FlhF and Its GTPase Activity Are Required for Distinct Processes in Flagellar Gene Regulation and Biosynthesis in Campylobacter jejuni

FlhF proteins are putative GTPases that are often necessary for one or more steps in flagellar organelle development in polarly flagellated bacteria. In Campylobacter jejuni, FlhF is required for σ⁵⁴-dependent flagellar gene expression and flagellar biosynthesis, but how FlhF influences these processes is unknown. Furthermore, the GTPase activity of any FlhF protein and the requirement of this speculated activity for steps in flagellar biosynthesis remain uncharacterized. We show here that C. jejuni FlhF hydrolyzes GTP, indicating that these proteins are GTPases. C. jejuni mutants producing FlhF proteins with reduced GTPase activity were not severely defective for σ⁵⁴-dependent flagellar gene expression, unlike a mutant lacking FlhF. Instead, these mutants had a propensity to lack flagella or produce flagella in improper numbers or at nonpolar locations, indicating that GTP hydrolysis by FlhF is required for proper flagellar biosynthesis. Additional studies focused on elucidating a possible role for FlhF in σ⁵⁴-dependent flagellar gene expression were conducted. These studies revealed that FlhF does not influence production of or signaling between the flagellar export apparatus and the FlgSR two-component regulatory system to activate σ⁵⁴. Instead, our data suggest that FlhF functions in an independent pathway that converges with or works downstream of the flagellar export apparatus-FlgSR pathway to influence σ⁵⁴-dependent gene expression. This study provides corroborative biochemical and genetic analyses suggesting that different activities of the C. jejuni FlhF GTPase are required for distinct steps in flagellar gene expression and biosynthesis. Our findings are likely applicable to many polarly flagellated bacteria that utilize FlhF in flagellar biosynthesis processes.Flagellar biosynthesis in bacteria is a complex process that requires expression of more than 50 genes in a sequential manner to ensure that the encoded proteins are secreted and interact in a proper order to construct a flagellar organelle (8). Formation of a flagellum to impart swimming motility is often an essential determinant for many bacteria to infect hosts or reside in an environmental niche. As such, flagella and flagellar motility are required for Campylobacter jejuni to initiate and maintain a harmless intestinal colonization in many wild and agriculturally important animals (16, 17, 19, 35, 47, 49), which leads to large reservoirs of the bacterium in the environment and the human food supply (13). In addition, flagellar motility is essential for the bacterium to infect human hosts to cause a diarrheal disease, which can range from a mild, watery enteritis to a severe, bloody diarrheal syndrome (4). Due to its prevalence in nature and in the food supply, C. jejuni is a leading cause of enteritis in humans throughout the world (7).C. jejuni belongs to a subset of motile bacteria that produce polarly localized flagella, which includes important pathogens of humans, such as Helicobacter, Vibrio, and Pseudomonas species. These bacteria have some commonalities in mechanisms for flagellar gene expression and biosynthesis, such as using both alternative σ factors, σ²⁸ and σ⁵⁴, for expression of distinct sets of flagellar genes (1, 6, 9, 11, 18, 20-22, 26, 36, 40, 44, 45, 49). In addition, these bacteria produce the putative FlhF GTPase, which is required in each bacterium for at least one of the following: expression of a subset of flagellar genes, biosynthesis of flagella, or the polar placement of the flagella. For instance, FlhF is required for expression of some σ⁵⁴- and σ²⁸-dependent flagellar genes and for production of flagella in the classical biotype of Vibrio cholerae (10). However, V. cholerae flhF mutants of another biotype can produce a flagellum in a minority of cells, but the flagellum is at a lateral site (14). Similar lateral flagella were found in flhF mutants of Pseudomonas aeruginosa and Pseudomonas putida (34, 37). FlhF of Vibrio alginolyticus may also be involved in the polar formation of flagella and may possibly influence the number of flagella produced (28, 29). Demonstration that FlhF is polarly localized in some of these species and the fact that FlhF has been observed to assist the early flagellar MS ring protein, FliF, in localizing to the old pole in one biotype of V. cholerae give credence that FlhF may be involved in the polar placement of flagella in the respective organisms (14, 29, 34).Bioinformatic analysis indicates that the FlhF proteins belong to the SIMIBI class of NTP-binding proteins (30). More specifically, the GTPase domains of FlhF proteins are most similar to those of the signal recognition particle (SRP) pathway GTPases, such as Ffh and FtsY. Because of the homology of the GTPase domains, these three proteins may form a unique subset within the SIMIBI proteins. Whereas the GTPase activities of the interacting Ffh and FtsY proteins have been extensively characterized (32, 38, 39, 42), little is known about the GTP hydrolysis activity of FlhF. Structural determination of FlhF of Bacillus subtilis indicates that the potential GTPase activity of FlhF is likely varied relative to those of Ffh and FtsY (2). However, no biochemical analysis has been performed to verify or characterize the ability of an FlhF protein to hydrolyze GTP. As such, no studies have correlated the biochemical activity of FlhF in relation to GTP hydrolysis with the role that FlhF performs in flagellar gene expression or biosynthesis.Through previous work, we have delineated the regulatory cascades governing flagellar gene expression in C. jejuni. We have found that formation of the flagellar export apparatus (FEA), a multiprotein inner membrane complex (consisting of the proteins FlhA, FlhB, FliF, FliO, FliP, FliQ, and FliR) that secretes most of the flagellar proteins out of the cytoplasm to form the flagellum, is required to activate the FlgS sensor kinase to begin a phosphorelay to the cognate FlgR response regulator (23, 24). Once activated by phosphorylation, FlgR likely interacts with σ⁵⁴ in RNA polymerase to initiate expression of many flagellar genes encoding components of the flagellar basal body, rod, and hook (20, 24). After formation of the hook, flaA, encoding the major flagellin, is expressed via σ²⁸ and RNA polymerase to generate the flagellar filament and complete flagellar biosynthesis (6, 18, 20, 21, 49). In two separate genetic analyses, we found that flhF mutants of C. jejuni are nonmotile and show a more than 10-fold reduction in expression of σ⁵⁴-dependent flagellar genes, indicating that FlhF is required for both flagellar gene expression and biosynthesis (20). However, it is unclear how FlhF influences expression of σ⁵⁴-dependent flagellar genes. Furthermore, it is unknown if the GTPase activity of FlhF is required for flagellar gene expression or biosynthesis in C. jejuni.We have performed experiments to determine that C. jejuni FlhF specifically hydrolyzes GTP, confirming that FlhF is a GTPase. Whereas the FlhF protein is required for motility, flagellar biosynthesis, and expression of σ⁵⁴-dependent flagellar genes, the GTPase activity of the protein significantly influences only proper biosynthesis of flagella. These results suggest that multiple biochemical activities of FlhF (including GTPase activity and likely other, as yet uncharacterized activities mediated by other domains) are required at distinct steps in flagellar gene expression and biosynthesis. In addition, we provide biochemical and genetic evidence that FlhF likely functions in a pathway separate from the FEA-FlgSR pathway in C. jejuni to influence expression of σ⁵⁴-dependent flagellar genes. This study provides corroborative genetic and biochemical analysis of FlhF to indicate that FlhF has multiple inherent activities that function at different steps in development of the flagellar organelle, which may be applicable to many polarly flagellated bacteria.     

