FliK-Driven Conformational Rearrangements of FlhA and FlhB Are Required for Export Switching of the Flagellar Protein Export Apparatus

The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be efficiently built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, but the mechanism remains a mystery. By analyzing a slow-cleaving flhB(P270A) mutant, we provide evidence that an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation.

ABSTRACT

FlhA and FlhB are transmembrane proteins of the flagellar type III protein export apparatus, and their C-terminal cytoplasmic domains (FlhA_C and FlhB_C) coordinate flagellar protein export with assembly. FlhB_C undergoes autocleavage between Asn-269 and Pro-270 in a well-conserved NPTH loop located between FlhB_CN and FlhB_CC polypeptides and interacts with the C-terminal domain of the FliK ruler when the length of the hook has reached about 55 nm in Salmonella. As a result, the flagellar protein export apparatus switches its substrate specificity, thereby terminating hook assembly and initiating filament assembly. The mechanism of export switching remains unclear. Here, we report the role of FlhB_C cleavage in the switching mechanism. Photo-cross-linking experiments revealed that the flhB(N269A) and flhB(P270A) mutations did not affect the binding affinity of FlhB_C for FliK. Genetic analysis of the flhB(P270A) mutant revealed that the P270A mutation affects a FliK-dependent conformational change of FlhB_C, thereby inhibiting the substrate specificity switching. The flhA(A489E) mutation in FlhA_C suppressed the flhB(P270A) mutation, suggesting that an interaction between FlhB_C and FlhA_C is critical for the export switching. We propose that the interaction between FliK_C and a cleaved form of FlhB_C promotes a conformational change in FlhB_C responsible for the termination of hook-type protein export and a structural remodeling of the FlhA_C ring responsible for the initiation of filament-type protein export.

IMPORTANCE The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be efficiently built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, but the mechanism remains a mystery. By analyzing a slow-cleaving flhB(P270A) mutant, we provide evidence that an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation.

INTRODUCTION

The bacterial flagellum is responsible for swimming motility in liquid media and for swarming motility on solid surfaces for many bacterial species. The flagellum is composed of a basal body (rotary motor), a hook (universal joint), and a filament (helical propeller). Flagellar assembly begins with the basal body, followed by the hook and finally the filament (1). For assembly of the hook and filament from the cell surface, a type III protein export apparatus transports these component proteins from the cytoplasm to the distal end of the growing structure. The type III protein export apparatus is composed of five transmembrane proteins, FlhA, FlhB, FliP, FliQ, and FliR and three cytoplasmic proteins, FliH, FliI, and FliJ (2).

The length of the hook is controlled, extending about 55 nm from the cell surface in wild-type cells of Salmonella enterica serovar Typhimurium (here referred to Salmonella) (3). The flagellar type III protein export apparatus uses the FliK ruler to measure the hook length and facilitates the switching of export specificity from hook-type substrates (FlgD, FlgE, and FliK) to filament-type ones (FlgK, FlgL, FlgM, FliC, and FliD) upon hook completion. The substrate specificity switch terminates hook assembly and initiates filament assembly. FlhB and FlhA are directly involved in export switching of the type III protein export apparatus (4).

FliK consists of N-terminal (FliK_N) and C-terminal (FliK_C) domains (5, 6). FliK_N acts as a molecular ruler to measure the length of the hook through interactions of FliK_N with the hook-capping protein FlgD and the hook protein FlgE (7–11), whereas FliK_C interacts with the C-terminal cytoplasmic domain of FlhB (FlhB_C) to promote substrate specificity switching of the flagellar type III protein export apparatus (12–15).

FlhB_C consists of FlhB_CN and FlhB_CC polypeptides with molecular weights of 8.5 and 11.5 kDa, respectively (16, 17). A conserved hydrophobic patch formed by Ala-286, Pro-287, Ala-341, and Leu-344 residues in FlhB_CC is involved in the interaction of FlhB_C with the N-terminal segments of FlgD, FlgE, and FliK, which contain an export signal recognized by the flagellar type III protein export apparatus (18). Autocatalytic cleavage between Asn-269 and Pro-270, which is located within a highly conserved NPTH loop between FlhB_CN and FlhB_CC, is critical for export switching of the protein export apparatus (19, 20). It has been reported that autocleavage of the C-terminal cytoplasmic domain of SpaS, which is a FlhB homologue of the Salmonella SPI-1 injectisome, allows the proper conformation of SpaS_C to render it competent for its switching function (21), but it remains unclear how its cleaved form flips export specificity of the type III protein export apparatus of the Salmonella SPI-1 injectisome.

FlhA forms a homo-nonamer through its C-terminal cytoplasmic domain (FlhA_C) in the flagellar type III protein export apparatus (22). FlhA_C interacts with flagellar chaperones in complex with their cognate filament-type substrates to promote the export of filament-type proteins to form the flagellar filament at the hook tip (23–26). Interactions of a flexible linker of FlhA (FlhA_L) with its neighboring FlhA_C subunit in the ring structure induce the remodeling of the FlhA_C ring structure upon completion of the hook structure, thereby allowing the flagellar chaperones in complex with their cognate substrates to bind to the FlhA_C ring to facilitate the export of filament-type proteins (27, 28). However, it remains unknown which protein triggers the structural remodeling of the FlhA_C ring structure.

