar components by binding to their Data deposition: The atomic coordinates for the structure have been deposited in the C-terminal, amphipathic oligomerization domains (11–13). They Protein Data Bank, www.pdb.org (PDB ID code 3MIX). also seem to be involved in the entry of subunits into the T3SS 1G. Bange and N.K. contributed equally to this work. – (14 16), but the molecular details are not known. 2To whom correspondence should be addressed. E-mail: irmi.sinning@bzh. T3SSs are used for the export of flagella building blocks (17) as uni-heidelberg.de. well as for the transmission of bacterial virulence effectors into This article contains supporting information online at www.pnas.org/lookup/suppl/ host cells (18). The inner membrane part of the T3SS is formed doi:10.1073/pnas.1001383107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1001383107 PNAS ∣ June 22, 2010 ∣ vol. 107 ∣ no. 25 ∣ 11295–11300 Downloaded by guest on September 23, 2021 Fig. 1. FlhA-C interacts with late flagellar substrate-chaperone complexes. (A) Scheme of FlhA, flagellin, and FliD constructs used in this study. The numbers correspond to amino acid positions. The FliS and FliT binding sites at flagellin and FliD, respectively, are indicated. (B) Binding of native flagellin to immobilized GST-FlhA-C in the absence (lane 1) and presence of recombinant FliS (lane 2). Flagellin and FliS, which were confirmed by mass spectrometry, are indicated by asterisks. (C) Binding of recombinantly expressed and purified flagellin variants to GST-tagged FlhA-C in the presence or absence of FliS. Lanes 3, 6, and 9 show the quality of the employed flagellin variants. Binding of flagellin/FliS and D0-C/FliS to FlhA-C is indicated by asterisks. (D) Binding of recombinantly expressed and purified FliD variants to GST-tagged FlhA-C in the presence or absence of FliT. Lanes 3 and 5 show the size and quality of the employed FliD variants. Binding of FliD/FliT to FlhA-C is indicated by asterisks. B–D show Coomassie-stained SDS/PAGE.