The Flagellar Basal Body-Associated Protein FlgT Is Essential for a Novel Ring Structure in the Sodium-Driven Vibrio Motor

In Vibrio alginolyticus, the flagellar motor can rotate at a remarkably high speed, ca. three to four times faster than the Escherichia coli or Salmonella motor. Here, we found a Vibrio-specific protein, FlgT, in the purified flagellar basal body fraction. Defects of FlgT resulted in partial Fla⁻ and Mot⁻ phenotypes, suggesting that FlgT is involved in formation of the flagellar structure and generating flagellar rotation. Electron microscopic observation of the basal body of ΔflgT cells revealed a smaller LP ring structure compared to the wild type, and most of the T ring was lost. His₆-tagged FlgT could be coisolated with MotY, the T-ring component, suggesting that FlgT may interact with the T ring composed of MotX and MotY. From these lines of evidence, we conclude that FlgT associates with the basal body and is responsible to form an outer ring of the LP ring, named the H ring, which can be distinguished from the LP ring formed by FlgH and FlgI. Vibrio-specific structures, e.g., the T ring and H ring might contribute the more robust motor structure compared to that of E. coli and Salmonella.The bacterial flagellar motor is a rotary nanomotor, which converts the electrochemical potential difference of the coupling ion (H⁺ or Na⁺) into rotational energy. Escherichia coli and Salmonella spp. have H⁺-driven motors, and Vibrio alginolyticus has Na⁺-driven motors. The rotation speed of the Vibrio motor is remarkably fast, 1,100 Hz on average and up to 1,700 Hz maximum, which is more than four times faster than that of the E. coli motor (24, 27).The flagellum is coordinately and hierarchically constructed from more than 30 related proteins and is composed of rotor, stator, universal joint (hook), and helical filament (22, 43). The rotor part (also called the basal body) contains several rings and a drive shaft, which are named the L, P, MS, and C rings and the rod (1, 14). The L, P, MS, and C rings are thought to be located in positions corresponding to the outer membrane, peptidoglycan layer, cytoplasmic membrane, and cytoplasm, respectively (Fig. ​(Fig.1).1). Because the LP ring is thought to be a bushing for rotation of the rod, the LP ring seems not to rotate. Analyses of the basal body components of Salmonella were carried out in detail, thereby identifying all of the gene products that are responsible for the substructures. The L, P and MS rings are composed of FlgH, FlgI, and FliF, respectively, while the C ring is composed of three different proteins, FliG, FliM, and FliN, and the rod is composed of FlgB, FlgC, FlgF, and FlgG (14, 17, 18, 39, 44).Open in a separate windowFIG. 1.Model of the flagellar basal body in Vibrio. The H ring and the T ring are shown in dark gray. The LP ring and the other basal body parts are shown in light gray. The PomA/B complex is shown in the medium gray. OM, outer membrane; PG, peptidoglycan layer; IM, inner membrane.The stator part is responsible for torque generation. The torque generation unit of the stator is composed of MotA and MotB in E. coli or PomA and PomB in Vibrio spp. and is a hexamer of four A subunits and two B subunits. They assemble around the rotor and transfer the coupling ions (H⁺ in E. coli and Na⁺ in Vibrio) across the membrane due to the electrochemical potential (2, 4, 11, 15, 37, 38, 40, 41). MotX and MotY are species-specific (e.g., Vibrio and Shewanella spp.) stator proteins, and defects in these proteins result in a mot phenotype in which flagellar morphogenesis is normal but the flagella cannot rotate (21, 30, 31, 33, 36). Pseudomonas spp. have only MotY but not MotX; MotY is required for flagellar rotation (12). In Vibrio alginolyticus it has been shown that MotX and MotY are produced as precursor proteins with signal sequences and are translocated to the periplasmic space by a general secretion pathway (35). MotX and MotY form a ring structure called the T ring in addition to the LP ring (Fig. ​(Fig.1).1). The N-terminal domain of MotY has been suggested to directly associate with the basal body, probably the P ring and MotX (23, 42), and MotX has been suggested to interact with PomB (34). Based on these lines of evidence, the T ring was proposed to be involved in the incorporation and/or stabilization of the PomA/B complex into the motor and provide a connection between the rotor and PomA/B in Vibrio (42).When flagellar basal bodies were purified from various species, the basic structures were similar but the details were different. When we compared the structures from Vibrio cells and E. coli cells, the Vibrio LP rings were bigger than those of E. coli (42). We speculated that additional proteins were present in the Vibrio LP rings. In the present study, we recognized a novel ring structure on the basal body of V. alginolyticus, and it was composed of the product of a recently identified motility gene, flgT. It was reported in that in Vibrio cholerae FlgT is somehow involved in motility and flagellar formation (9, 29). Furthermore, V. cholerae strains with defects in FlgT develop outer membrane blebbing and release the flagellum into the medium, suggesting that FlgT is involved in anchoring the flagellar base on the cell surface (29). We found that FlgT is necessary to form an outer ring of the LP ring, named the H ring (for holding ring of the flagellar base on the cell surface). The H ring is thought to be involved in assembly of MotX and MotY to the basal body.     