The flhB(N269A) mutation inhibits not only autocleavage of FlhB_C but also substrate specificity switching of the flagellar type III protein export apparatus (19), leading to a plausible hypothesis that an interaction between FliK_C and a cleaved form of FlhB_C may induce the structural remodeling of the FlhA_C ring responsible for the substrate specificity switching. To clarify this hypothesis, we analyzed a slow-cleaving flhB(P270A) mutant in detail and provide evidence suggesting that the binding of FliK_C to a cleaved form of FlhB_C not only terminates hook polymerization but also induces conformational rearrangements of the FlhA_C ring to initiate filament assembly at the hook tip.

RESULTS

Effect of the flhB(N269A) and flhB(P270A) mutations on photo-cross-linking between FliK_C and FlhB_C.

A well-conserved Ile-304 residue of FliK is critical for substrate specificity switching of the flagellar type III protein export apparatus (14). Recently, it has been shown that Ile-304 of FliK_C is in relatively close proximity to FlhB_C, allowing FliK to form a photo-cross-linked product with FlhB_CC (15). To investigate whether autocleavage of FlhB_C is required for the interaction of FlhB_C with FliK_C, photo-cross-linking experiments were conducted using a photoreactive phenylalanine, p-benzoyl-phenylalanine (pBPA), that was introduced into an amber codon at position 304 of FliK using the amber suppressor tyrosyl tRNA and the engineered tyrosyl-tRNA synthetase (29). In agreement with a previous report (15), UV irradiation of FliK(I304pBPA) reproducibly produced a ca. 53-kDa photo-cross-linked product with FlhB_CC when coexpressing with wild-type FlhB_C (Fig. 1, lane 2). Photo-cross-linked products were also seen when FliK(I304pBPA) was coexpressed with FlhB_C(N269A) and FlhB_C(P270A) (Fig. 1, lanes 4 and 6). In contrast to wild-type FlhB_C, which is cleaved into FlhB_CN and FlhB_CC polypeptides in an autocatalytic manner, FlhB_C(N269A) and FlhB_C(P270A) do not undergo autocleavage. As a result, the molecular mass of the FliK-FlhB_C heterodimer is estimated to be ca. 64 kDa, which is the sum of the molecular masses of FliK (42 kDa) and noncleaved FlhB_C (22 kDa), respectively. This mass is almost the same as that of the cross-linked products detected on immunoblots. Therefore, we suggest that the flhB(N269A) and flhB(P270A) mutations do not interfere with the interaction of FlhB_C with FliK.

FIG 1.

Effect of the flhB(N269A) and flhB(P270A) mutations on photo-cross-linking between FliK_C and FlhB_C. E. coli BL21 Star(DE3) cells coexpressing FliK(I304pBPA) with FlhB_C, FlhB_C(N269A), or FlhB_C(P270A) were UV irradiated for 5 min (+) or not irradiated (–) and then analyzed by immunoblotting with polyclonal anti-FliK antibody. Photo-cross-linked FliK-FlhB_C products are indicated by red dots. The position of free FliK is indicated by an arrow. Molecular mass markers (in kilodaltons) are shown on the left.

Effect of the flhB(N269A) and flhB(P270A) mutations on hook length.

The flhB(N269A) and flhB(P270A) mutants produce polyhooks (10, 19). We found that the flhB(N269A) and flhB(P270A) mutations do not affect the interaction of FlhB_C with FliK, raising the question of whether the FliK-FlhB_C interaction influences the rate of hook assembly. To clarify this question, we purified polyhook-basal bodies from these two flhB mutants and measured the length of each polyhook. The average hook lengths of the flhB(N269A) and flhB(P270A) mutants were 127.4 ± 85.6 nm (n = 237) and 125.6 ± 77.9 nm (n = 251), respectively, which were much longer than the wild-type length with a wide size distribution (53.5 ± 7.5 nm, n = 103) but shorter than the length of the polyhook produced by the fliK null mutant (418.5 ± 228.5 nm, n = 332) (Fig. 2). When a ΔfliK::tetRA allele was introduced into the flhB(N269A) and flhB(P270A) mutants by P22-mediated transduction, the average lengths of the polyhooks produced by the flhB(N269A) ΔfliK::tetRA and flhB(P270A) ΔfliK::tetRA mutants were 331 ± 198.9 nm (n = 316) and 319.1 ± 169.9 nm (n = 318) (Fig. 2). These results suggest that the FliK-FlhBc interaction reduces the hook polymerization rate significantly.

FIG 2.

Effect of flhB(N269A) and flhB(P270A) mutations on hook length control. Electron micrograms of hook-basal bodies isolated from SJW1103 (wild type, indicated as WT), TH13763 [flhB(N269A), indicated as N269A], TH12499 [flhB(P270A), indicated as P270A], MM1103iK (ΔfliK), TH13763iK (N269A ΔfliK), and TH12499iK (P270A ΔfliK) are shown. The average hook length and standard deviations are shown. N, number of hook-basal bodies and polyhook-basal bodies, which were measured. Scale bars, 100 nm.

