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.     

Self-assembly and type III protein export of the bacterial flagellum

The bacterial flagellum is a supramolecular structure consisting of a basal body, a hook and a filament. Most of the flagellar components are translocated across the cytoplasmic membrane by the flagellar type III protein export apparatus in the vicinity of the flagellar base, diffuse down the narrow channel through the nascent structure and self-assemble at its distal end with the help of a cap structure. Flagellar proteins synthesized in the cytoplasm are targeted to the export apparatus with the help of flagellum-specific chaperones and pushed into the channel by an ATPase, whose activity is controlled by its regulator to enable the energy of ATP hydrolysis to be efficiently coupled to the translocation reaction. The export apparatus switches its substrate specificity by monitoring the state of flagellar assembly in the cell exterior, allowing this huge and complex macromolecular assembly to be built efficiently by a highly ordered and well-regulated assembly process.     

Flipping the switch: bringing order to flagellar assembly

The bacterial flagellum is a complex self-assembling nanomachine that contains its own type III protein export apparatus. Upon completion of early flagellar structure, this apparatus switches substrate specificity to export late structural subunits, thereby coupling sequential flagellar gene expression with flagellar assembly. The switch is achieved by a conformational change of the export apparatus component FlhB driven by the flagellar hook-length control protein FliK. Two basic models of FliK- and FlhB-based switching are currently being pursued, together with the investigation of another factor, Flk, which prevents premature export of late substrates. Here, we review in detail each of these three export switch components and present the current understanding of how they work in concert in the making of a flagellum.     

Mechanisms of type III protein export for bacterial flagellar assembly

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

Molecular motors of the bacterial flagella

The bacterial flagellum, which is responsible for motility, is a biological nanomachine consisting of a reversible rotary motor, a universal joint, a helical screw, and a protein export apparatus dedicated for flagellar assembly. The motor is fueled by an inward-directed electrochemical gradient of protons or sodium ions across the cytoplasmic membrane. The motor consists of a rotor, a drive shaft, a bushing, and about a dozen stator units. The flagellar protein export apparatus is located at the cytoplasmic side of the rotor. Interactions between the rotor and the stators and those between soluble and membrane components of the export apparatus are highly dynamic. The structures of flagellar basal body components including those of the export apparatus, being revealed at high resolution by X-ray crystallography and electron cryomicroscopy and cryotomography, are giving insights into their mechanisms.     

Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis.

During flagellar morphogenesis in Salmonella typhimurium, the genes involved in filament assembly are expressed fully only after completion of hook-basal body assembly. This coupling of gene expression to morphogenesis is achieved by exporting the flagellum-specific anti-sigma factor, FlgM, out of the cell through the mature hook-basal body structure. Therefore, the flagellum-specific export apparatus must be able to sense the assembly state of the flagellar structure and to turn on FlgM export at a specific stage of hook assembly. It has been suggested that FlhB may act as the molecular switch which mediates this ordered export. Here, I report genetic evidence that in addition to FlhB, the product of a newly identified gene, rflH, is involved in the negative regulation of FlgM export. FlgM is released through the basal body structure lacking the hook and the filament only when the flhB and rflH genes are both defective. Therefore, the export gate for FlgM should be double locked by FlhB and RflH. The rflH gene is located at around 52 min, where no flagellum-related gene has been found. I propose a revised model of the export-switching machinery which consists of two systems, the hook-length signal transduction pathway and the double-locked gate for FlgM export.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.     

Assembly of the switch complex onto the MS ring complex of Salmonella typhimurium does not require any other flagellar proteins.

The cytoplasmic portion of the bacterial flagellum is thought to consist of at least two structural components: a switch complex and an export apparatus. These components seem to assemble around the MS ring complex, which is the first flagellar basal body substructure and is located in the cytoplasmic membrane. In order to elucidate the process of assembly of cytoplasmic substructures, the membrane localization of each component of the switch complex (FliG, FliM, and FliN) in various nonflagellated mutants was examined by immunoblotting. It was found that all these switch proteins require the MS ring protein FliF to associate with the cell membrane. FliG does not require FliM and FliN for this association, but FliM and FliN associate cooperatively with the membrane only through FliG. Furthermore, all three switch proteins were detected in membranes isolated from fliE, fliH, fliI, fliJ, fliO, fliP, fliQ, fliR, flhA, flhB, and flgJ mutants, indicating that the switch complex assembles on the MS ring complex without any other flagellar proteins involved in the early stage of flagellar assembly. The relationship between the switch complex and the export apparatus is discussed.     