Identification and Characterization of a Phosphodiesterase That Inversely Regulates Motility and Biofilm Formation in Vibrio cholerae

Vibrio cholerae switches between free-living motile and surface-attached sessile lifestyles. Cyclic diguanylate (c-di-GMP) is a signaling molecule controlling such lifestyle changes. C-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain. We constructed in-frame deletions of all V. cholerae genes encoding proteins with GGDEF and/or EAL domains and screened mutants for altered motility phenotypes. Of 52 mutants tested, four mutants exhibited an increase in motility, while three mutants exhibited a decrease in motility. We further characterized one mutant lacking VC0137 (cdgJ), which encodes an EAL domain protein. Cellular c-di-GMP quantifications and in vitro enzymatic activity assays revealed that CdgJ functions as a PDE. The cdgJ mutant had reduced motility and exhibited a small decrease in flaA expression; however, it was able to produce a flagellum. This mutant had enhanced biofilm formation and vps gene expression compared to that of the wild type, indicating that CdgJ inversely regulates motility and biofilm formation. Genetic interaction analysis revealed that at least four DGCs, together with CdgJ, control motility in V. cholerae.Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger in bacteria. It is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain (46, 48, 50). The receptors of c-di-GMP, which can be proteins or RNAs (riboswitches), bind to c-di-GMP and subsequently transmit the signal to downstream targets (22). C-di-GMP signaling is predicted to occur via a common or localized c-di-GMP pool(s) through so-called c-di-GMP signaling modules harboring DGCs and PDEs, receptors, and targets that affect cellular function (22).C-di-GMP controls various cellular functions, including the transition between a planktonic lifestyle and biofilm lifestyle. In general, high concentrations of c-di-GMP promote the expression of adhesive matrix components and result in biofilm formation, while low concentrations of c-di-GMP result in altered motility upon changes in flagellar or pili function and/or production (reviewed in reference 25). C-di-GMP inversely regulates motility and biofilm formation by implementing control at different levels through gene expression or through posttranslational mechanisms (reviewed in reference 25).Vibrio cholerae, the causative agent of the disease cholera, uses c-di-GMP signaling to undergo a motile-to-sessile lifestyle switch that is important for both environmental and in vivo stages of the V. cholerae life cycle. The survival of the pathogen in both natural aquatic environments and during infection depends on the appropriate regulation of motility, surface attachment, and colonization factors (26). The V. cholerae genome encodes a total of 62 putative c-di-GMP metabolic enzymes: 31 with a GGDEF domain, 12 with an EAL domain, 10 with both GGDEF and EAL domains, and 9 with an HD-GYP domain (21). V. cholerae contains a few known or predicted c-di-GMP receptors: two riboswitches (53), five PilZ domain proteins (43), VpsT (31), and CdgG (6). C-di-GMP regulates virulence, motility, biofilm formation, and the smooth-to-rugose phase variation in V. cholerae (6, 8, 9, 12, 30, 33, 43, 45, 54, 56, 57). However, particular sets of proteins have not been matched to discrete cellular processes.Some of the DGCs and PDEs involved in regulating motility in V. cholerae have been identified: rocS and cdgG mutants exhibit a decrease in motility (45), while cdgD and cdgH mutants exhibit an increase in motility (6). In addition, VieA (PDE) positively regulates motility in the V. cholerae classical biotype but not in the El Tor biotype (7). AcgA (PDE) positively regulates motility at low concentrations of inorganic phosphate (42). In this study, we investigated the role of each putative gene encoding DGCs and PDEs in controlling cell motility. In addition to the already-characterized proteins CdgD, CdgH, and RocS, we identified two putative DGCs (CdgK and CdgL) that negatively control motility and a putative PDE (CdgJ) that positively controls motility. We further characterized CdgJ and showed that it functions as a PDE and inversely regulates motility and biofilm formation. Genetic interaction studies revealed that DGCs CdgD, CdgH, CdgL, and CdgK and PDE CdgJ form a c-di-GMP signaling network to control motility in V. cholerae.     

