The type III flagellar export specificity switch is dependent on FliK ruler and a molecular clock

Salmonella flagellar hook length is controlled at the level of export substrate specificity of the FlhB component of the type III flagellar export apparatus. FliK is believed to be the hook length sensor and interacts with FlhB to change its export specificity upon hook completion. To find properties of FliK expected of such a molecular ruler, we assayed binding of FliK to the hook and found that the N-terminal domain of FliK (FliK(N)) bound to the hook-capping protein FlgD with high affinity and to the hook protein FlgE with low affinity. To investigate a possible role of FlgE in hook length control, flgE mutants with partially impaired motility were isolated and analyzed. Eight flgE mutants obtained all formed flagellar filaments. The mutants produced significantly shorter hooks while the hook-type substrates such as FlgE, FliK and FlgD were secreted in large amounts, suggesting defective hook assembly with the mutant FlgE proteins. Upon overexpression, mutant FlgEs produced hooks of normal length and wild-type FlgE produced longer hooks. These results suggest that hook length is dependent on the hook polymerization rate and that the start of hook polymerization initiates a "time countdown" for the specificity switch to occur or for significant slow down of rod/hook-type export after hook length reaches around 55 nm for later infrequent FliK(C)-FlhB(C) interaction. We propose that FliK(N) acts as a flexible tape measure, but that hook length is also dependent on the hook elongation rate and a switch timing mechanism.     

The role of the FliK molecular ruler in hook‐length control in Salmonella enterica

A molecular ruler, FliK, controls the length of the flagellar hook. FliK measures hook length and catalyses the secretion‐substrate specificity switch from rod‐hook substrate specificity to late substrate secretion, which includes the filament subunits. Here, we show normal hook‐length control and filament assembly in the complete absence of the C‐ring thus refuting the previous ‘cup’ model for hook‐length control. Mutants of C‐ring components, which are reported to produce short hooks, show a reduced rate of hook–basal body assembly thereby allowing for a premature secretion‐substrate specificity switch. Unlike fliK null mutants, hook‐length control in an autocleavage‐defective mutant of flhB, the protein responsible for the switch to late substrate secretion, is completely abolished. FliK deletion variants that retain the ability to measure hook length are secreted thus demonstrating that FliK directly measures rod‐hook length during the secretion process. Finally, we present a unifying model accounting for all published data on hook‐length control in which FliK acts as a molecular ruler that takes measurements of rod‐hook length while being intermittently secreted during the assembly process of the hook–basal body complex.     

N-terminal signal region of FliK is dispensable for length control of the flagellar hook

The length of the flagellar hook is regulated; it is 55 +/- 6 nm long in Salmonella. Five genes involved in hook-length regulation are fliK, flhB, fliG, fliM and fliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented a fliK null (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system by the FliK C-terminal domain, and that the N-terminal region is dispensable for hook length control.     

An infrequent molecular ruler controls flagellar hook length in Salmonella enterica

The bacterial flagellum consists of a long external filament connected to a membrane-embedded basal body at the cell surface by a short curved structure called the hook. In Salmonella enterica, the hook extends 55 nm from the cell surface. FliK, a secreted molecular ruler, controls hook length. Upon hook completion, FliK induces a secretion-specificity switch to filament-type substrate secretion. Here, we demonstrate that an infrequent ruler mechanism determines hook length. FliK is intermittently secreted during hook polymerization. The probability of the specificity switch is an increasing function of hook length. By uncoupling hook polymerization from FliK expression, we illustrate that FliK secretion immediately triggers the specificity switch in hooks greater than the physiological length. The experimental data display excellent agreement with a mathematical model of the infrequent ruler hypothesis. Merodiploid bacteria expressing simultaneously short and long ruler variants displayed hook-length control by the short ruler, further supporting the infrequent ruler model. Finally, the velocity of FliK secretion determines the probability of a productive FliK interaction with the secretion apparatus to change secretion substrate specificity.     