are involved in the binding of flagellin/FliS to FlhA-C (Fig. 1C). Right and 1A). D1a consists of two α-helices that pack against In the absence of FliS binding of flagellin to FlhA-C was not ob- a four-stranded, mixed β-sheet. D1b is only poorly ordered in served (Fig. 1C). A flagellin variant lacking the FliS binding site the electron density map and seems therefore highly flexible (35, 36) (ΔD0-C, amino acid residues 1–244, Fig. 1A) was unable and/or unstructured (see Table S2). D2 (residues 485–571) con- to interact with FlhA-C even in the presence of FliS (Fig. 1C). sists of five α-helices (α6–α10) and interacts with D1a by a pre- This experiment also revealed that empty FliS does not bind dominantly hydrophobic interface with a total buried surface area 2 to FlhA-C. A complex of FliS and its binding domain in flagellin of ∼1350 Å (Fig. 2B). Disruption of this interface by mutation of (D0-C, residues 244–304, Fig. 1A) was sufficient for binding to the conserved Gly380 impairs the function of FlhA in vivo (38) FlhA-C (Fig. 1C). Taken together we show that the flagellin/FliS and in vitro (Fig. 2C). Thus, the hydrophobic D1a-D2 contact complex binds to FlhA-C but not the individual proteins. seems to be essential for the binding of substrate-chaperone com- To investigate whether other late substrate-chaperone complexes to FlhA-C. D3 (residues 578–677) consists of a four- plexes also bind to FlhA-C, the filament-capping protein FliD stranded β-sheet (β5–β8), which is surrounded by three α-helices and its chaperone FliT were chosen. Like flagellin/FliS, purified (α11–α13). Helices α11 and α12 are connected by a partially dis- FliD and FliT form a heterodimer (Fig. 1A and Fig. S1), which ordered loop (amino acids 596–603). D2 and D3 interact by a binds to FlhA-C (Fig. 1D). A FliD variant lacking the C-terminal bipartite network of electrostatic and hydrophobic contacts with 2 FliT binding site (FliD-N403, Fig. 1A) was unable to associate a total buried surface area of ∼1;500 Å (Fig. 2D). Helix α10 with with FlhA-C in the presence or absence of FliT (Fig. 1D). FliT a kink of about 80° at leucine 563 is part of the D2-D3 interface. alone did not bind to FlhA-C (Fig. 1D). Taken together, targeting Two conserved glutamate residues in the interface are essential of late flagella building blocks (FliD, flagellin) to FlhA-C follows for flagellar export in Salmonella typhimurium (Glu547 and a general mechanism and relies on the recognition of the secre- Glu676, Fig. S4) (38). Analysis of the corresponding mutants tion substrate by its cognate chaperone. Thereby, FlhA receives of B. subtilis FlhA-C (Glu528 and Glu662) in our in vitro assay flagella building blocks in a state competent for translocation. was hindered, because both mutant proteins tend to degrade during expression and purification. This implies that the D2-D3 The Crystal Structure of FlhA-C Shows Three Subdomains with a Novel interface is crucial for the stability of FlhA-C. Taken together, Fold. In order to understand how FlhA-C receives substrate-cha- the arrangement of the D1, D2 and D3 domains of FlhA-C and perone complexes we determined its crystal structure at 2.3 Å the specific interdomain interactions observed in the crystal resolution (Table S1, Fig. S2). The structure with the overall di- structure are essential for the correct function of FlhA. mensions of 45 Å × 55 Å × 50 Å comprises amino acid residues 318–677 (with only two disordered regions: residues 420–461 and D1b Is Essential for Binding of Late Substrate-Chaperone Complexes 596–603). FlhA-C consists of three subdomains, named D1, D2, to FlhA-C. Guided by the X-ray structure we tested whether the and D3 that form a groove (Fig. 2A Left and Fig. S3). D1, D2, and linker/D1 (residues 302–482) or D2/D3 (ΔN481, residues D3 have novel folds as determined by the DALI server (37). D1 482–677) regions alone would be sufficient to bind flagellin/FliS can be divided into two parts: a well-folded D1a part (residues or FliD/FliT (Fig. 3A). None of the two FlhA-C variants was able 340–484) and a disordered D1b part (residues 420–461) that is to bind late substrate-chaperone complexes showing that all three inserted into D1a just before helix α5. The N terminus of D1 FlhA-C subdomains are required (Fig. 3B and Fig. S5). Only the is connected to the FlhA transmembrane domain by a linker linker region of FlhA-C (ΔN339, residues 302–339) is dispensable region (residues 318–342) that contains two helices (Figs. 2A for FlhA-C interaction with flagellin/FliS (Fig. 3B). In S. typhi-

11296 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001383107 Bange et al. Downloaded by guest on September 23, 2021 Fig. 2. Crystal structure of FlhA-C. (A) Cartoon representation of the crystal structure of B. subtilis FlhA-C. The domains D1a, D1b, D2, and D3 are indicated and rainbow colored from the N terminus to the C terminus. The secondary structure elements are labeled with α and β for α-helix and β-strand, respectively. (B) Close-up of the D1a/D2 interface. The majority of hydrophobic residues are provided by helices α6, α8, and α5 of D2 and D1a, respectively. The conserved glycine (Gly380) is indicated by a red ball. In S. typhimurium, replacement of the corresponding Gly399 by arginine abolishes the secretion of flagellar proteins at nonpermissive temperature (38). (C) The Gly380Arg variant of B. subtilis FlhA-C does not bind the flagellin/FliS complex in our in vitro assay. Replacement of Gly380 (and Gly399 in S. typhimurium) by arginine most likely destroys the hydrophobic interface between D1a and D2 which is conserved also in S. typhimurium. The figure shows a Coomassie-stained SDS/PAGE. (D) Close-up of the D2/D3 interface. D2 and D3 interact via an extended, bipartite interface of electrostatic and hydrophobic interactions. A central part of the interface is formed by the kinked helix α10. The residues forming the interface are conserved among FlhA proteins of different bacterial species (Fig. S3).