Identification of Flagellar Motility Genes in Yersinia ruckeri by Transposon Mutagenesis

Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.Yersinia ruckeri is the etiologic agent of enteric redmouth disease, a disease of salmonid fish species that is found worldwide in areas where salmonid fish species are farmed (3, 6, 18, 20). Vaccines for enteric redmouth disease have been used successfully for nearly 3 decades and consist of immersion-applied, killed whole-cell preparations of motile serovar 1 Y. ruckeri strains (22). Recently though, outbreaks have been reported in vaccinated fish at trout farms in the United Kingdom (2), Spain (9), and the United States (1). The Y. ruckeri strains isolated from these outbreaks are uniformly atypical serovar 1 isolates lacking both flagellar motility and secreted lipase activity. These variants have been classified as Y. ruckeri biotype 2 (BT2) and are believed to have a reduced sensitivity to immersion vaccination (2). The objective of this study was to obtain a better understanding of the emergence of BT2 Y. ruckeri by identifying genetic elements necessary for expression of the Y. ruckeri flagellum and determining the role that the flagellum plays in virulence by using a rainbow trout infection model.     

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24, 26, 34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34, 38, 54).Type IV pili are typically composed of major pilins, the primary structural components of the pilus, and several minor pilin-like proteins that play important roles in pilus assembly or function (15, 17, 46). Pilin precursor proteins are transported across the cytoplasmic membrane via the Sec translocation pathway (7, 20). Most Sec substrates contain either a class I or a class II signal peptide that is cleaved at a recognition site that lies subsequent to the hydrophobic portion of the signal peptide (18, 43). However, the precursors of type IV pilins contain class III signal peptides, which are processed at recognition sites that precede the hydrophobic domain by a prepilin-specific peptidase (SPase III) (38, 43, 45). Similarly, archaeal flagellin precursors contain a class III signal peptide that is processed by a prepilin-specific peptidase homolog (FlaK/PibD) (3, 8, 10, 11). Moreover, flagellar assembly involves homologs of components involved in the biosynthesis of bacterial type IV pili, including FlaI, an ATPase homologous to PilB, and FlaJ, a multispanning membrane protein that may provide a platform for flagellar assembly, similar to the proposed role for PilC in pilus assembly (38, 44, 53, 54). These genes, as well as a number of others that encode proteins often required for either flagellar assembly or function (flaCDEFG and flaH), are frequently coregulated with the flg genes (11, 26, 44, 54).Interestingly, most sequenced archaeal genomes also contain diverse sets of genes that encode type IV pilin-like proteins with little or no homology to archaeal flagellins (3, 39, 52). While often coregulated with pilB and pilC homologs, these genes are never found in clusters containing the motility-specific flaCDEFG and flaH homologs; however, the proteins they encode do contain class III signal peptides (52). Several of these proteins have been shown to be processed by an SPase III (4, 52). Moreover, in Sulfolobus solfataricus and Methanococcus maripaludis, some of these archaeal type IV pilin-like proteins were confirmed to form surface filaments that are distinct from the flagella (21, 22, 56). These findings strongly suggest that the genes encode subunits of pilus-like surface structures that are involved in functions other than swimming motility.In bacteria, type IV pili are multifunctional filamentous protein complexes that, in addition to facilitating twitching motility, mediate adherence to abiotic surfaces and make close intercellular associations possible (15, 17, 46). For instance, mating between Escherichia coli in liquid medium has been shown to require type IV pili (often referred to as thin sex pili), which bring cells into close proximity (29, 30, 57). Recent work has shown that the S. solfataricus pilus, Ups, is required not only for efficient adhesion to surfaces of these crenarchaeal cells but also for UV-induced aggregation (21, 22, 58). Frols et al. postulate that autoaggregation is required for DNA exchange under these highly mutagenic conditions (22). Halobacterium salinarum has also been shown to form Ca²⁺-induced aggregates (27, 28). Furthermore, conjugation has been observed in H. volcanii, which likely requires that cells be held in close proximity for a sustained period to allow time for the cells to construct the cytoplasmic bridges that facilitate DNA transfer between them (35).To determine the roles played by haloarchaeal flagella and other putative type IV pilus-like structures in swimming and surface motility, surface adhesion, autoaggregation, and conjugation, we constructed and characterized two mutant strains of H. volcanii, one lacking the genes that encode the flagellins and the other lacking pibD. Our analyses indicate that although this archaeon was previously thought to be nonmotile (14, 36), wild-type (wt) H. volcanii can swim in a flagellum-dependent manner. Consistent with the involvement of PibD in processing flagellins, the peptidase mutant is nonmotile. Unlike nonhalophilic archaea, however, the flagellum mutant can adhere to glass as effectively as the wild type. Conversely, the ΔpibD strain fails to adhere to glass surfaces, strongly suggesting that in H. volcanii surface adhesion involves nonflagellar, type IV pilus-like structures.     

Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA_C) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA_C. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA_C consists of at least three subdomains, a flexible linker, FlhA_CN, and FlhA_CC, and that FlhA_CN becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA_C not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.The bacterial flagellum, which is responsible for motility, is a supramolecular complex of about 30 different proteins, and it consists of at least three substructures: the basal body, the hook, and the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. Many of the flagellar component proteins are translocated into the central channel of the growing flagellar structure and then to the distal end of the structure for self-assembly by the flagellar type III protein export apparatus (11, 16, 22). This export apparatus consists of six integral membrane proteins, FlhA, FlhB, FliO, FliP, FliQ, and FliR, and three soluble proteins, FliH, FliI, and FliJ (18, 21). These protein components show significant sequence and functional similarities to those of the type III secretion systems of pathogenic bacteria, which directly inject virulence factors into their host cells (11, 16).FliI is an ATPase (4) and forms an FliH₂-FliI complex with its regulator, FliH, in the cytoplasm (20). FliI self-assembles into a homo-hexamer and hence exhibits full ATPase activity (1, 8, 17). FliH and FliI, together with FliJ and the export substrate, bind to the export core complex, which is composed of the six integral membrane proteins, to recruit export substrates from the cytoplasm to the core complex (14) and facilitate the initial entry of export substrates into the export gate (23). FliJ not only prevents premature aggregation of export substrates in the cytoplasm (13) but also plays an important role in the escort mechanism for cycling export chaperones during flagellar assembly (3). The export core complex is believed to be located in the central pore of the basal body MS ring (11, 16, 22). In fact, it has been found that FlhA, FliP, and FliR are associated with the MS ring (5, 9). The FliR-FlhB fusion protein is partially functional, suggesting that FliR and FlhB interact with each other within the MS ring (29). The export core complex utilizes a proton motive force across the cytoplasmic membrane as the energy source to drive the successive unfolding of export substrates and their translocation into the central channel of the growing flagellum (23, 27). Here we refer to the export core complex as the “export gate,” as we have previously (8, 16, 23, 24).FlhA is a 692-amino-acid protein consisting of two regions: a hydrophobic N-terminal transmembrane region with eight predicted α-helical transmembrane spans (FlhA_TM) and a hydrophilic C-terminal cytoplasmic region (FlhA_C) (12, 15). FlhA_TM is responsible for the association with the MS ring (9). FlhA_C interacts with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB (6, 12, 21, 24) and plays a role in the initial export process with these proteins (28). It has been shown that the V404M mutation in FlhA_C increases not only the probability of FliI binding to the export gate in the absence of FliH (14) but also the efficiency of substrate translocation through the export gate in the absence of FliH and FliI (23). Recently, it has been shown that FlhA_C is also required for substrate recognition (7). These observations suggest that an interaction between FlhA_C and FliI is coupled with substrate entry, although it is not clear how.In order to understand the mechanism of substrate entry into the export gate, we characterized a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, whose mutation blocks the flagellar protein export process at 42°C (28). We show here that this mutation severely inhibits translocation of flagellar proteins through the export gate after the FliH-FliI-FliJ complex binds to the FlhA-FlhB platform of the gate and that the impaired ability of the flhA(G368C) mutant to export flagellar proteins is restored almost to wild-type levels by intragenic second-site mutations that may alter the interactions between subdomains of FlhA_C for possible rearrangement for the export function.     

Quadruplex Real-Time PCR Assay for Detection and Identification of Vibrio cholerae O1 and O139 Strains and Determination of Their Toxigenic Potential

Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non-V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.Occasional outbreaks and pandemics caused by the bacterium Vibrio cholerae indicate that cholera is still a global threat to public health (1, 2, 6, 13, 14). The disease may become life-threatening if appropriate therapy is not undertaken quickly. Of the more than 200 serogroups of V. cholerae that have been identified (28), two serogroups, O1 and O139, cause epidemic and pandemic cholera (14), whereas non-O1, non-O139 serogroups are associated only with sporadic, isolated outbreaks of diarrhea (3, 23). O1 and O139 strains are also categorized as toxin-producing and non-toxin-producing strains. The toxin-producing strains cause life-threatening secretory diarrhea, while the non-toxin-producing isolates elicit only mild diarrhea. These differences among the serogroups of V. cholerae demand rapid diagnostic tests capable of both distinguishing O1 and O139 from other serogroups and differentiating toxin-producing from nonproducing isolates (20).PCR has become a molecular alternative to culture, microscopy, and biochemical testing for the identification of bacterial species (27). Many PCR methods have been developed for characterization of serogroups (O1 and/or O139), biotypes, and the toxigenic potential of V. cholerae strains (7, 11, 15, 19, 21, 22, 24-26). However, these conventional PCR methods require gel electrophoresis for product analysis and are therefore not suitable for routine use due to the risk of carryover contamination, low throughput, and intensive labor.Real-time PCR allows detection of amplification product accumulation through fluorescence intensity changes in a closed-tube setting, which is faster and more sensitive than conventional PCR and has become increasingly popular in clinical microbiology laboratories. Moreover, when multicolor fluorophore-labeled probes and/or melting curve analysis is used, multiplex real-time PCR can be designed to simultaneously detect many different target genes in a single reaction tube (8). So far, the majority of published real-time PCR assays for V. cholerae detect no more than two genes simultaneously (4, 8, 18), which precludes their use for simultaneous serogroup and toxin status determination. Recent reports show that multiplex real-time PCR greatly improves specificity and sensitivity for the detection of V. cholerae through either melting curve analysis (9) or using differently fluorophore-labeled probes (10).In the present work, we report the development of a quadruplex real-time PCR assay that enables simultaneous serogroup differentiation and toxigenic potential detection. By using four different fluorophore-labeled probes, which target hlyA, O1-specfic rfb, O139-specific rfb, and ctxA, the quadruplex assay can reveal whether the target is an O1, O139, or non-O1/non-O139 strain and whether the bacterium detected is capable of producing toxins. We report that by alleviating primer dimer formation by use of a homotag-assisted nondimer system (HANDS) (5), we were able to retain the analytical sensitivity of uniplex PCR and successfully differentiated serogroups and toxigenic potentials from aquatic animal and environmental samples.     

