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.     

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.     

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.     

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.     

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.     

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.     

Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export 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.     

FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium.

FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum. We have overproduced and purified FlgD and raised an antibody against it. By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies from flgA, flgE, flgH, flgI, flgK, and fliK mutants, in much smaller amounts in those from the wild type and flgL, fliA, fliC, fliD, and fliE mutants, and not at all in those from flgB, flgD, flgG, and flgJ mutants. In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip. Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook. Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament. However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein. When FlgD was added to the culture medium of a flgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously. When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant. Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export.     

Subdivision of flagellar region III of the Escherichia coli and Salmonella typhimurium chromosomes and identification of two additional flagellar genes.

The many genes involved in flagellar structure and function in Escherichia coli and Salmonella typhimurium are located in three major clusters on the chromosome: flagellar regions I, II and III. We have found that region III does not consist of a contiguous set of flagellar genes, as was thought, but that in E. coli there is almost 7 kb of DNA between the filament cap gene, fliD, and the next known flagellar gene, fliE; a similar situation occurs in S. typhimurium. Most of this DNA is unrelated to flagellar function, since a mutant in which 5.4 kb of it had been deleted remained fully motile and chemotactic as judged by swarming on semi-solid agar. We have therefore subdivided flagellar region III into two regions, IIIa and IIIb. The known genes in region IIIa are fliABCD, all of which are involved in filament structure and assembly, while region IIIb contains genes fliEFGHIJKLMNOPQR, all of which are related to formation of the hook (basal-body)-complex or to even earlier assembly events. We have found that fliD, the last known gene in region IIIa, is immediately followed by two additional genes, both necessary for flagellation, which we have designated fliS and fliT. They encode small proteins with deduced molecular masses of about 15 kDa and 14 kDa, respectively. The functions of FliS and FliT remain to be determined, but they do not appear to be members of the axial family of structural proteins to which FliD belongs.     

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.     

Molecular characterization of the Salmonella typhimurium flhB operon and its protein products.

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, may constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.     

Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium.

The flaAII.2, flaQ, and flaN genes of Salmonella typhimurium are important for assembly, rotation, and counterclockwise-clockwise switching of the flagellar motor. Paralyzed and nonchemotactic mutants were subjected to selection pressure for partial acquisition of motility and chemotaxis, and the suppressor mutations of the resulting pseudorevertants were mapped and isolated. Many of the intergenic suppressor mutations were in one of the other two genes. Others were in genes for cytoplasmic components of the chemotaxis system, notably cheY and cheZ; one of the mutations was found in the cheA gene and one in a motility gene, motB. Suppression among the three fla genes was allele specific, and many of the pseudorevertants were either cold sensitive or heat sensitive. We conclude that the FlaAII.2, FlaQ, and FlaN proteins form a complex which determines the rotational sense, either counterclockwise or clockwise, of the motor and also participates in the conversion of proton energy into mechanical work of rotation. This switch complex is probably mounted to the base of the flagellar basal body and, via binding of the CheY and CheZ proteins, receives sensory information and uses it to control flagellar operation.