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.     

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.     

MotD of Sinorhizobium meliloti and related alpha-proteobacteria is the flagellar-hook-length regulator and therefore reassigned as FliK

The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates.     

FliK regulates flagellar hook length as an internal ruler

The mechanism of length control of the flagellar hook is under debate between two theories. One claims that the FliK directly measures the hook length as a molecular ruler, while the other claims that the cytoplasmic substructure measures the amount of hook subunits to determine the hook length. Both agree that the FliK C-terminal domain catalyses the substrate-specificity switch to terminate hook elongation. In this study, we systematically created fliK mutants with deletions and insertions at various sites within the FliK N-terminal domain and analysed their effects on the final hook length. Insertions of peptide fragments from the Yersinia YscP into FliK gave rise to hooks with defined lengths, which was proportional to the molecular size of the FliK-YscP chimeras. Among fliK deletion mutants, only those with small truncations in three specific sites of FliK produced hooks of a defined, shortened length. For the majority of deletion mutants, FliK was secreted, but hook length was not controlled. On the other hand, for some deletion mutants FliK was not secreted, but the hook length was controlled, indicating that FliK secretion is not necessary for hook-length control. We conclude that FliK regulates hook length as an internal molecular ruler.     

Interaction of FliK with the bacterial flagellar hook is required for efficient export specificity switching

FliK–FlhB interaction switches export specificity of the bacterial flagellar protein export apparatus to stop hook protein export at an appropriate timing for hook length control. The hook structure is required for the productive FliK–FlhB interaction to flip the switch but it remains unknown how it works. Here, we characterize the role of FliK in the switching probability in the absence of the hook. When RflH/Flk was missing in the hook mutants, the switching occurred at a low probability. Overproduction of FliK significantly increased the switching probability although not at the wild-type level. An in-frame deletion of residues 129 through 159 of FliK weakened the interaction with the hook protein but not with the hook-capping protein, producing polyhooks with filaments attached. We suggest that temporary association of FliK with the inner surface of the hook during FliK secretion results in a pause in the secretion process to allow the C-terminal switch domain of FliK to be positioned and appropriately oriented near FlhB for catalysing the switch and that RflH/Flk interferes with premature switch by preventing access of cytoplasmic FliK to FlhB and even that of FliK during its secretion until hook length reaches 55 nm; only then FliK_C passes the RflH/Flk block.     

Domain organization and function of Salmonella FliK, a flagellar hook-length control protein

Salmonella hook-length control protein FliK, which consists of 405 amino acid residues, switches substrate specificity of the type III flagellar protein export apparatus from rod/ hook-type to filament-type by causing a conformational change in the cytoplasmic domain of FlhB (FlhB(C)) upon completion of the hook assembly. An N-terminal region of FliK contains an export signal, and a highly conserved C-terminal region consisting of amino acid residues 265-405 (FliK((265-405))) is directly involved in the switching of FlhB(C). Here, we have investigated the structural properties of FliK. Gel filtration chromatography, multi-angle light scattering and analytical ultracentrifugation showed that FliK is monomeric in solution and has an elongated shape. Limited proteolysis showed that FliK consists of two domains, the N-terminal (FliK(N)) and C-terminal domains (FliK(C)), and that the first 203 and the last 35 amino acid residues are partially unfolded and subjected to proteolysis. Both FliK(N) and FliK(C) are more globular than full-length FliK, suggesting that these domains are connected in tandem. Overproduced His-FliK((199-405)) failed to switch export specificity of the export apparatus. Affinity blotting revealed that FlhB(C) binds to FliK and FliK((1-147)), but not to FliK((265-405)). Based on these results, we propose that FliK(N) within the central channel of the hook-basal body during the export of FliK is the sensor and transmitter of hook completion information and that the binding interaction of FliK(C) to FlhB(C) is structurally regulated by FliK(N) so as to occur only when the hook has reached a preset length. The conformational flexibility of FliK(C) may play a role in interfering with switching at an inappropriate point of flagellar assembly.     