Isolation of Basal Bodies with C-Ring Components from the Na+-Driven Flagellar Motor of Vibrio alginolyticus

To investigate the Na⁺-driven flagellar motor of Vibrio alginolyticus, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.The basal body, which is the part of the rotor, is composed of four rings and a rod that penetrates them. Three of these rings, the L, P, and MS rings, are embedded in the outer membrane, peptidoglycan layer and in the inner membrane, respectively (1), while the C-ring of Salmonella species is attached to the cytoplasmic side of the basal body (3). The C-ring is composed of the proteins FliG, FliM, and FliN (25), and genetic evidence indicates that the C-ring is important for flagellar assembly, torque generation, and regulation of rotational direction (33, 34). FliG, 26 molecules of which are incorporated into the motor, appears to be the protein that is most directly involved in torque generation (15). Mutational analysis suggests that electrostatic interactions between conserved charged residues in the C-terminal domain of FliG and the cytoplasmic domain of MotA are important in torque generation (14), although this may not be the case for the Na⁺-type motor of Vibrio alginolyticus (32, 35, 36). FliM interacts with the chemotactic signaling protein CheY in its phosphorylated form (CheY-P) to regulate rotational direction (30). It has been reported that 33 to 35 copies of FliM assemble into a ring structure (28, 29). FliN contributes mostly to forming the C-ring structure (37). The crystal structure of FliN revealed a hydrophobic patch formed by several well-conserved hydrophobic residues (2). Mutational analysis showed that this patch is important for flagellar assembly and rotational switching (23, 24). The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. Thermotoga maritima FliN exists primarily as a dimer in solution, and T. maritima FliN and FliM together formed a stable FliM₁-FliN₄ complex (2). The spatial distribution of these proteins in the C-ring of Salmonella species was investigated using three-dimensional reconstitution analysis with electron microscopy (28). However, the correct positioning has still not been clarified.The Na⁺-driven motor requires two additional proteins, MotX and MotY, for torque generation (19-21, 22). These proteins form a unique ring structure, the T ring, located below the LP ring in the polar flagellum of V. alginolyticus (9, 26). It has been suggested that MotX interacts with MotY and PomB (11, 27). Unlike peritrichously flagellated Escherichia coli and Salmonella species, V. alginolyticus has two different flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum is used for motility in low-viscosity environments such as seawater (18). As described above, it is driven by a Na⁺-type motor. However, in high-viscosity environments, such as the mucus-coated surfaces of fish bodies, cells induce numerous unsheathed lateral flagella that have H⁺-driven motors (7, 8). We have been focusing on the Na⁺-driven polar flagellar motor, since there are certain advantages to studying its mechanism of torque generation over the H⁺-type motor: sodium motive force can be easily manipulated by controlling the Na⁺ concentration in the medium, and motor rotation can be specifically inhibited using phenamil (10). Moreover, its rotation rate is surprisingly high, up to 1,700 rps (compared to ∼200 rps and ∼300 rps for Salmonella species flagella and E. coli flagella, respectively) (12, 16, 17).Although understanding the C-ring structure and function is essential for clarifying the mechanism of motor rotation, there is no information about the C-ring of the polar flagellar motor of Vibrio species or the flagella of any genus other than Salmonella. Since Vibrio species have all of the genes coding for C-ring components, we would expect its location to be on the cytoplasmic side of the MS ring, as in Salmonella species. In this study, we attempted to isolate the polar flagellar basal body with the C-ring attached and investigate whether it is organized similarly to the H⁺-driven flagellar motor of Salmonella enterica serovar Typhimurium.     