murium FlhA, two temperature-sensitive mutants (Leu449Arg FliT and FliD/FliT Compete for a Common Binding Site at FliJ. Isolated and Gly451Asp), which are unable to export flagellar proteins FliJ was previously reported to bind FliT, but not FliS or at restrictive temperature (20, 38), localize within D1b (Fig. 2A substrate/chaperone complexes (24). We now show that FliJ binds Left and Fig. S3). We hypothesized that D1b might participate in binding substrate-chaperone complexes to FlhA-C and generated different FlhA-C variants with five amino acid deletions in D1b. While all deletion variants behaved like the full-length FlhA-C in purification, indicating proper folding, none of them was able to bind flagellin/FliS (Fig. 3C) or FliD/FliT (Fig. S5) in our in vitro assay. Therefore, D1b is essential for the interaction of late substrate-chaperone complexes with FlhA-C.

FliJ Enhances FliD/FliT Binding to FlhA-C. We noticed that the binding BIOCHEMISTRY efficiencies of the two substrate-chaperone complexes to FlhA-C differ significantly. While flagellin/FliS binds almost stoichiome- trically to FlhA-C, FliD/FliT binds substoichiometrically (Fig. 4A, compare lanes 1 and 3). Therefore, the interaction with of FliD/ FliT with FlhA-C might require an additional factor. In S. typhimurium an interaction between FlhA-C and FliJ was described (30, 32, 39). We confirm this interaction for the B. subtilis homo- logues (Fig. 4B). Deletion of the linker region (ΔN339) abolishes Fig. 3. Functional analysis of FlhA-C. (A) Scheme of FlhA-C variants used in the interaction (Fig. 4B), indicating that the linker region is this study. The numbers correspond to amino acid positions. (B) Analysis of required for binding FliJ to FlhA-C. We then tested whether FliJ association of FlhA-C variants with flagellin/FliS. FlhA-C and FlhA-ΔN339 are influences the ability of FlhA-C to interact with flagellin/FliS or able to bind flagellin/FliS. FlhA-302-482 and ΔN481, which represent the D1 FliD/FliT. The presence of FliJ did not influence the binding of and D2/D3, respectively, are unable to bind flagellin/FliS. (C) Binding studies of flagellin/FliS to “five amino acid” deletion variants in D1b of FlhA-C. flagellin/FliS (Fig. 4A, lane 2), but a significant increase in FliD/ All mutants failed to bind flagellin/FliS. The asterisk indicates the negative FliT binding was observed (Fig. 4A, Lane 4). Therefore, FliJ control: GST-FlhA-C incubated with flagellin in the absence of FliS. B and selectively enhances association of FliD/FliT with FlhA-C. C show Coomassie-stained SDS/PAGE.

Bange et al. PNAS ∣ June 22, 2010 ∣ vol. 107 ∣ no. 25 ∣ 11297 Downloaded by guest on September 23, 2021 Fig. 4. FliJ selectively enhances interaction of FliD/FliT with FlhA-C. (A) Binding of flagellin/FliS and FliD/FliT to FlhA-C in the absence and the presence of FliJ. FliJ does not influence binding of flagellin/FliS to FlhA-C (lanes 1 and 2). The presence of FliJ significantly increases binding of FliD/FliT to FlhA-C (lanes 3 and 4). (B) Association of FliJ and FlhA-C requires the linker region that connects that the transmembrane domain of FlhA with its cytoplasmic domain. FliJ is indicated by an asterisk. FlhA-ΔN339, which lacks the linker region, is unable to bind FliJ. (C) FliT binds to FliJ in the context of FlhA-C. Lanes 3–5 show the quality of FlhA-C, FliT, and FliJ, respectively. (D) FliT and FliD/FliT compete for a common binding site at FliJ bound to FlhA-C. FliT does not influence FliD/FliT binding to FlhA-C in the absence of FliJ (lane 1). In the presence of FliJ, increasing amounts of FliT lead to a decreased binding of FliD/FliT (lanes 3–5). A–D show Coomassie- stained SDS/PAGE.