Isolation of pseudorevertants form the flhB(P270A) mutant.

To clarify the role of autocleavage of FlhB_C on substrate specificity switching of the flagellar type III protein export apparatus, we isolated five pseudorevertants from the flhB(P270A) mutant. The motility of these pseudorevertants was better than that of their flhB(P270A) mutant, although it was not as good as that of the wild-type strain (Fig. 3A). P22-mediated genetic mapping showed that the gain-of-function mutations were located within the flhBAE operon. DNA sequencing identified two missense mutations, T268I (isolated two times) and E314A (isolated two times) in FlhB_C, and a missense mutation, A489E, in FlhA_C (Fig. 3B).

FIG 3.

Isolation of pseudorevertants from the flhB(P270A) mutant. (A) Motilities of SJW1103 (WT), TH12499 (P270A), MMB12499-SP1 [flhB(P270A/T268I), indicated as P270A/T268I], MMB12499-SP2 [flhB(P270A/E314A), indicated as P270A/E314A], and MMB12499-SP3 [flhB(P270A) flhA(A489E), indicated as P270A/FlhA(A489E)] in soft agar. Plates were incubated at 30°C for 7.5 h. (B) Location of intragenic flhB(T268I) and flhB(E314A) suppressor mutations in the crystal structure of FlhB_C (PDB ID 3B0Z) and an extragenic flhA(A489E) suppressor mutation in the crystal structure of FlhA_C (PDB ID 3A5I). FlhB_C undergoes autocatalytic cleavage between Asn-269 and Pro-270 residues and so is composed of two distinct FlhB_CN (magenta) and FlhB_CC (rainbow) polypeptides. Well-conserved Asp-456, Phe-459, and Thr-490 residues of FlhA form part of the flagellar chaperone-binding site. The C-α backbone is color-coded from blue to red, going through the rainbow colors from the N terminus to the C terminus. (C) Electron micrograms of hook-basal bodies and polyhook-basal bodies isolated from the MMB12499-SP2 and MMB12499-SP3 strains. The average hook lengths and standard deviations are shown. Scale bars, 100 nm. (D) Secretion assays of FlgD, FlgE, FlgK, and FliC. Immunoblotting using polyclonal anti-FlgD (first row), anti-FlgE (second row), anti-FlgK (third row), or anti-FliC (fourth row) antibody of whole-cell proteins (Cell) and culture supernatants (Sup) from the strains described above. Arrowheads indicate positions of FlgD, FlgE, FlgK, and FliC. The positions of molecular mass markers (in kilodaltons) are given on the left. (E) Effect of flhB(T268I) and flhB(E314A) suppressor mutations on autocleavage of FlhB_C(P270A). Coomassie blue-stained SDS-PAGE gels of whole-cell proteins prepared from E. coli BL21 Star(DE3) cells transformed with pET19b (V), pYI124 (N-terminally His-tagged FlhB_C, indicated as WT), pYI140 [His-FlhB_C(P270A), indicated as P270A], pYI140-SP1 [His-FlhB_C(P270A/T268I), indicated as P270A/T268I], or pYI140-SP2 [His-FlhB_C(P270A/E314A), indicated as P270A/E314A] are shown. The positions of the intact form and the cleaved His-FlhB_CN and FlhB_CC polypeptides are indicated by arrowheads.

To test whether these suppressor mutations shorten the length of the polyhooks produced by the flhB(P270A) mutant, we isolated flagella from the flhB(P270A/E314A) and flhB(P270A) flhA(A489E) suppressor mutants and measured the hook length. The hook lengths of the flhB(P270A/E314A) and flhB(P270A) flhA(A489E) suppressor mutants were 97.7 ± 65.5 nm (n = 129) and 102.6 ± 62.8 nm (n = 138) (Fig. 3C) compared to 53.5 ± 7.5 nm (n = 103) for wild-type cells and 125.6 ± 77.9 nm (n = 251) for the flhB(P270A) cells (Fig. 2). This indicates that these two suppressor mutations slightly shorten the polyhook length. This suggests that these suppressor mutants still cannot properly terminate the export of FlgE. Consistently, there was no significant difference in the secretion levels of hook-type proteins such as FlgE and FlgD between the flhB(P270A) mutant and the suppressor mutants (Fig. 3D, first and second rows).

To investigate the export-switching efficiency of the flhB(P270A/T268I), flhB(P270A/E314A), and flhB(P270A) flhA(A489E) suppressor mutants, we analyzed the secretion levels of FlgK and FliC by immunoblotting with polyclonal anti-FlgK and anti-FliC antibodies, respectively. The levels of FlgK and FliC secreted by these suppressor mutants were higher than those seen in their parent flhB(P270A) mutant and were almost the same as the wild-type levels (Fig. 3D, third and fourth rows). These results suggest that the flhB(T268I), flhB(E314A), and flhA(A489E) suppressor mutations allow the flhB(P270A) mutant to initiate the assembly of the filament efficiently.