MotX and MotY Are Required for Flagellar Rotation in Shewanella oneidensis MR-1

The single polar flagellum of Shewanella oneidensis MR-1 is powered by two different stator complexes, the sodium-dependent PomAB and the proton-driven MotAB. In addition, Shewanella harbors two genes with homology to motX and motY of Vibrio species. In Vibrio, the products of these genes are crucial for sodium-dependent flagellar rotation. Resequencing of S. oneidensis MR-1 motY revealed that the gene does not harbor an authentic frameshift as was originally reported. Mutational analysis demonstrated that both MotX and MotY are critical for flagellar rotation of S. oneidensis MR-1 for both sodium- and proton-dependent stator systems but do not affect assembly of the flagellar filament. Fluorescence tagging of MotX and MotY to mCherry revealed that both proteins localize to the flagellated cell pole depending on the presence of the basal flagellar structure. Functional localization of MotX requires MotY, whereas MotY localizes independently of MotX. In contrast to the case in Vibrio, neither protein is crucial for the recruitment of the PomAB or MotAB stator complexes to the flagellated cell pole, nor do they play a major role in the stator selection process. Thus, MotX and MotY are not exclusive features of sodium-dependent flagellar systems. Furthermore, MotX and MotY in Shewanella, and possibly also in other genera, must have functions beyond the recruitment of the stator complexes.Flagellum-mediated swimming motility is a widespread means of locomotion among bacteria. Flagella consist of protein filaments that are rotated at the filament''s base by a membrane-embedded motor (3, 39). Rotation is powered by electrochemical gradients across the cytoplasmic membrane. Thus far, two coupling ions, sodium ions and protons, have been described as energy sources for bacterial flagellar motors (4, 24, 48). Two major components confer the conversion of the ion flux into rotary motion. The first component forms a rotor-mounted ring-like structure at the base of the flagellar basal body and is referred to as the switch complex or the C ring; it is composed of the proteins FliG, FliM, and FliN. The second major component is the stator system, consisting of membrane-embedded stator complexes that surround the C ring (3). Each stator complex is composed of two subunits in a 4:2 stoichiometry. In Escherichia coli, MotA and MotB constitute the stator complex by forming a proton-specific ion channel; the Na⁺-dependent counterpart in Vibrio species consists of the orthologs PomA and PomB (1, 5, 49). MotA and PomA both have four transmembrane domains and are thought to interact with FliG via a cytoplasmic segment to generate torque (2, 50). Stator function is presumably made possible by a peptidoglycan-binding motif located at the C-terminal portion of MotB and PomB that anchors the stator complex to the cell wall (1, 8). In E. coli, at least 11 stator complexes can be synchronously involved in driving flagellar rotation (35). However, a single complex is sufficient for rotation of the filament (36, 40). Despite its tight attachment to the peptidoglycan, the stator ring system was found to form a surprisingly dynamic complex. It has been suggested that inactive precomplexes of the stators form a membrane-located pool before being activated upon incorporation into the stator ring system around the motor (13, 45). In E. coli, the turnover time of stator complexes can be as short as 30 s (21).In Vibrio species, two auxiliary proteins, designated MotX and MotY, are required for motor function of the Na⁺-driven polar flagellar system (22, 23, 28, 31). Recently, it was shown that the proteins associate with the flagellar basal body in Vibrio alginolyticus to form an additional structure, the T ring (42). MotX interacts with MotY and the PomAB stator complexes, and both proteins are thought to be crucial for the acquisition of the stators to the motor of the polar flagellum. (29, 30, 42). A MotY homolog is also associated with the proton-dependent motor system of the lateral flagella of V. alginolyticus that is induced under conditions of elevated viscosity (41).We recently showed that Shewanella oneidensis MR-1 uses two different stator systems to drive the rotation of its single polar flagellum, the Na⁺-dependent PomAB stator and the proton-driven MotAB stator. As suggested by genetic data, the MotAB stator has been acquired by lateral gene transfer, presumably in the process of adaptation from a marine to a freshwater environment (32). The two different stators are recruited to the motor in a way that depends on the sodium ion concentration in the medium. The Na⁺-dependent PomAB stator is present at the flagellated cell pole regardless of the sodium ion concentration, whereas the proton-dependent MotAB stator functionally localizes only under conditions of low sodium or in the absence of PomAB. It is still unclear how stator selection is achieved and whether additional proteins play a role in this process.Orthologs of motX and motY have been annotated in S. oneidensis MR-1. We thus hypothesized that MotX and MotY might play a role in stator selection in S. oneidensis MR-1. However, the originally published sequence of motY harbors a frameshift that would result in a drastically truncated protein lacking a functionally relevant putative peptidoglycan-binding domain at its C terminus (16, 18). This situation seemed inconsistent with a role for MotY in S. oneidensis MR-1.Here we describe a functional analysis of the MotX and MotY orthologs in S. oneidensis MR-1. We found that motY does not, in fact, contain a frameshift mutation, so that MotY is translated in its full-length form. Both MotX and MotY were essential for Na⁺-dependent and proton-dependent motility. Therefore, these proteins have a role in S. oneidensis MR-1 that differs from their function in Vibrio species. We also used fusions to the fluorescent protein mCherry for functional localization studies of MotX and MotY.     

