Information essential for cell-cycle-dependent secretion of the 591-residue Caulobacter hook protein is confined to a 21-amino-acid sequence near the N-terminus

Recent findings suggest that axial flagellar proteins and virulence proteins of Gram-negative bacteria are exported from the cytoplasm via conserved trans-location systems. To identify residues essential for secretion of flagellar axial proteins we examined the 591-residue Caulobacter crescentus flagellar hook protein. Western blot assays of the culture media of strains producing mutant hook proteins show that only residues 38–58 are essential for its secretion to the cell surface. We discuss the observation that this unprocessed 21-residue sequence is not conserved in other axial proteins and does not correspond to the SGL-, ANN LAN- and heptad repeat motifs that are located Just upstream of the essential secretion information in the hook protein and are conserved near the N-termini of other axial proteins. These motifs, for which an essential role in export or assembly has been proposed, are required for motility. However, we also demonstrate that hook protein can only be secreted when the flagellar basal body is present in the cell envelope. The cell-cycle regulation of hook protein secretion confirms the specificity of the assay used in these studies and suggests that the basal body itself may serve as a secretion channel for the hook protein.     

C‐ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation

The cytoplasmic C‐ring of the flagellum consists of FliG, FliM and FliN and acts as an affinity cup to localize secretion substrates for protein translocation via the flagellar‐specific type III secretion system. Random T‐POP transposon mutagenesis was employed to screen for insertion mutants that allowed flagellar type III secretion in the absence of the C‐ring using the flagellar type III secretion system‐specific hook–β‐lactamase reporter ( Lee and Hughes, 2006 ). Any condition resulting in at least a twofold increase in flhDC expression was sufficient to overcome the requirement for the C‐ring and the ATPase complex FliHIJ in flagellar type III secretion. Insertions in known and unknown flagellar regulatory loci were isolated as well as chromosomal duplications of the flhDC region. The twofold increased flhDC mRNA level coincided in a twofold increase in the number of hook‐basal bodies per cell as analysed by fluorescent microscopy. These results indicate that the C‐ring functions as a nonessential affinity cup‐like structure during flagellar type III secretion to enhance the specificity and efficiency of the secretion process.     

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.     

Subunit Organization and Reversal-associated Movements in the Flagellar Switch of Escherichia coli

Bacterial flagella contain a rotor-mounted protein complex termed the switch complex that functions in flagellar assembly, rotation, and clockwise/counterclockwise direction control. In Escherichia coli and Salmonella, the switch complex contains the proteins FliG, FliM, and FliN and corresponds structurally with the C-ring in the flagellar basal body. Certain features of subunit organization in the switch complex have been deduced previously, but details of subunit organization in the lower part of the C-ring and the molecular movements responsible for motor switching remain unclear. In this study, we use cross-linking, binding, and mutational experiments to examine subunit organization in the bottom of the C-ring and to probe movements that occur upon switching. The results show that FliN tetramers alternate with FliM C-terminal domains to form the bottom of the C-ring in an arrangement that closely reproduces the major features observed in electron microscopic reconstructions. When motors were switched to clockwise rotation by a repellent stimulus, cross-link yields were altered in a pattern indicating relative movement of FliN and FliM_C. These results are discussed in the framework of a structurally grounded hypothesis for the switching mechanism.     

DegU-phosphate activates expression of the anti-sigma factor FlgM in Bacillus subtilis

The bacterial flagellum is a complex molecular machine that is assembled by more than 30 proteins and is rotated to propel cells either through liquids or over solid surfaces. Flagellar gene expression is extensively regulated to co-ordinate flagellar assembly in both space and time. In Bacillus subtilis, the proteins of unknown function, SwrA and SwrB, and the alternative sigma factor σ(D) are required to activate expression of the flagellar filament protein, flagellin. Here we determine that in the absence of SwrA and SwrB, the phosphorylated form of the response regulator DegU inhibits σ(D) -dependent gene expression indirectly by binding to the P(flgM) promoter region and activating expression of the anti-sigma factor FlgM. We further demonstrate that DegU-P-dependent activation of FlgM is essential to inhibit flagellin expression when flagellar basal body assembly is disrupted. Regulation of FlgM is poorly understood outside of Salmonella, and differential control of FlgM expression may be a common means of coupling flagellin expression to flagellar assembly.     

FliN is a Major Structural Protein of the C-ring in theSalmonella typhimuriumFlagellar Basal Body

TheSalmonella typhimuriumFliN protein has been proposed to form a mutually interacting complex with FliG and FliM, the switch complex, that is required for flagellar morphogenesis and function. We have used affinity chromatography for purification of extended flagellar basal bodies sufficient for quantitative analysis of their protein composition. The belled, extended structure is predominantly comprised of the switch complex proteins; with FliN present in the most copies (111±13). This explains why single, missensefliN,fliGorfliMmutations, found in many non-motile strains, can alter the belled morphology. Cell lysates from these strains contained the wild-type complement of FliG, FliM and FliN; but the basal bodies lacked the outer, cytoplasmic(C)-ring of the bell and were separated by sedimentation from FliM and FliN. The amount of FliG present in basal bodies from wild-type and one such mutant, FliN100LP, was comparable. These data show that: (1) the mutations define a FliG and FliMFliN multiple contact interface important for motility. (2) FliG is responsible for the increased size of the membrane-embedded MS-ring complex of belled relative to acid-treated basal bodies. (3) FliN, together with FliM, account for most of the C-ring. As a major component of the C-ring, FliN is distinct from the other proteins implicated in axial flagellar protein export. Inner, cytoplasmic rod basal substructure, seen by negative-stain and quick- freeze replica electron microscopy, may gate such export. Lack of connectivity between the cytoplasmic rod and ring substructures places contacts between FliG and FliMFliN at the periphery of the basal body, proximal to the flagellar intramembrane ring particles. This topology is consistent with models where torque results from interaction of circumferential arrays of the switch complex proteins with the ring particles.     