We tested whether intragenic flhB(T268I) and flhB(E314A) suppressor mutations promote autocleavage of FlhB_C(P270A). Only the intact form of FlhB_C was detected in whole-cell lysates prepared from E. coli BL21 Star(DE3) cells overexpressing His-FlhB_C(P270A) (Fig. 3E). In contrast, significant amounts of cleaved forms of FlhB_C were detected in BL21 Star(DE3) cells overexpressing His-FlhB_C(P270A/T268I) or His-FlhB_C(P270A/E314A) in a way similar to wild-type FlhB_C although their intact forms were also seen (Fig. 3E). These results indicate that the intragenic flhB(T268I) and flhB(E314A) suppressor mutations induce a conformational change of the NPTH loop, allowing FlhB_C to undergo autocatalytic cleavage into two distinct FlhB_CN and FlhB_CC polypeptides. Therefore, we conclude that the autocleavage of FlhB_C is required not only for efficient termination of hook-type protein export but also for the efficient export of filament-type proteins.

Effect of second-site flhB and flhA mutations on motility.

To test whether second-site flhB and flhA mutations by themselves affect the export-switching function of the flagellar type III protein export apparatus, we introduced these second-site mutations into wild-type FlhB and FlhA by site-directed mutagenesis and analyzed their motility. The motilities of the flhB(T268A), flhB(E314A), and flhA(E314A) mutants were almost the same as that of wild-type cells (see Fig. S1A and B in the supplemental material), indicating that these second-site mutations display no significant motility phenotype.

Effect of FliK defect on the export-switching function of the flagellar type III protein export apparatus.

To test whether the export-switching function of the flhB(P270A/T268I), flhB(P270A/E314A), and flhB(P270A) flhA(A489E) suppressor mutants is dependent on FliK, we introduced a ΔfliK::tetRA allele into these three suppressor mutants by P22-mediated transduction and analyzed the secretion levels of FlgE and FliC. A deletion of the fliK gene totally inhibited the export of FliC but not that of FlgE (Fig. S2). This indicates that these suppressor mutations do not induce autonomous export switching of the flagellar type III protein export apparatus in the absence of FliK.

Effect of the flhA(A489E) mutation on the interaction of FlhA_C with flagellar chaperones in complex with their cognate substrates.

Asp-456, Phe-459, and Thr-490 of FlhA are directly involved in the interaction of the FlgN, FliS, and FliT chaperones in complex with their cognate export substrates, FlgK and FlgL, FliC, and FliD, respectively (24–26). The flhA(A489E) mutation is located in the chaperone-binding site of FlhA_C (Fig. 3B), raising the question of whether the flhA(A489E) suppressor mutation affects the interaction of FlhA_C with the chaperone/filament-type export substrate complexes. To clarify this question, we carried out pulldown assays by glutathione S-transferase (GST) affinity chromatography. FlhA_C coeluted with the GST-FlgN/FlgK and GST-FliS/FliC complexes from a GST column (Fig. 4A, second and fourth rows) but not with GST alone (first row), in agreement with previous reports (24, 25). However, the flhA(A489E) mutation reduced its binding affinities for the FlgN/FlgK and FliS/FliC complexes (third and fifth rows).

FIG 4.

Effect of flhA(A489E) mutation on the export of filament-type proteins. (A) Effect of the flhA(A489E) mutation on the interaction of FlhA_C with the FlgN/FlgK and FliS/FliC complexes. Mixtures (L) of purified His-FlhA_C or His-FlhA_C(A489E) with GST (first row), GST-FlgN/FlgK (second and third rows), or GST-FliS/FliC (fourth and fifth rows) were purified by GST affinity chromatography. The flowthrough fraction (F.T.), wash fractions, and elution fractions were analyzed by Coomassie brilliant blue staining. Molecular mass markers (in kilodaltons) are given on the left. (B) Secretion assays of FlgD and FliC. Immunoblotting with polyclonal anti-FlgD (first row) or anti-FliC (second row) antibody of whole-cell proteins (Cell) and culture supernatants (Sup) prepared from the Salmonella NH001 strain carrying pTrc99AFF4 (indicated as ΔflhA), pMM130 (indicated as WT), or pYI130-SP3 [indicated as flhA(A489E)] and the Salmonella NH004 strain (ΔfliHI flhB* ΔflhA) transformed with pTrc99AFF4 (indicated as ΔfliHI flhB* ΔflhA), pMM130 (indicated as ΔfliHI flhB*), or pYI130-SP3 [indicated as ΔfliHI flhB* flhA(A489E)] was performed. Arrowheads indicate the positions of FlgD and FliC.

