Plasticity of the domain structure in FlgJ, a bacterial protein involved in flagellar rod formation

Bacterial flagellar rod structure is built across the peptidoglycan (PG) layer. A Salmonella enterica flagellar protein FlgJ is believed to consist of two functional domains, the N-terminal half acting as a scaffold or cap essential for rod assembly and the C-terminal half acting as a PG hydrolase (PGase) that makes a hole in the PG layer to facilitate rod penetration. In this study, molecular data analyses were conducted on FlgJ data sets sampled from a variety of bacterial species, and three types of FlgJ homologs were identified: (i) "canonical dual-domain" type found in beta- and gamma-proteobacteria that has a domain for one of the PGases, acetylmuramidase (Acm), at the C terminus, (ii) "non-canonical dual-domain" type found in the genus Desulfovibrio (delta-proteobacteria) that bears a domain for another PGase, M23/M37-family peptidase (Pep), at the C terminus and (iii) "single-domain" type found in phylogenetically diverged lineages that lacks the Acm or Pep domain. FlgJ phylogeny, together with the domain architecture, suggested that the single-domain type was the original form of FlgJ and the canonical dual-domain type had evolved from the single-domain type by fusion of the Acm domain to its C terminus in the common ancestor of beta- and gamma-proteobacteria. The non-canonical dual-domain type may have been formed by fusion of the Pep domain to the single-domain type in the ancestor of Desulfovibrio. In some lineages of gamma-proteobacteria, the Acm domain appeared to be lost secondarily from the dual-domain type FlgJ to yield again a single-domain type one. To rationalize the underlying mechanism that gave rise to the two different types of dual-domain FlgJ homologs, we propose a model assuming the lineage-specific co-option of flagellum-specific PGase from diverged housekeeping PGases in bacteria.     

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 in flagellar rod assembly of the N-terminal domain of Salmonella FlgJ, a flagellum-specific muramidase

The C-terminal half of the Salmonella flagellar protein FlgJ has peptidoglycan hydrolyzing activity and it has been suggested that it is a flagellum-specific muramidase which locally digests the peptidoglycan layer to permit assembly of the rod structure to proceed through the periplasmic space. It was also suggested that FlgJ might be involved in rod formation itself, although there was no direct evidence for this. We purified basal body structures from SJW1437(flgJ) transformed with plasmids encoding various mutant FlgJ proteins and found that these basal bodies possessed the periplasmic P ring but lacked the outer membrane L ring; they also lacked a hook at their distal end. All of these mutant FlgJ proteins had an altered or missing C-terminal domain but had at least the first 151 amino acid residues of the N-terminal domain. Immunoblotting analysis of fractionated cell extracts revealed that a rod/hook export class protein, FlgD, was exported to the periplasm but not to the culture supernatant in these mutants. FlgJ was shown to physically interact with several proteins, and especially FliE and FlgB, which are believed to reside at the cell-proximal end of the rod. On the basis of these results, we conclude that the N-terminal 151 amino acid residues of FlgJ are directly involved in rod formation and that the muramidase activity of FlgJ, though needed for formation of the L ring and subsequent events such as hook formation, is not essential for rod or P ring formation. In contrast, muramidase activity alone does not support rod assembly.     

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.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

Length Control of the Flagellar Hook in a Temperature-Sensitive flgE Mutant of Salmonella enterica Serovar Typhimurium

The flagellar hook is a short, curved, extracellular structure located between the basal body and the filament. The hook is composed of the FlgE protein. In this study, we analyzed flagellum assembly in a temperature-sensitive flgE mutant of Salmonella enterica serovar Typhimurium. When the mutant cells were grown at 30°C, they produced flagella of a normal length (71% of the population) and short hooks without filaments (26%). At 37°C, 70% of the basal bodies lacked hooks, and intact flagella made up only 6% of the population. Mutant cells secreted monomeric FlgE in abundance at 37°C, suggesting that the mutant FlgE protein might be defective in polymerization at higher temperatures. The average length of the hooks in intact filaments was 55 nm, whereas after acid treatment, it was 45 nm. SDS-PAGE analysis of the hook-basal body showed that HAP1 was missing in the mutant but not in the wild type. We concluded that hook length in the mutant is controlled in the same way as in the wild type, but the hook appeared short after acid treatment due to the lack of HAP1. We also learned that the true length of the hook is possibly 45 nm, not 55 nm, as has been believed.     

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 triangular loop of domain D1 of FlgE is essential for hook assembly but not for the mechanical function

The bacterial flagellar hook is a short, curved tubular structure made of FlgE. The hook connects the basal body as a rotary motor and the filament as a helical propeller and functions as a universal joint to smoothly transmit torque produced by the motor to the filament. Salmonella FlgE consists of D0, Dc, D1 and D2 domains. Axial interactions between a triangular loop of domain D1 (D1-loop) and domain D2 are postulated to be responsible for hook supercoiling. In contrast, Bacillus FlgE lacks the D1-loop and domain D2. Here, to clarify the roles of the D1-loop and domain D2 in the mechanical function, we carried out deletion analysis of Salmonella FlgE. A deletion of the D1-loop conferred a loss-of-function phenotype whereas that of domain D2 did not. The D1-loop deletion inhibited hook polymerization. Suppressor mutations of the D1-loop deletion was located within FlgD, which acts as the hook cap to promote hook assembly. This suggests a possible interaction between the D1-loop of FlgE and FlgD. Suppressor mutant cells produced straight hooks, but retained the ability to form a flagellar bundle behind a cell body, suggesting that the loop deletion does not affect the bending flexibility of the Salmonella hook.     

Substrate specificity classes and the recognition signal for Salmonella type III flagellar export

Most flagellar proteins of Salmonella are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ). In this study, we have measured the amounts of these proteins exported before and after hook completion. Their amounts in the culture supernatant from a flgE mutant (which is still at the hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export.     

Components of the Salmonella flagellar export apparatus and classification of export substrates

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.     

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.     

Formation of flagella lacking outer rings by flaM, flaU, and flaY mutants of Escherichia coli.

Among flagellar mutants of Escherichia coli, flaM or flaU mutants form basal bodies lacking the outer P and L rings, whereas flaY mutants predominantly form basal bodies lacking the L ring. In these mutants, hooks and filaments are occasionally assembled onto these incomplete basal bodies. When the hook protein gene, flaFV, of Salmonella typhimurium was cloned on the multicopy plasmid pBR322 and introduced into these mutants, the efficiency with which cells assembled hooks and filaments onto the incomplete basal bodies increased significantly. Such cells formed characteristic dotted swarms on semisolid plates, indicating that cells carrying flagella without the outer rings are weakly motile because of poor function of their flagella, a low flagellar number per cell, or both of these defects. FlaV mutants also produced incomplete basal bodies lacking the outer rings, but assembly of hooks and filaments did not occur in these mutants even after introduction of the plasmid carrying flaFV of S. typhimurium. The failure in the case of flaV mutants was attributed to their inability to modify the rod tip to the structure competent for assembly of hook protein.     

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.     

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.     

Peptidoglycan-hydrolyzing activity of the FlgJ protein, essential for flagellar rod formation in Salmonella typhimurium

Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in Salmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from S. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJ mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.