FliT also when associated with FlhA-C (Fig. 4C). To address a FlhA via FliJ (step 3). Assuming that FlhA is an oligomer (1, 19), possible competition between FliT and FliD/FliT for FliJ at the stoichiometry of FlhA could define the number of binding sites FlhA-C, increasing amounts of FliT were added to the in vitro (step 4). Thereby, the number of secreted FliD molecules would be assay. FliT decreased the interaction of FliD/FliT with FlhA-C simply restricted by the number of FliT molecules that stay asso- in the presence of FliJ (Fig. 4D) indicating that FliTand FliD/FliT ciated with FliJ. Because efficient binding of FliD/FliT to FlhA-C compete for a common binding site at FliJ. requires a vacant FliJ, while that of Flagellin/FliS does not, the occupation of FliJ might switch the T3SS to the high-throughput A Model for Coordinated Substrate-Chaperone Binding to FlhA-C. secretion of the filament component flagellin (>20;000 copies). After export of FliD and flagellin subunits by the T3SS machin- Reduction in complexity elegantly ensures filament assembly with ery, these proteins travel through a central channel formed by the a minimal regulatory effort and low error probability. However, already established hook and hook-filament structures. FliD this model needs to be further validated by the determination stably assembles into a pentameric cap structure at the nascent of the stoichiometry of the integral membrane protein FlhA. end of the growing flagellum, which is essential for the subsequent polymerization of flagellin into the filament (6, 40). Mutant The export of defined protein copy numbers for establishing strains of S. typhimurium with a nonfunctional FliD fail to assem- extracellular superstructures is a unique problem in biology ble the filament, leading to the diffusion of flagellin monomers (e.g., the bacterial injectosome) (18), and we imagine that a similar into the culture medium (reviewed in ref. 7). Once the FliD mechanism might be employed. pentamer is completed, it cannot accept another monomer in Methods Summary the cap. Current electron-microscopic data on the FliD cap of A detailed description of the applied methods can be found in the filament (41) suggest that accessory FliD monomers would SI Methods. Shortly, all genes were amplified by polymerase chain presumably block the central channel and, therefore, subsequent filament assembly. In this respect a weak association of FliD/FliT reaction (PCR) from the B. subtilis genome and cloned into with FlhA-C in the absence of FliJ might be necessary to prevent pET24d (Novagen), pET16b (Novagen) or pGAT2 (EMBL). Pro- uncontrolled FliD binding and secretion. In order to assure the teins were expressed in Escherichia coli BL21(DE3) and purified precise stoichiometry of the cap structure it would be advanta- by Ni-ion affinity and size exclusion chromatography. Crystalliza- geous if the T3SS restricted the number of secreted FliD molecules tion, structure determination, and crystallographic statistics are in to five. Based on our data, it is tempting to propose the following SI Methods and Table S1. The GST-pulldown assay is described in working model (Fig. 5): FliJ modifies the binding abilities of detail in SI Methods. The biophysical properties of FlhA-C FlhA-C or provides an additional binding site for the FliD/FliT variants were analyzed by static light scattering (Minidawn complex in close proximity to the T3SS pore (step 1–2). After Tristar, Wyatt) and refractive index measurements (ΔN1000, the secretion of FliD, empty FliTchaperones stay associated with Dr. Bures).

11298 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001383107 Bange et al. Downloaded by guest on September 23, 2021 Fig. 5. Model for the delivery of late flagella building blocks to FlhA-C. The transmembrane (TM) protein FlhA resides in the cytosolic membrane (CM). Its cytoplasmic part (orange) consists of three main domains (D1, D2, and D3). The color code is: FliJ (blue), FliD/FliT complex (gray/red), Flagellin/FliS complex (gray/green). The flexible nature of D1b is indicated by a dashed line. The oligomeric nature of FlhA is indicated by a bracket, and “n” refers to the still unknown stoichiometry.