FlhA requires the support of FliH and FliI to fully exert its export function (30–32). When FliH and FliI are missing, much larger amounts of FliC molecules are leaked out into the culture media because the hook-filament junction structure is not formed at the hook tip properly by FlgK and FlgL (32). Although the flhA(A489E) mutation reduced the binding affinities of FlhA_C for the FlgN/FlgK and FliS/FliC complexes, the flhA(A489E) mutation increased the secretion levels of FlgK and FliC significantly even in the presence of the fllhB(P270A) mutation, raising the possibility that FlhA(A489E) requires FliH and FliI to exert its export function. Therefore, we analyzed the effect of the flhA(A489E) mutation on the filament-type protein export in a ΔfliH-fliI flhB(P28T) (ΔfliH-fliI flhB*) mutant background, of which second-site flhB(P28T) mutation considerably increases the probability of flagellar formation in the absence of FliH and FliI (33). The motility of the ΔfliH-fliI flhB(P28T) flhA(A489E) mutant was worse than that of the ΔfliH-fliI flhB(P28T) mutant (Fig. S1C). Consistently, the secretion level of FliC was lower in the ΔfliH-fliI flhB(P28T) flhA(A489E) mutant than in the ΔfliH-fliI flhB(P28T) mutant (Fig. 4B, right panel, second row). However, there was no difference in the secretion level of FlgD between these two mutant strains (Fig. 4B, right panel, first row). When FliH and FliI were present, the flhA(A489E) mutation does not affect the secretion level of FliC at all (Fig. 4C, left panel, second row). These results indicate that FlhA(A489E) requires FliH and FliI to efficiently promote the export of filament-type proteins upon completion of hook assembly. Because the flagellar chaperones in complex with their cognate filament-type substrates bind to the open form of FlhA_C in the FlhA_C ring structure but not to the closed form (26, 28), we propose that the FlhA_C(A489E) ring structure may adopt the open form to allow the flagellar type III protein export apparatus to promote the export of filament-type proteins in the presence of the flhB(P270A) mutation.

Effect of flhB(G293R), flhB(G293V), and flhB(A298V) mutations on export switching.

The flhB(G293R), flhB(G293V), and flhB(A298V) mutations in FlhB_CC were originally isolated as extragenic fliK suppressor mutations to support filament assembly even in the absence of FliK (13). Motility of the flhB(G293R), flhB(G293V), and flhB(A298V) mutants is worse than the wild-type level and much better than that of the flhB(N269A) and flhB(P270A) mutants (Fig. 5A). The flhB(G293R), flhB(G293V), and flhB(A298V) mutations significantly affect autocleavage of FlhB_C (16), raising a question about the impact of these flhB mutations on the export switching of the flagellar type III protein export apparatus. To clarify this question, we analyzed the levels of filament-type proteins secreted by the flhB(G293R), flhB(G293V), and flhB(A298V) mutants. These three flhB mutations reduced the secretion levels of FlgK and FliC by approximately 60% of the wild-type levels (Fig. 5B), confirming that FlhB_C cleavage is required for the efficient export of filament-type proteins.

FIG 5.

Effect of flhB(G293R), flhB(G293V), and flhB(A298V) mutations on substrate specificity switching of the flagellar type III protein export apparatus. (A) Motility of SJW1103 (WT), MKM50 (ΔflhB), MMB2714 [flhB(G293R), indicated as G293R], MMB3201 [flhB(G293V), indicated as G293V], MMB2701 [flhB(A298V), indicated as A298V], TH13763 [flhB(N269A), indicated as N269A], and TH12499 [flhB(P270A), indicated as P270A] in soft agar. Plates were incubated at 30°C for 6 h. (B) Secretion assays of FlgE, FlgK, and FliC. Immunoblotting with polyclonal anti-FlgE (first row), anti-FlgK (second row), or anti-FliC (third row) antibody of whole-cell proteins (Cell) and culture supernatants (Sup) prepared from the above strains was performed. Arrowheads indicate the positions of FlgE, FlgK, and FliC. Molecular mass markers (in kilodaltons) are given on the left. (C) Electron micrograms of hook-basal bodies and polyhook-basal bodies isolated from the MMB2714, MMB3201, and MMB2701 strains. The average hook length and standard deviations are shown. Scale bars, 100 nm.

To investigate whether the flhB(G293R), flhB(G293V), and flhB(A298V) mutations affect hook length control, we prepared hook-basal bodies from these flhB mutants and measured their hook length. The average hook lengths of the flhB(G293R) and flhB(A298V) mutants were 63.4 ± 20.2 nm (n = 107) and 66.4 ± 28.1 nm (n = 128) (Fig. 5C) compared to (53.5 ± 7.5 nm, n = 103) for the wild-type (Fig. 2), suggesting that the flhB(G293R) and flhB(A298V) mutants cannot terminate the export of FlgE at an appropriate timing of hook assembly, thereby causing a loose hook length control. Consistently, the levels of FlgE secreted by the flhB(G293R) and flhB(A298V) mutants were about 1.4-fold higher than the wild-type level (Fig. 5B). In contrast, hook length control was not affected by the flhB(G293V) mutation (Fig. 5C), suggesting that the flhB(G293V) mutation affects the initiation of filament formation but not the termination of hook assembly.

DISCUSSION

An interaction between FliK_C and FlhB_C triggers substrate specificity switching of the flagellar type III protein export apparatus from the hook protein to the filament protein when the length of the hook has reached about 55 nm in Salmonella (4). Autocatalytic cleavage between Asn-269 and Pro-270 within the conserved NPTH loop occurs through a chemical reaction involving cyclization of Asp-269 (20, 34). A conformational change of FlhB_CC through a remodeling of hydrophobic interaction networks in FlhB_CC is postulated to be responsible for the substrate specificity switching (35). However, little is known about the role of the autocleavage of FlhB_C in the export-switching mechanism. Here, we showed that the flhB(N269A) and flhB(P270A) mutations did not affect the interaction of FlhB_C with FliK_C (Fig. 1). We also found that the lengths of polyhooks produced by the flhB(N269A) and flhB(P270A) mutants were shorter than that of the fliK null mutant (Fig. 2). Introduction of the fliK null mutation into these two flhB mutants increased the length of the polyhook considerably (Fig. 2). Therefore, we propose that the binding of FliK_C to FlhB_C induces conformational rearrangements of FlhB_CC to terminate the export of hook-type proteins and that the autocleavage of FlhB_C is required for proper conformational changes of FlhB_C in a FliK-dependent manner.