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

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

FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly

In wild-type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at approximately 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild-type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook-filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N-terminal region of FliK, but not by C-terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook-length control and the switching of export substrate specificity.     

Targeting early proximal-rod component substrate FlgB to FlhB for flagellar-type III secretion in Salmonella

The Salmonella flagellar secretion apparatus is a member of the type III secretion (T3S) family of export systems in bacteria. After completion of the flagellar motor structure, the hook-basal body (HBB), the flagellar T3S system undergoes a switch from early to late substrate secretion, which results in the expression and assembly of the external, filament propeller-like structure. In order to characterize early substrate secretion-signals in the flagellar T3S system, the FlgB, and FlgC components of the flagellar rod, which acts as the drive-shaft within the HBB, were subject to deletion mutagenesis to identify regions of these proteins that were important for secretion. The β-lactamase protein lacking its Sec-dependent secretion signal (Bla) was fused to the C-terminus of FlgB and FlgC and used as a reporter to select for and quantify the secretion of FlgB and FlgC into the periplasm. Secretion of Bla into the periplasm confers resistance to ampicillin. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 of FlgB decreased FlgB secretion levels while deleting amino acid 6 through 14 diminished FlgC secretion levels. Further PCR-directed mutagenesis indicated that amino acid F45 of FlgB was critical for secretion. Single amino acid mutagenesis revealed that all amino acid substitutions at F45 of FlgB position impaired rod assembly, which was due to a defect of FlgB secretion. An equivalent F49 position in FlgC was essential for assembly but not for secretion. This study also revealed that a hydrophobic patch in the cleaved C-terminal domain of FlhB is critical for recognition of FlgB at F45.     

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.     

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

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

Flagellar assembly in Salmonella typhimurium

The bacterial flagellum is a motility apparatus in which a long helical filament - the propeller - is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed.     

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

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

Interactions between C ring proteins and export apparatus components: a possible mechanism for facilitating type III protein export

The flagellar switch proteins of Salmonella, FliG, FliM and FliN, participate in the switching of motor rotation, torque generation and flagellar assembly/export. FliN has been implicated in the flagellar export process. To address this possibility, we constructed 10-amino-acid scanning deletions and larger truncations over the C-terminal domain of FliN. Except for the last deletion variant, all other variants were unable to complement a fliN null strain or to restore the export of flagellar proteins. Most of the deletions showed strong negative dominance effects on wild-type cells. FliN was found to associate with FliH, a flagellar export component that regulates the ATPase activity of FliI. The binding of FliM to FliN does not interfere with this FliN-FliH interaction. Furthermore, a five-protein complex consisting of FliG, His-tagged FliM, FliN, FliH and FliI was purified by nickel-affinity chromatography. FliJ, a putative general chaperone, is bound to FliM even in the absence of FliH. The importance of the C ring as a possible docking site for export substrates, chaperones and FliI through FliH for their efficient delivery to membrane components of the export apparatus is discussed.     

Flagellar assembly in Salmonella typhimurium: analysis with temperature-sensitive mutants.

The process of flagellar assembly in Salmonella typhimurium was investigated by using temperature-sensitive mutants. The mutants were grown at the restrictive temperature and then at the permissive temperature, with radiolabel supplied in the first phase of the experiment and not the second, or vice versa. Flagellar hook-basal body complexes were then purified and analyzed by gel electrophoresis and autoradiography. The extent to which a given protein was labeled in the two phases of the experiment provided information as to whether it preceded or followed the block caused by the mutant protein. We conclude the following concerning flagellar assembly. The M-ring protein (FliF) is stably incorporated in the earliest stage detected, along with two previously unknown proteins, with apparent molecular masses of 23 and 26 kilodaltons, respectively, and possibly one of the switch components, FliG. Independent of that event and all other events, the P-ring and L-ring proteins (FlgI and FlgH) are synthesized and exported to the periplasm and outer membrane by the primary cellular export pathway. Rod assembly occurs by export (via the flagellum-specific pathway) of subunits of four proteins, FlgB, FlgC, FlgF, and FlgG, and their incorporation, probably in that order, into the rod structure; this stage requires the flhA and fliI genes, perhaps because they encode part of the export apparatus. Once rod assembly is complete, the FlgI and FlgH proteins assemble around the rod to form the P and L rings. The rod structure, which is only metastable while it is being constructed, becomes stable upon P-ring addition. Export (via the flagellum-specific pathway) and assembly of hook protein, hook-associated proteins, and filament protein then occur successively. A number of flagellar proteins, whose genetic origin and structural role are not yet known, were identified on the basis of their dependence on the flagellar master operon for expression.     

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.