ACKNOWLEDGMENTS. We thank Astrid Hendricks for excellent technical European Synchrotron Radiation Facility, Grenoble. This work was supported assistance, Jürgen Kopp from our Crystallization Platform (BZH/Cluster of by a collaborative research grant from the Deutsche Forschungsgemeinschaft Excellence:CellNetworks) for excellent support, and Dieter Kressler for stimu- (SFB 638) and by the interdisciplinary PhD program “Molecular Machines: lating discussion. We thank Hans Helferdorfer (Die Graphiker, Heidelberg) Mechanisms and Functional Interconnections” of the Land Baden- for support in preparing the cartoon. Data collection was performed at the Württemberg.

1. Macnab RM (2003) How bacteria assemble flagella. Annu Rev Microbiol 57:77–100. 17. Macnab RM (2004) Type III flagellar protein export and flagellar assembly. Biochim 2. Chevance FF, Hughes KT (2008) Coordinating assembly of a bacterial macromolecular Biophys Acta 1694:207–217. machine. Nat Rev Microbiol 6:455–465. 18. Moraes TF, Spreter T, Strynadka NC (2008) Piecing together the type III injectisome of 3. McCarter LL (2006) Regulation of flagella. Curr Opin Microbiol 9:180–186. bacterial pathogens. Curr Opin Struct Biol 18:258–266. 4. Ottemann KM, Miller JF (1997) Roles for motility in bacterial-host interactions. Mol 19. Fan F, Ohnishi K, Francis NR, Macnab RM (1997) The FliP and FliR proteins of Salmonella Microbiol 24:1109–1117. typhimurium, putative components of the type III flagellar export apparatus, are 5. Moens S, Vanderleyden J (1996) Functions of bacterial flagella. Crit Rev Microbiol located in the flagellar basal body. Mol Microbiol 26:1035–1046. – 22:67 100. 20. Minamino T, Macnab RM (1999) Components of the Salmonella flagellar export “ ” J Mol Biol 6. Ikeda T, Asakura S, Kamiya R (1985) Cap on the tip of Salmonella flagella. apparatus and classification of export substrates. J Bacteriol 181:1388–1394. – 184:735 737. 21. Fan F, Macnab RM (1996) Enzymatic characterization of FliI. An ATPase involved in 7. Minamino T, Namba K (2004) Self-assembly and type III protein export of the bacterial flagellar assembly in Salmonella typhimurium. J Biol Chem 271:31981–31988. J Mol Microb Biotech – flagellum. 7:5 17. 22. Minamino T, MacNab RM (2000) FliH, a soluble component of the type III flagellar 8. Minamino T, Namba K (2008) Distinct roles of the FliI ATPase and proton motive force export apparatus of Salmonella, forms a complex with FliI and inhibits its ATPase in bacterial flagellar protein export. Nature 451:485–488. BIOCHEMISTRY activity. Mol Microbiol 37:1494–1503. 9. Paul K, Erhardt M, Hirano T, Blair DF, Hughes KT (2008) Energy source of flagellar type 23. Stafford GP, et al. (2007) Sorting of early and late flagellar subunits after docking at III secretion. Nature 451:489–492. the membrane ATPase of the type III export pathway. J Mol Biol 374:877–882. 10. Galan JE (2008) Energizing type III secretion machines: What is the fuel? Nat Struct Mol 24. Evans LD, Stafford GP, Ahmed S, Fraser GM, Hughes C (2006) An escort mechanism for Biol 15:127–128. cycling of export chaperones during flagellum assembly. Proc Natl Acad Sci USA 11. Auvray F, Thomas J, Fraser GM, Hughes C (2001) Flagellin polymerisation control by a 103:17474–17479. cytosolic export chaperone. J Mol Biol 308:221–229. 25. Evans LD, Hughes C (2009) Selective binding of virulence type III export chaperones by 12. Bennett JC, Thomas J, Fraser GM, Hughes C (2001) Substrate complexes and domain FliJ escort orthologues InvI and YscO. FEMS Microbiol Lett 293:292–297. organization of the Salmonella flagellar export chaperones FlgN and FliT. Mol Micro- biol 39:781–791. 26. Kihara M, Minamino T, Yamaguchi S, Macnab RM (2001) Intergenic suppression 13. Fraser GM, Bennett JC, Hughes C (1999) Substrate-specific binding of hook-associated between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane J Bacteriol – proteins by FlgN and FliT, putative chaperones for flagellum assembly. Mol Microbiol component of its export apparatus. 183:1655 1662. 32:569–580. 27. Hueck CJ (1998) Type III protein secretion systems in bacterial pathogens of animals Microbiol Mol Biol Rev – 14. Thomas J, Stafford GP, Hughes C (2004) Docking of cytosolic chaperone-substrate and plants. 62:379 433. complexes at the membrane ATPase during flagellar type III protein export. Proc Natl 28. McMurry JL, Van Arnam JS, Kihara M, Macnab RM (2004) Analysis of the cytoplasmic Acad Sci USA 101:3945–3950. domains of Salmonella FlhA and interactions with components of the flagellar export 15. Akeda Y, Galan JE (2005) Chaperone release and unfolding of substrates in type III machinery. J Bacteriol 186:7586–7592. secretion. Nature 437:911–915. 29. Carpenter PB, Ordal GW (1993) Bacillus subtilis FlhA: A flagellar protein related to a 16. Gauthier A, Finlay BB (2003) Translocated intimin receptor and its chaperone interact new family of signal-transducing receptors. Mol Microbiol 7:735–743. with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli. 30. Minamino T, MacNab RM (2000) Interactions among components of the Salmonella J Bacteriol 185:6747–6755. flagellar export apparatus and its substrates. Mol Microbiol 35:1052–1064.