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.     

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.     

Molecular Characterization of the Flagellar Hook in Bacillus subtilis

The structure of the Gram-positive flagellum is poorly understood, and Bacillus subtilis encodes three proteins homologous to the flagellar hook protein from Salmonella enterica. Here we generated a modified B. subtilis hook protein that could be fluorescently stained using a cysteine-reactive dye. We used the fluorescently labeled hook to demonstrate that FlgE is the hook structural protein and that FliK regulated hook length. We further demonstrate that two proteins of unknown function, FlhO and FlhP, and the putative hook cap, FlgD, were required for hook assembly, such that when flhO, flhP, or flgD was mutated, hook protein was secreted into the supernatant. All mutants defective in hook completion resulted in homogeneously reduced σ(D)-dependent gene expression due to the action of the anti-sigma factor FlgM.     

Bringing order to a complex molecular machine: The assembly of the bacterial flagella

The bacterial flagellum is an example of elegance in molecular engineering. Flagella dependent motility is a widespread and evolutionarily ancient trait. Diverse bacterial species have evolved unique structural adaptations enabling them to migrate in their environmental niche. Variability exists in the number, location and configuration of flagella, and reflects unique adaptations of the microorganism. The most detailed analysis of flagellar morphogenesis and structure has focused on Escherichia coli and Salmonella enterica. The appendage assembles sequentially from the inner to the outer-most structures. Additionally the temporal order of gene expression correlates with the assembly order of encoded proteins into the final structure. The bacterial flagellar apparatus includes an essential basal body complex that comprises the export machinery required for assembly of the hook and flagellar filament. A review outlining the current understanding of the protein interactions that make up this remarkable structure will be presented, and the associated temporal genetic regulation will be briefly discussed.     

Robustness and the evolution of length control strategies in the T3SS and flagellar hook

Bacterial cells construct many structures, such as the flagellar hook and the type III secretion system (T3SS) injectisome, that aid in crucial physiological processes such as locomotion and pathogenesis. Both of these structures involve long extracellular channels, and the length of these channels must be highly regulated in order for these structures to perform their intended functions. There are two leading models for how length control is achieved in the flagellar hook and T3SS needle: the substrate switching model, in which the length is controlled by assembly of an inner rod, and the ruler model, in which a molecular ruler controls the length. Although there is qualitative experimental evidence to support both models, comparatively little has been done to quantitatively characterize these mechanisms or make detailed predictions that could be used to unambiguously test these mechanisms experimentally. In this work, we constructed a mathematical model of length control based on the ruler mechanism and found that the predictions of this model are consistent with experimental data—not just for the scaling of the average length with the ruler protein length, but also for the variance. Interestingly, we found that the ruler mechanism allows for the evolution of needles with large average lengths without the concomitant large increase in variance that occurs in the substrate switching mechanism. In addition to making further predictions that can be tested experimentally, these findings shed new light on the trade-offs that may have led to the evolution of different length control mechanisms in different bacterial species.     

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

The bacterial flagellar export switching machinery consists of a ruler protein, FliK, and an export switch protein, FlhB and switches substrate specificity of the flagellar type III export apparatus upon completion of hook assembly. An interaction between the C‐terminal domain of FliK (FliK_C) and the C‐terminal cytoplasmic domain of FlhB (FlhB_C) is postulated to be responsible for this switch. FliK_C has a compactly folded domain termed FliK_T3S4 (residues 268–352) and an intrinsically disordered region composed of the last 53 residues, FliK_CT (residues 353–405). Residues 301–350 of FliK_T3S4 and the last five residues of FliK_CT are critical for the switching function of FliK. FliK_CT is postulated to regulate the interaction of FliK_T3S4 with FlhB_C, but it remains unknown how. Here we report the role of FliK_CT in the export switching mechanism. Systematic deletion analyses of FliK_CT revealed that residues of 351–370 are responsible for efficient switching of substrate specificity of the export apparatus. Suppressor mutant analyses showed that FliK_CT coordinates FliK_T3S4 action with the switching. Site‐directed photo‐cross‐linking experiments showed that Val‐302 and Ile‐304 in the hydrophobic core of FliK_T3S4 bind to FlhB_C. We propose that FliK_CT may induce conformational rearrangements of FliK_T3S4 to bind to FlhB_C.     