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.     

Architectural Features of theSalmonella typhimuriumFlagellar Motor Switch Revealed by Disrupted C-Rings

The three-dimensional surface topology of rapid-frozenSalmonella typhimuriumflagellar hook basal body complexes was studied by stereo-examination of thin-film metal replicas. The complexes contained the extended cytoplasmic structure, composed of the switch complex proteins; FliG, FliM, and FliN. Distinct nanometer-scale element arrays, separated by grooves, defined the outer surface of the cytoplasmic (C-) ring. The number of array elements was comparable to previously determined FliG and FliM copy numbers in the basal body. In addition to basal body complexes lacking C-rings, complexes containing incomplete C-rings were identified. The incomplete C-rings had lost segments of the proximal array. Basal bodies with the distal C-ring array alone were not found. These findings are compatible with the spatial organization of the flagellar switch suggested by previous biochemical data.     

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.     

Flagellar filament elongation can be impaired by mutations in the hook protein FlgE of Salmonella typhimurium: a possible role of the hook as a passage for the anti-sigma factor FlgM

Among motile revertants isolated from flagellar hook-deficient ( flgE ) mutants of Salmonella typhimurium one produced only short flagellar filaments in L broth, despite the fact that flagellin itself has the ability to polymerize into long filaments in vitro . This pseudorevertant has an intragenic suppressor, resulting in a two-amino-acid substitution (Asp-Gln→Ala-Arg) in the C-terminal region of the hook protein, FlgE. The flagellation of the pseudorevertant was greatly affected by the concentration of NaCl in the culture media: we observed no filaments in the absence of NaCl, short filaments in 1% NaCl and full-length filaments in 2% NaCl. Electron microscopy of osmotically shocked cells showed that the number of hook–basal bodies on cells was constant under various NaCl conditions. Furthermore, we found that the mutant hook was straight rather than curved. We monitored the cellular flagellin level of this pseudorevertant under various NaCl concentrations by immunoblotting. It was revealed that little flagellin was present under NaCl-free conditions in contrast with the ordinary amounts of flagellin present in 2% NaCl. As the expression of flagellin is regulated by competitive interaction of a sigma factor, FliA, and a corresponding anti-sigma factor, FlgM, we also observed the effect of NaCl on the secretion of FlgM. FlgM was secreted into the media in more than 1% NaCl but accumulated inside the cells in the absence of NaCl, indicating that the failure of secretion of FlgM in the absence of salt was the cause of the impaired elongation of filaments.     

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.     

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.     

A Spef1-interacting microtubule quartet protein in Trypanosoma brucei promotes flagellar inheritance by regulating basal body segregation

The human parasite Trypanosoma brucei contains a motile flagellum that determines the plane of cell division, controls cell morphology, and mediates cell–cell communication. During the cell cycle, inheritance of the newly formed flagellum requires its correct positioning toward the posterior of the cell, which depends on the faithful segregation of multiple flagellum-associated cytoskeletal structures including the basal body, the flagellar pocket collar, the flagellum attachment zone, and the hook complex. A specialized group of four microtubules termed the microtubule quartet (MtQ) originates from the basal body and runs through the flagellar pocket collar and the hook complex to extend, along the flagellum attachment zone, toward the anterior of the cell. However, the physiological function of the MtQ is poorly understood, and few MtQ-associated proteins have been identified and functionally characterized. We report here that an MtQ-localized protein named NHL1 interacts with the microtubule-binding protein TbSpef1 and depends on TbSpef1 for its localization to the MtQ. We show that RNAi-mediated knockdown of NHL1 impairs the segregation of flagellum-associated cytoskeletal structures, resulting in mispositioning of the new flagellum. Furthermore, knockdown of NHL1 also causes misplacement of the cell division plane in dividing trypanosome cells, halts cleavage furrow ingression, and inhibits completion of cytokinesis. These findings uncover a crucial role for the MtQ-associated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei.     

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body

Most of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum-specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are FliP (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS-ring protein FliF and immunoblotting isolated hook basal body complexes with anti-FLAG monoclonal antibody, we estimate (using the FLAG-tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti-FLAG antibody and gold–protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains FliP and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.     

FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.     

ATPase-Independent Type-III Protein Secretion in Salmonella enterica

Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.     

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.     

Isolation and Ultrastructure of the Flagella of Methanococcus thermolithotrophicus and Methanospirillum hungatei

The flagella of the archaebacteria Methanococcus thermolithotrophicus and Methanospirillum hungatei enter the cells in regions with ultrastructure resembling that of the polar organelles found in a variety of eubacteria. Flagella of both organisms consist of a filament, a hook, and a basal body with two rings similar to those of gram-positive eubacteria. The integrity of the flagella of M. thermolithotrophicus is lost in the absence of high salt concentrations, and those of both organisms are unstable at high pH. The flagellar filaments of M. hungatei are composed of two flagellins of 24 and 26 kilodaltons.