Bange et al. PNAS ∣ June 22, 2010 ∣ vol. 107 ∣ no. 25 ∣ 11299 Downloaded by guest on September 23, 2021 31. Ghelardi E, et al. (2002) Requirement of flhA for swarming differentiation, flagellin 37. Holm L, Sander C (1995) Dali: A network tool for protein structure comparison. Trends export, and secretion of virulence-associated proteins in Bacillus thuringiensis. Biochem Sci 20:478–480. J Bacteriol – 184:6424 6433. 38. Saijo-Hamano Y, Minamino T, Macnab RM, Namba K (2004) Structural and functional 32. Fraser GM, Gonzalez-Pedrajo B, Tame JR, Macnab RM (2003) Interactions of FliJ with analysis of the C-terminal cytoplasmic domain of FlhA, an integral membrane the Salmonella type III flagellar export apparatus. J Bacteriol 185:5546–5554. component of the type III flagellar protein export apparatus in Salmonella. J Mol Biol 33. Minamino T, et al. Role of the C-terminal cytoplasmic domain of FlhA in bacterial 343:457–466. flagellar type III protein export. J Bacteriol, in press. 34. Rust M, et al. (2009) The Helicobacter pylori anti-sigma factor FlgM is predominantly 39. Gonzalez-Pedrajo B, Fraser GM, Minamino T, Macnab RM (2002) Molecular dissection cytoplasmic and cooperates with the flagellar basal body protein FlhA. J Bacteriol of Salmonella FliH, a regulator of the ATPase FliI and the type III flagellar protein 191:4824–4834. export pathway. Mol Microbiol 45:967–982. 35. Evdokimov AG, et al. (2003) Similar modes of polypeptide recognition by export 40. Homma M, Iino T (1985) Locations of hook-associated proteins in flagellar structures of Nat Struct Biol – chaperones in flagellar biosynthesis and type III secretion. 10:789 793. Salmonella typhimurium. J Bacteriol 162:183–189. 36. Ozin AJ, Claret L, Auvray F, Hughes C (2003) The FliS chaperone selectively binds the 41. Yonekura K, et al. (2000) The bacterial flagellar cap as the rotary promoter of flagellin disordered flagellin C-terminal D0 domain central to polymerisation. FEMS Microbiol self-assembly. Science 290:2148–2152. Lett 219:219–224.

11300 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001383107 Bange et al. Downloaded by guest on September 23, 2021