The Flagellar Soluble Protein FliK Determines the Minimal Length of the Hook in Salmonella enterica Serovar Typhimurium

The length of the flagellar hook is controlled by the soluble protein FliK. FliK is structurally divided into two halves with distinct functions; the N-terminal half determines hook length, while the C-terminal half switches the secretion substrate specificity, consequently terminating hook elongation. FliK properly achieves both functions only when it is secreted. In a previous paper, we showed that a temperature-sensitive flgE mutant of Salmonella enterica serovar Typhimurium, SJW2219, produced basal bodies with short hooks (average length, 25 nm) at 37°C. In this study, we show that the mutant cells grown at 37°C secrete FliK but not flagellin (FliC), indicating that FliK is abortively secreted into the medium when the hook is shorter than 30 nm. In contrast, FliK unfailingly switches the gate modes when the hook is longer than 30 nm. Taking the FliC, FliK, and FlgM secretion patterns into account, we conclude that FliK determines the minimal length of the hook. We will discuss how FliK detects the critical switching point of the secretion gate.     

Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics

The bacterial flagellum contains its own type III secretion apparatus that coordinates protein export with assembly at the distal end. While many interactions among export apparatus proteins have been reported, few have been examined with respect to the differential affinities and dynamic relationships that must govern the mechanism of export. FlhB, an integral membrane protein, plays critical roles in both export and the substrate specificity switching that occurs upon hook completion. Reported herein is the quantitative characterization of interactions between the cytoplasmic domain of FlhB (FlhB_C) and other export apparatus proteins including FliK, FlhA_C and FliI. FliK and FlhA_C bound with micromolar affinity. K_D for FliI binding in the absence of ATP was 84 nM. ATP-induced oligomerization of FliI induced kinetic changes, stimulating fast-on, fast-off binding and lowering affinity. Full length FlhB purified under solubilizing, nondenaturing conditions formed a stable dimer via its transmembrane domain and stably bound FliH. Together, the present results support the previously hypothesized central role of FlhB and elucidate the dynamics of protein-protein interactions in type III secretion.     

Characterization of the flagellar hook length control protein fliK of Salmonella typhimurium and Escherichia coli.

During flagellar morphogenesis in Salmonella typhimurium and Escherichia coli, the fliK gene product is responsible for hook length control. A previous study (M. Homma, T. Iino, and R. M. Macnab, J. Bacteriol. 170:2221-2228, 1988) had suggested that the fliK gene may generate two products; we have confirmed that both proteins are products of the fliK gene and have eliminated several possible explanations for the two forms. We have determined the DNA sequence of the fliK gene in both bacterial species. The deduced amino acid sequences of the wild-type FliK proteins of S. typhimurium and E. coli correspond to molecular masses of 41,748 and 39,246 Da, respectively, and are fairly hydrophilic. Alignment of the sequences gives an identity level of 50%, which is low for homologous flagellar proteins from S. typhimurium and E. coli; the C-terminal sequence is the most highly conserved part (71% identity in the last 154 amino acids). The central and C-terminal regions are rich in proline and glutamine residues, respectively. Linker insertion mutagenesis of the conserved C-terminal region completely abolished motility, whereas disruption of the less conserved N-terminal and central regions had little or no effect. We suggest that the N-terminal (or N-terminal and central) and C-terminal regions may constitute domains. For several reasons, we consider it unlikely that FliK is functioning as a molecular ruler for determining hook length and conclude that it is probably employing a novel mechanism.