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.     

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.     

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.     

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.     

Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium.

The length of flagellar hooks isolated from wild-type and mutant cells with various hook lengths were measured on electron micrographs. The length of the wild-type hook showed a narrow distribution with a peak (+/- standard deviation) at 55.0 +/- 5.9 nm, whereas fliK mutants (so-called polyhook mutants) showed a broad distribution of hook lengths ranging from 40 to 900 nm, strongly indicating that FliK is involved in hook length determination. Among pseudorevertants isolated from such polyhook mutants, fliK intragenic suppressors gave rise to polyhook filaments. However, intergenic suppressors mapping to flhB also gave rise to hooks of abnormal length, albeit they were much shorter than polyhooks. Furthermore, double mutations of flhB and flgK (the structural gene for hook-associated protein 1; HAP1) resulted in polyhooks, suggesting another way in which hook length can be affected. The roles of FliK, FlhB, and HAP1 in hook length determination are discussed.     

Kinetic analysis of the growth rate of the flagellar hook in Salmonella typhimurium by the population balance method.

The growth rate of flagellar hooks in Salmonella typhimurium was analyzed by computer-aided simulation of the length distributions of mutant hooks of uncontrolled length (polyhooks). The wild-type hook has a relatively well-controlled length, with an average of 55 nm and a standard deviation of 6 nm. Mutations in the fliK gene give rise to polyhooks. A histogram of the lengths of polyhooks from a fliK mutant shows a peak at 55 nm with a long monotonic tail extending out to 1 microm. To analyze the growth rate, we employed the population balance method. Regression analysis showed that the histogram could fit a combination of two theoretical curves. In the first phase of growth, the hook starts with a very fast growth rate (40 nm/min), and then the rate exponentially slows until the length reaches 55 nm. In the second phase of growth, where the hook length is over 55 nm, the hook grows at a constant rate of 8 nm/min. Second mutations in either the fliK or flhB genes, as found in pseudorevertants from fliK mutants, give rise to polyhook filaments (phf). The ratio between the numbers of hooks with and without filament was 6:4. The calculated probability of filament attachment to polyhooks was low so that the proportion of hooks that start filament growth was only 2% per minute. The lengths of polyhooks with and without filaments were measured. A histogram of hook length in phf's was the same as that for polyhooks in single-site fliK mutants, against the expectation that the distribution would shift to a shorter average. The role of FliK in hook length control is discussed.     

Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook of Salmonella

The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a fliC (flagellin) mutant, or a fliD (hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.     

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.     

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.     

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 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.     

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.     

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.     

Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium.

Mutations in the fliK gene of Salmonella typhimurium commonly cause failure to terminate hook assembly and initiate filament assembly (polyhook phenotype). Polyhook mutants give rise to pseudorevertants which are still defective in hook termination but have recovered the ability to assemble filament (polyhook-filament phenotype). The polyhook mutations have been found to be either frameshift or nonsense, resulting in truncation of the C terminus of FliK. Intragenic suppressors of frameshift mutations were found to be ones that restored the original frame (and therefore the C-terminal sequence), but in most cases with substantial loss of natural sequence and sometimes the introduction of artificial sequence; in no cases did intragenic suppression occur when significant disruption remained within the C-terminal region. By use of a novel PCR protocol, in-frame deletions affecting the N-terminal and central regions of FliK were constructed and the resulting phenotypes were examined. Small deletions resulted in almost normal hook length control and almost wild-type swarming. Larger deletions resulted in loss of control of hook length and poor swarming. The largest deletions severely affected filament assembly as well as hook length control. Extragenic suppressors map to an unlinked gene, flhB, which encodes an integral membrane protein (T. Hirano, S. Yamaguchi, K. Oosawa, and S.-I. Aizawa, J. Bacteriol. 176:5439-5449, 1994; K. Kutsukake, T. Minamino, and T. Yokoseki, J. Bacteriol. 176:7625-7629, 1994). They were either point mutations in the C-terminal cytoplasmic region of FlhB or frameshift or nonsense mutations close to the C terminus. The processes of hook and filament assembly and the roles of FliK and FlhB in these processes are discussed in light of these and other available data. We suggest that FliK measures hook length and, at the appropriate point, sends a signal to FlhB to switch the substrate specificity of export from hook protein to late proteins such as flagellin.     

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.     

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.     

Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus

The switch in export specificity of the type III flagellar protein export apparatus from rod/hook type to filament type is believed to occur upon completion of hook assembly by way of an interaction of the type III secretion substrate specificity switch (T3S4) domain of the hook-length control protein FliK, with the integral membrane export apparatus component FlhB. The T3S4 domain of FliK (FliKT3S4) consisting of amino acid residues 265-405 has an unstable and flexible conformation in its last 35 residues (FliKCT). To investigate the role of FliKT3S4 in substrate specificity switching, we studied the effect of deletions and point mutations within this domain and characterized suppressor mutations. Deletions of ten amino acid residues within the region of residues 301-350 and five amino acids of residues 401-405 abolished switching of export specificity. Site directed mutagenesis showed that highly conserved residues, Val302, Ile304, Leu335, Val401 and Ala405, are essential, and that the five C terminal residues (401-405) are restricted in conformation for the switching process. Suppressor mutant analysis of the fliK(S319Y) mutant, which produces extended hooks with filaments attached due to delayed switching, suggested that FliKT3S4 interacts with the C terminal half of the cytoplasmic domain of FlhB (FlhBC). We propose a two step binding model of FliKT3S4 and FlhBC, in which residues 301-350 of FliK bind to FlhBC upon hook assembly completion at about 55 nm, and then unfolded FliKCT binds to FlhBC to trigger the switch in substrate specificity.