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.     

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.     

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed “ruler” protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a “ball-and-chain” architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein''s N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.     

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.     

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.     

The FliK protein and flagellar hook-length control

The bacterial flagellum is a highly complex prokaryotic organelle. It is the motor that drives bacterial motility, and despite the large amount of energy required to make and operate flagella, motile organisms have a strong adaptive advantage. Flagellar biogenesis is both complex and highly coordinated and it typically involves at least three two-component systems. Part of the flagellum is a type III secretion system, and it is via this structure that flagellar components are exported. The assembly of a flagellum occurs in a number of stages, and the "checkpoint control" protein FliK functions in this process by detecting when the flagellar hook substructure has reached its optimal length. FliK then terminates hook export and assembly and transmits a signal to begin filament export, the final stage in flagellar biosynthesis. As yet the exact mechanism of how FliK achieves this is not known. Here we review what is known of the FliK protein and discuss the evidence for and against the various hypotheses that have been proposed in recent years to explain how FliK controls hook length, FliK as a molecular ruler, the measuring cup theory, the role of the FliK N terminus, the infrequent molecular ruler theory, and the molecular clock theory.     

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.     

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.     

A molecular ruler mechanism for length control of extended protein structures in bacteria

The lengths of the hook structure of flagellar motors and of the needle of the injectosome are both carefully controlled, by apparently similar mechanisms. In this paper we propose a novel mechanism for this length control and develop a mathematical model of this process which shows excellent agreement with published data on hook lengths.The proposed mechanism for length control (described using biochemical nomenclature appropriate for hooks) is as follows: Hook growth is terminated when the C-terminus of the length control molecule FliK interacts with FlhB, the secretion gatekeeper. The probability of this interaction is an increasing function of the length of the hook for two reasons. First, FliK is secreted through the hook intermittently during hook growth. Second, the probability of interaction with FlhB is a function of the amount of time the C-terminus of a secreted FliK spends in the vicinity of FlhB. This time is short when the hook is short because the folding of FliK exiting the distal end of the hook acts to pull the FliK molecule through the hook rapidly. In contrast, this time is much longer when the hook is longer than the unfolded FliK polymer since movement through the tube is not enhanced by folding. Thus, it is much more likely that interaction will occur when the hook is long than when the hook is short.     

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.     

Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica

The length of the needle ending the Yersinia Ysc injectisome is determined by YscP, a protein acting as a molecular ruler. In addition, YscP is required for Yop secretion. In the present paper, by a systematic deletion analysis, we localized accurately the region required for Yop secretion between residues 405 and 500. As this C-terminal region of YscP has also been shown to control needle length it probably represents the substrate specificity switch of the machinery. By a bioinformatics analysis, we show that this region has a globular structure, an original alpha/beta fold, a P-x-L-G signature and presumably no catalytic activity. In spite of very limited sequence similarities, this structure is conserved among the proteins that are presumed to control the needle length in many different injectisomes and also among members of the FliK family, which control the flagellar hook length. This region thus represents a new protein domain that we called T3S4 for Type III secretion substrate specificity switch. The T3S4 domain of YscP can be replaced by the T3S4 domain of AscP (Aeromonas salmonicida) or PscP (Pseudomonas aeruginosa) but not by the one from FliK, indicating that in spite of a common global structure, these domains need to fit their partner proteins in the secretion apparatus.     

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.     

Gap compression/extension mechanism of bacterial flagellar hook as the molecular universal joint

Bacterial flagellar hook acts as a molecular universal joint, transmitting torque produced by the flagellar basal body, a rotary motor, to the flagellar filament. The hook forms polymorphic supercoil structures and can be considered as an assembly of 11 circularly arranged protofilaments. We investigated the molecular mechanism of the universal joint function of the hook by a approximately two-million-atom molecular dynamics simulation. On the inner side of the supercoil, protein subunits are highly packed along the protofilament and no gaps remain for further compression, whereas subunits are slightly separated and are hydrogen bonded through one layer of water molecules on the outer side. As for the intersubunit interactions between protofilaments, subunits are packed along the 6-start helix in a left-handed supercoil whereas they are highly packed along the 5-start helix in a right-handed supercoil. We conclude that the supercoiled structures of the hook in the left- and right-handed forms make maximal use of the gaps between subunits, which we call "gap compression/extension mechanism". Mutual sliding of subunits at the subunit interface accompanying rearrangements of intersubunit hydrogen bonds is interpreted as a mechanism to allow continuous structural change of the hook during flagellar rotation at low energy cost.     

Length control of extended protein structures in bacteria and bacteriophages

The length of the tail of bacteriophages is controlled by a protein which acts as a molecular ruler. The needle of the injectisome, which is assembled by the polymerization of subunits that are exported through the nascent injectisome, is functionally related to the tail of bacteriophages. Interestingly, its length is controlled by a protein, which is itself exported and acts as a molecular ruler that is coupled to a substrate specificity switch. The bacterial flagellum is evolutionarily related to the injectisome. The length of the hook is also controlled by a secreted protein. This protein acts as a substrate specificity switch and, possibly, also as a ruler.     

Interactions of bacterial flagellar chaperone–substrate complexes with FlhA contribute to co‐ordinating assembly of the flagellar filament

Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C‐terminal cytoplasmic domain of FlhA (FlhA_C), a membrane component of the export apparatus, provides a binding‐site for these chaperone–substrate complexes but it remains unknown how it co‐ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhA_C is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhA_C for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14‐fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhA_C for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.     

Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates

A remarkable feature of the flagellar‐specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook‐basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N‐terminal secretion signal, mRNA secretion signals within the 5′‐untranslated region (5′‐UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational β‐lactamase fusions to probe the secretion efficacy of the N‐terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early‐type substrates FlgD, FlgF, FlgE and the late‐type substrate FlgL were analysed in detail. Analysing the role of the 5′‐UTR in secretion of flgB and flgE revealed that the native 5′‐UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate‐specific chaperones allowing for targeted secretion of flagellar substrates.     

Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium.

A flagellum of Salmonella typhimurium and Escherichia coli consists of three structural parts, a basal body, a hook, and a filament. Because the fliK mutants produce elongated hooks, called polyhooks, lacking filament portions, the fliK gene product has been believed to be involved in both the determination of hook length and the initiation of the filament assembly. In the present study, we isolated two mutants from S. typhimurium which can form flagella even in the absence of the fliK gene product. Flagellar structures were fractionated from these suppressor mutants and inspected by electron microscopy. The suppressor mutants produced polyhook-filament complexes in the fliK mutant background, while they formed flagellar structures apparently indistinguishable from those of the wild-type strain in the fliK+ background. Genetic and sequence analyses of the suppressor mutations revealed that they are located near the 3'-end of the flhB gene, which has been believed to be involved in the early process of the basal body assembly. On the basis of these results, we discuss the mechanism of suppression of the fliK defects by the flhB mutations and propose a hypothesis on the export switching machinery of the flagellar proteins.     

Characterization of the type III export signal of the flagellar hook scaffolding protein FlgD of Escherichia coli

Transport of flagellar structural proteins beyond the cytoplasmic membrane is accomplished by a type III secretory pathway [flagellar type III secretion system (fTTSS)]. The mechanism of substrate recognition by the fTTSS is still enigmatic. Using the hook scaffolding protein FlgD of Escherichia coli as a model substrate, it is demonstrated that the export signal is contained within the N-terminal 71 amino acids of FlgD. Analysis of frame-shift mutations and alterations of the nucleotide sequence suggest a proteinaceous nature of the signal. Furthermore, the physicochemical properties of the first about eight amino acids are crucial for export.