The C-terminal cytoplasmic domain EscU (EscU_C), which is a FlhB homologue of the injectisome of pathogenic E. coli, undergoes autocleavage into EscU_CN and EscU_CC in a way similar to FlhB_C (34). Structural comparison between EscU_C and EscU_C(P263A) has revealed that the escU(P263A) mutation does not induce a large conformational change of the entire EscU_C structure (Fig. S3A) (34). However, Ala-263 makes hydrophobic contacts with Lys-261, Asn-262, and Glu-307, whereas Pro-263 does not (see Fig. S3A in the supplemental material), suggesting that the conformational flexibility of the conserved NPTH loop connecting between EscU_CN and EscU_CC could be restricted by a replacement of Pro-270 by Ala, thereby suppressing the autocleavage of EscU_C. Here, we found that the intragenic flhB(T268I) and flhB(E314A) suppressor mutations facilitated the autocleavage of FlhB_C(P270A) to a significant degree (Fig. 3E). Thr-268 and Glu-314 of FlhB_C correspond to Lys-261 and Glu-307 of EscU_C, suggesting that the flhB(T268I) and flhB(E314A) suppressor mutations affect hydrophobic interactions of Ala-270 with Thr-268, Asn-269, and Glu-307 to allow Asn-269 to be exposed to solvent to induce the autocleavage of FlhB_C (Fig. 3B). Recently, it has been shown that the flhB(R320A) mutation affecting hydrophobic interaction networks in FlhB_CC inhibits the autocleavage of FlhB_C significantly, causing a loose hook length control (35). The flhB(G293R), flhB(G293V), and flhB(A298V) mutations, which reduce the rate of the autocleavage process of FlhB_C considerably (16), seem to affect hydrophobic interaction networks in FlhB_CC in a way similar to the flhB(R320A) mutation (Fig. S3B). The flhB(293R), flhB(G293V), and flhB(A298V) mutations reduced the secretion levels of filament-type proteins (Fig. 5B). Interestingly, the flhB(G293R) and flhB(A298V) mutants produced longer hooks compared to wild-type cells (Fig. 5C). Because we found that the flhB(T268I) and flhB(E314A) suppressor mutations significantly increased the probability of filament assembly in the flhB(P270A) mutant (Fig. 3), we propose that the autocleavage of FlhB_C induces a conformational flexibility of FlhB_CN-FlhB_CC boundary and that such a flexibility is important not only for the initiation of the export of filament-type proteins but also for the termination of the export of hook-type proteins at an appropriate timing of hook assembly.

How does the FliK_C-FlhB_C interaction terminate the export of hook-type proteins? The C-terminal domain of YscP (YscP_C), which is a FliK homologue of the Yersinia injectisome, directly binds to the C-terminal domain of YscU (YscU_C), which is a FlhB homologue. Ala-335 of YscU_C, which corresponds to Ala-341 of FlhB_C, is critical for the binding to YscP_C, and Leu-280 of YscU_C, which corresponds to Ala-286 of FlhB_C, contributes to this binding (36). Ala-286, Pro-287, Ala-341, and Leu-344 form a well-conserved hydrophobic patch on the molecular surface of FlhB_CC, and this hydrophobic patch is directly involved in the recognition of the export signals of hook-type proteins (18). Therefore, we propose that the binding of FliK_C to FlhB_CC may induce a conformational change of the hydrophobic patch, thereby terminating the export of the hook-type proteins.

Flk, which is a transmembrane protein with a large cytoplasmic domain, interferes with premature switching of the substrate specificity of the flagellar type III protein export apparatus during hook-basal body assembly, and this inhibitory effect of Flk on the export of filament-type proteins is released after completion of hook assembly (9, 37–39). The flhB(N269A) and flhB(P270A) mutations interfere with the export of filament-type proteins (19), raising the possibility that these two mutations affect the negative impact of Flk on filament-type protein export. A disruption of the flk gene significantly increased the secretion level of FliC in the flgE mutant (Fig. S4), in agreement with a previous report (9). However, deletion of the flk gene did not increase the secretion level of FliC in the flhB(N269A) and flhB(P270A) mutants (Fig. S4), suggesting that the inability of FlhB(N269A) and FlhB(P270A) to switch export specificity does not result from failure to release the inhibitory effect of Flk on the switching. Therefore, we conclude that the autocleavage of FlhB_C is critical for the flagellar type III protein export apparatus to promote the initiation of filament-type protein export.