Essential Role of the SycP Chaperone in Type III Secretion of the YspP Effector

The Ysa type III secretion (T3S) system enhances gastrointestinal infection by Yersinia enterocolitica bv. 1B. One effector protein targeted into host cells is YspP, a protein tyrosine phosphatase. It was determined in this study that the secretion of YspP requires a chaperone, SycP. Genetic analysis showed that deletion of sycP completely abolished the secretion of YspP without affecting the secretion of other Ysps by the Ysa T3S system. Analysis of the secretion and translocation signals of YspP defined the first 73 amino acids to form the minimal region of YspP necessary to promote secretion and translocation by the Ysa T3S system. Function of the YspP secretion/translocation signals was dependent on SycP. Curiously, when YspP was constitutively expressed in Y. enterocolitica bv. 1B, it was recognized and secreted by the Ysc T3S system and the flagellar T3S system. In these cases, the first 21 amino acids were sufficient to promote secretion, and while SycP did enhance secretion, it was not essential. However, neither the Ysc T3S system nor the flagellar T3S system translocated YspP into mammalian cells. This supports a model where SycP confers secretion/translocation specificities for YspP by the Ysa T3S system. A series of biochemical approaches further established that SycP specifically interacts with YspP and protected YspP degradation in the cell prior to secretion. Collectively, the evidence suggests that YspP secretion by the Ysa T3S system is a posttranslational event.Many gram-negative bacteria have evolved sophisticated delivery systems termed type III secretion (T3S) systems to transport effector proteins into the cytosols of eukaryotic host cells (10, 21, 22). The translocated effectors manipulate host cell activities in various ways, thereby permitting the establishment of a pathogenic or symbiotic interaction (20). T3S systems are ancestrally related to the flagellar T3S system, having in common a basal body spanning the inner and outer bacterial membranes responsible for the appropriate selection of polypeptides delivered into a hollow channel leading out of the bacterium. At the outer surface, flagellar polypeptides travel the length of the adjoining hook and filament, but in T3S systems, the secreted polypeptides pass through a special hollow needle that extends away from the bacterium to the targeted host cell (10, 21, 22). Heterologous multimeric proteins localized to the tip of the needle form the translocon, a porelike channel that is assembled in the eukaryotic plasma membrane, enabling the injection of bacterial effectors (24, 48, 51).Two terminologies are distinctly used to describe protein transport by T3S systems. While “secretion” is a transport event for proteins from the bacterial cytosol into the extracellular milieu, “translocation” is a transport event for proteins from the bacterial cytosol into the eukaryotic host''s cytosol. Generally, secretion but not translocation is mediated by the first 20 amino acids of effector proteins (41, 46, 47), albeit mRNA sequences at the N terminus of some proteins have been also considered to function as the secretion signals (3, 44). This secretion event is independent of the presence of cognate effector chaperones (46, 59). Despite no conservation of the amino acids among the secretion signals, amphipathic or disordered secondary structures of the peptides are thought to function as the secretion signals recognized by the T3S apparatuses (22, 34, 35). In contrast, translocation usually requires both the secretion (the first 20 amino acids) and the translocation (amino acids 20 to 100) signals (46, 47, 59). This translocation event is efficiently mediated by the presence of the cognate chaperones (9, 14, 30), and the chaperone-effector complexes have been proposed to function as the three-dimensional signals recognized by the T3S apparatuses (5, 33, 38, 49, 50).Many T3S effectors employ cognate chaperones in the bacterial cytoplasm (43, 57). The effector chaperones have been categorized into two subgroups, class 1A and class 1B, primarily based on the substrate properties (and the gene locations) (13, 43). Class 1A chaperones commonly bind to one effector, and most of them are encoded by genes located adjacent to the gene encoding the cognate effectors. In contrast, class 1B chaperones bind to multiple effectors and are encoded by genes located within operons that code for structural components of the T3S apparatus that are distant to the cognate effector genes. Evolutionally, this subgroup of chaperones is thought to be an archetype of effector chaperones. Although T3S effector chaperones lack primary sequence similarity even in same subgroup, overall the effector chaperones whose three-dimensional structures are solved share similar folds, consisting of three α-helices and five β-strands (5, 36, 38, 49, 54). Similarly, effector chaperones share the common biochemical characteristics of acidic properties (pI 4 to 5) and low molecular masses (12 to 15 kDa), with a tendency to form homodimers (43). These homodimers recognize the chaperone binding domains (CBD) of the cognate effectors, which are usually located in the amino-terminal 20 to 100 amino acids (translocation signal) of the effector (19, 30, 59). Despite the wealth of information about individual chaperones, a universally accepted model for the mechanisms by which they promote secretion is lacking. One study shows that the guidance of chaperone-effector complexes toward the T3S apparatus is provided by the affinity of their chaperones to the ATPase of the T3S apparatus, whereby the ATPase releases the chaperones from the complexes and then unfolds the cognate effector for secretion (2). Several additional functions of T3S effector chaperones have been reported, including the prevention of effector aggregation prior to delivery to the secretion system, limitation of premature interactions, and protection of effectors from protease degradation in bacterial cells (17, 43). When an organism has multiple T3S pathways, as is the case for some Yersinia spp., there is the opportunity to gain new insight into how a given chaperone might influence T3S system specificity for substrates. Without direct testing of the aforementioned mechanistic models, the role of a chaperone in T3S and how it affects the overall sequence of pathogenic events is, at best, a conjecture.Highly virulent strains of Yersinia enterocolitica bv. 1B have a total of three T3S systems. The first T3S system (Ysc) is encoded by the virulence plasmid, and it secretes six effectors termed Yops. Ysc T3S is important for systemic infection (11, 12, 42). This T3S system is common to all Yersinia species pathogenic to humans, including another enteropathogen, Yersinia pseudotuberculosis, and the plague pathogen Yersinia pestis. The second system (Ysa) is encoded by a cluster of genes mapping to the Ysa pathogenicity island (25, 53). The Ysa T3S system secretes a set of eight effectors termed Ysps and, interestingly, also secretes three Yops, YopE, YopN, and YopP/YopJ (39, 58, 61). This Ysa T3S system is restricted to clinical isolates of Y. enterocolitica bv. 1B and promotes the initial establishment of infection in gastrointestinal tissue (39, 55). The third T3S system is an integral part of the flagellum and secretes proteins termed Fops to the extracellular milieu (64).Previously, we identified the suite of Ysp proteins secreted by the Ysa T3S system (39). However, little is known about the detailed mechanism by which these proteins are secreted and translocated by this system. Among the Ysp proteins identified, YspP is a protein tyrosine phosphatase (PTPase) whose activity is required for full virulence (39). Here, we found a small open reading frame (ORF) immediately downstream of yspP and designated it sycP. The SycP protein was demonstrated to be a YspP-specific chaperone essential for both the secretion and the translocation of YspP by the Ysa T3S system. In addition, we also examined the secretion specificity requirements for YspP secretion by three different T3S systems as model cases. Interestingly, our data suggest that the mechanisms by which the secretion and translocation signals are recognized are different, depending on the type of T3S system examined.