Asp-456, Phe-459, and Thr-490 of FlhA_C are involved in the interactions with the FlgN, FliS, and FliT chaperones in complex with their cognate substrates (Fig. 3B) (24–26). FlhA_C adopts two distinct, open and closed conformations (40, 41), and the flagellar chaperones in complex with their cognate substrates bind to the open form of FlhA_C but not to the closed form (26, 28). Interactions of FlhA_L with its neighboring FlhA_C subunit convert the entire FlhA_C ring structure from a closed to an open form to facilitate the export of filament-type proteins (27, 28). Although it has been shown FlhB_C binds to FlhA_C with a micromolar affinity in solution (42), it remained unknown how FlhB_C could induce a conformational change of FlhA_C and promote filament-type protein export upon completion of hook assembly. Here, we found that the extragenic flhA(A489E) suppressor mutation facilitated the export of filament-type proteins even in the presence of the flhB(P270A) mutation (Fig. 3D). Interestingly, the flhA(A489E) mutation is located in the chaperone-binding site of FlhA_C (Fig. 3B). Pulldown assays by GST affinity chromatography revealed that the flhA(A489E) mutation reduced the binding affinities of FlhA_C for the FlgN/FlgK and FliS/FliC complexes (Fig. 4A). However, FliH and FliI overcame the defect of FlhA_C(A489E) in the interaction with these chaperone/substrate complexes (Fig. 4B), thereby allowing the flhB(P270A) flhA(A489E) mutant to transport FlgK and FliC at wild-type levels (Fig. 3D). Therefore, we suggest that the flhA(A489E) mutation allows FlhA_C to adopt a certain conformation mimicking a chaperone-bound state of FlhA_C even in the presence of the flhB(P270A) mutation. Introduction of the fliK null mutation into the flhB(P270A) flhA(A489E) suppressor mutant abolished the export of FliC but not that of FlgE (Fig. S2), suggesting that FliK is required for conformational rearrangements of the FlhA_C(A489E) ring structure to initiate the export of filament-type proteins. Therefore, we propose that the binding of FliK_C to a cleaved form of FlhB_C may promote the FlhB_C-FlhA_C interaction to allow the FlhA_C ring structure to undergo a structural transition from a closed to an open form, thereby promoting efficient docking of flagellar chaperones in complex with their cognate substrates to the FlhA_C ring structure to facilitate subsequent export of filament-type proteins (Fig. 6).

FIG 6.

Model for export switching of the flagellar type III protein export apparatus. During hook assembly, the flagellar type III protein export apparatus transports the hook protein (FlgE) from the cytoplasm to the distal end of the nascent flagellar structure. The export apparatus also secretes the FliK ruler to measure the hook length during hook assembly. FlhB_CC provides a binding site for FlgE and FliK for their efficient export. When the hook length has reached about 55 nm, the C-terminal domain of FliK (FliK_C) binds to FlhB_CC to induce a conformational change of FlhB_C, thereby terminating the export of FlgE. Then, cleaved FlhB_C binds to FlhA_C to induce a structural remodeling of the FlhA_C ring structure, allowing the FlgN, FliT, and FliS chaperones in complex with their cognate substrates to bind to the FlhA_C ring to initiate the export of filament-type proteins to form the filament at the hook tip.

MATERIALS AND METHODS

Bacterial strains, plasmids, transduction, and media.

Bacterial strains and plasmids used in this study are listed in Table 1 . P22-mediated transduction was carried out as described previously (43). L broth and 0.35% soft agar plates were prepared as described previously (44, 45). Ampicillin was added at a final concentration of 100 μg/ml if needed.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid	Relevant characteristics	Source or reference
Strains
E. coli BL21 Star(DE3)	Overexpression of proteins	Novagen
Salmonella
SJW1103	Wild type for motility and chemotaxis	43
SJW1103RflH	Δflk::tetRA	9
MKM50	ΔflhB	19
MMB2701	flhB(A298V)	20
MMB2714	flhB(G293R)	20
MMB3201	flhB(G293V)	20
MM1103iK	ΔfliK::tetRA	This study
NH001	ΔflhA	30
NH004	ΔfliH-fliI flhB(P28T) ΔflhA	30
NME001	ΔflgE	9
NME001RflH	ΔflgE Δflk::tetRA	9
TH8426	ΔfliK	14
TH8426RflH	ΔfliK Δflk::tetRA	9
TH13763	flhB(N269A)	Kelly T. Hughes
TH13763iK	flhB(N269A) ΔfliK::tetRA	This study
TH13763RflH	flhB(N269A) Δflk::tetRA	This study
TH12499	flhB(P270A)	Kelly T. Hughes
TH12499iK	flhB(P270A) ΔfliK::tetRA	This study
TH12499RflH	flhB(P270A) Δflk::tetRA	This study
MMB12499-SP1	flhB(P270A/T268I)	This study
MMB12499-SP1iK	flhB(P270A/T268I) ΔfliK::tetRA	This study
MMB12499-SP2	flhB(P270A/E314A)	This study
MMB12499-SP2iK	flhB(P270A/E314A) ΔfliK::tetRA	This study
MMA12499-SP3	flhB(P270A) flhA(A489E)	This study
MMA12499-SP3iK	flhB(P270A) flhA(A489E) ΔfliK::tetRA	This study
Plasmids
pTrc99AFF4	Modified pTrc expression vector	48
pEVOL	For incorporation of pBPA into the amber codon	29
pGEX-6p-1	Expression vector	GE Healthcare
pMKGK2	pTrc99A/FlgK	49
pMKM1002	pGEX-6p-1/GST-FliS	25
pMM104	pET19b/His-FlhAC (residues 211 to 692)	45
pMM130	pTrc99AFF4/FlhA	50
pMMGN101	pGEX-6p-1/GST-FlgN	24
pMMK521	pETDuet-1/FliK(I304amber) + FlhBC	15
pMMK536	pETDuet-1/FliK(I304amber) + FlhBC(N269A)	This study
pMMK537	pETDuet-1/FliK(I304amber) + FlhBC(P270A)	This study
pYI101	pTrc99AFF4/FlhB	35
pYI101-SP1	pTrc99AFF4/FlhB(T268I)	This study
pYI101-SP2	pTrc99AFF4/FlhB(E314A)	This study
pYI124	pET19b/His-FlhBC (residues 211 to 383)	35
pYI140	pET19b/His-FlhBC(P270A)	35
pYI140-SP1	pET19b/His-FlhBC(P270A/T268I)	This study
pYI140-SP2	pET19b/His-FlhBC(P270A/E314A)	This study
pYI130-SP3	pTrc99A/FlhA(A489E)	This study
pYI104-SP3	pET19b/His-FlhAC(A489E)	This study

Site-directed mutagenesis.

Site-directed mutagenesis was carried out using the QuikChange site-directed mutagenesis method (Stratagene). DNA sequencing reactions were carried out using BigDye v3.1 (Applied Biosystems); the reaction mixtures were analyzed by a 3130 Genetic Analyzer (Applied Biosystems).

Photo-cross-linking.

E. coli BL21 Star(DE3) cells harboring pEVOL (29) and a pETDuet-based plasmid encoding both FliK(I304amber) and wild-type FlhB_C, FlhB_C(N269A), or FlhB_C(P270A) were exponentially grown at 30°C in L broth containing 1 mM p-benzoyl-phenylalanine (pBPA) and 100 μg/ml ampicillin. Then, 100 μM IPTG (isopropyl-β-d-thiogalactopyranoside) and 0.02% arabinose were added, and the incubation was continued until the culture density had reached an optical density at 600 nm (OD₆₀₀) of ca. 1.4 to 1.5. Photo-cross-linking experiments were performed as described previously (46). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting with polyclonal anti-FliK antibody was carried out as described previously (44). Detection was done with an ECL Prime Western blotting detection reagent (GE Healthcare). Chemiluminescence signals were captured by a Luminoimage analyzer LAS-3000 (GE Healthcare). At least three independent experiments were performed.

Electron microscopy.

Salmonella cells were grown in L broth at 30°C with shaking until the cell density had reached an OD₆₀₀ of ca. 1.0 to 1.3. The flagella were isolated by 20 to 50% (wt/wt) sucrose density gradient ultracentrifugation as described previously (32). After ultracentrifugation at 60,000 × g for 60 min, the pellets were suspended in 50 mM glycine (pH 2.5) and 0.1% Triton X-100, followed by incubation at room temperature for 30 min to depolymerize flagellar filaments. After ultracentrifugation, the pellets were resuspended in 50 μl of 10 mM Tris-HCl (pH 8.0), 5 mM EDTA, and 0.1% Triton X-100. Samples were negatively stained with 2% (wt/vol) uranyl acetate. Electron micrograms were recorded with a JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan) operated at 100 kV and equipped with a F415 charge-coupled device camera (TVIPS, Gauting, Germany). Hook length was measured by ImageJ, version 1.48 (National Institutes of Health).

Motility assay.

Fresh colonies were inoculated onto 0.35% soft agar plates and incubated at 30°C. At least six independent measurements were carried out.

Flagellar protein export assay.

Details of sample preparations have been described previously (47). Both whole-cell proteins and culture supernatants were normalized to a cell density of each culture to give a constant number of Salmonella cells. After heating at 95°C for 3 min, these protein samples were assessed by SDS-PAGE, followed by immunoblotting with polyclonal anti-FlgD, anti-FlgE, anti-FlgK, or anti-FliC antibody. More than five independent experiments were carried out.

Preparation of whole-cell proteins.

E. coli BL21 Star(DE3) cells harboring a pET19b-based plasmid encoding N-terminally His-tagged FlhB_C or its mutant variants were grown overnight in 5 ml of L broth containing ampicillin at 30°C with shaking. Then, 500 μl of each culture was transferred into a 1.5-ml Eppendorf tube. After centrifugation at 20,000 × g for 5 min, each cell pellet was suspended in 1× SDS loading buffer (the amount of loading buffer was equal to OD₆₀₀ × 250 μl) containing 1 μl of 2-mercaptoethanol and then heated at 95°C for 3 min. Proteins in whole-cell lysates were separated by SDS-PAGE, followed by Coomassie blue staining and immunoblotting with polyclonal anti-FlhB_C antibody.

Pulldown assays by GST chromatography.

Detailed protocols for pulldown assays by GST affinity chromatography have been described previously (25). At least three independent experiments were carried out.