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József Dobó János Varga Ráchel Sajó Barbara M. Végh Péter Gál Péter Závodszky Ferenc Vonderviszt 《Applied and environmental microbiology》2010,76(3):891-899
Recently, we have demonstrated that the 26-47 segment of Salmonella enterica serovar Typhimurium flagellin is capable of mediating flagellar export. In order to reveal whether other parts of the N-terminal region have any significant influence on secretion, a series of plasmids were constructed containing the lac promoter followed by the 26-47, 2-65, or 2-192 portion of Salmonella flagellin, to which various heterologous proteins of different size were fused (18 constructs overall). Essentially, all three segments could drive protein export; however, the nature of the attached polypeptide also had a significant effect on secretion efficiency. When low export efficiency was observed, it was mainly caused by inclusion body formation. Our data provide strong support for the idea that a short segment within the disordered N-terminal region of axial proteins is recognized by the flagellar type III export machinery. The 26-47 segment of flagellin contains all of the necessary information to direct translocation of attached polypeptide chains. This short (positions 26 to 47) flagellin segment attached to recombinant proteins can be used for secreted protein expression. Certain fusion proteins that are easily degraded within the cells were found to be intact in the medium, implying a potential application of this expression system for proteins with high proteolytic susceptibility.The bacterial flagellum is a biological nanomachine for locomotion. A membrane embedded molecular motor rotates a long helical filament that works as a propeller driving the bacterium through the liquid environment. The filamentous portion of flagellum extends from the cytoplasm to the cell exterior and involves several substructures: the rod, the hook, the hook-filament junction, the long helical filament, and a cap at the filament tip.The flagellar proteins forming the structures lying beyond the cytoplasmic membrane are synthesized in the cell and exported sequentially by the flagellum-specific protein export apparatus from the cytoplasm to the site of assembly at the distal end of the growing filament (23). Thousands of subunits must be translocated through the narrow (20 to 25 Å wide) central channel of the flagellum (42) in a mostly unfolded conformation. It is puzzling how subunits can be efficiently transported through the hollow core of filaments over large (10- to 15-μm) distances. The hydrophilic inner surface of the channel is supposed to be essential for rapid and efficient transport. It is an interesting question whether the export channel is specialized for the delivery of flagellar proteins, or whether it is capable of passing a wide variety of polypeptide chains.The flagellar protein export system is thought to exist at the cytoplasmic face of the basal body to distinguish flagellar proteins from other cytoplasmic proteins and to facilitate their transportation (28). Since the identification and enzymatic characterization of FliI ATPase as a component of the flagellar protein export system, it had been thought that the flagellar protein export is driven by the energy of ATP hydrolysis (37). Recent studies, however, have clearly shown that the proton motive force across the cytoplasmic membrane is responsible for driving the export process that involves unfolding of export substrate proteins and translocation of the unfolded chains with the help of the FliI hexamer ring complex (27, 31).The flagellar protein export system belongs to the family of the type III secretion systems (T3SSs) (4), which also include those for secretion of virulence factors by a wide variety of pathogenic bacteria (15). The nature of the signal directing flagellar protein secretion is still debated because the protein substrates have no cleavable signal sequences or do not share any obvious consensus sequence (12). It has been suggested that the recognition of flagellar export substrates may involve mRNA signals (25), but a growing amount of evidence indicates that the signal is located in the disordered N-terminal region of the secreted proteins (6, 11, 12, 19, 33, 36, 40). For example, residues 38 to 58 of Caulobacter crescentus flagellar hook protein were found to be essential for secretion (19). Similarly, it has been demonstrated that the export signal of the hook scaffolding protein FlgD of Escherichia coli is located exclusively within the N-terminal 71 amino acids (40). Single amino acid substitutions within the N-terminal region of the anti-sigma factor FlgM (6), which is also exported by the flagellum-specific export apparatus, severely impair its export.Our previous experiments have suggested that the 26-47 disordered segment of Salmonella flagellin contains the recognition signal for the flagellar export machinery (36). When this segment was attached to the small CCP2 domain of the human C1r complement protein, the fusion construct was secreted into the culture medium. The objective of the present study was to explore whether other N-terminal portions of flagellin have any significant influence on secretion. We also aimed to reveal whether the recognition signal can facilitate translocation of a wide variety attached foreign proteins through the flagellum-specific export pathway, which may open up the possibility to use the flagellar export system to secrete heterologous proteins overexpressed in bacteria. 相似文献
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An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpLM, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpLM and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpLM is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impLM mutants with substitutions or deletions in the Walker A motif failed to complement the impLM deletion mutant for Hcp secretion, which provided evidence that ImpLM may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpLM and another essential T6SS component, ImpKL. Topology and biochemical fractionation analyses suggested that ImpKL is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpLM-ImpKL interaction domains suggested that ImpLM interacts with ImpKL via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpLM interacts with ImpKL, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.Many pathogenic gram-negative bacteria employ protein secretion systems formed by macromolecular complexes to deliver proteins or protein-DNA complexes across the bacterial membrane. In addition to the general secretory (Sec) pathway (18, 52) and twin-arginine translocation (Tat) pathway (7, 34), which transport proteins across the inner membrane into the periplasm, at least six distinct protein secretion systems occur in gram-negative bacteria (28, 46, 66). These systems are able to secrete proteins from the cytoplasm or periplasm to the external environment or the host cell and include the well-documented type I to type V secretion systems (T1SS to T5SS) (10, 15, 23, 26, 30) and a recently discovered type VI secretion system (T6SS) (4, 8, 22, 41, 48, 49). These systems use ATPase or a proton motive force to energize assembly of the protein secretion machinery and/or substrate translocation (2, 6, 41, 44, 60).Agrobacterium tumefaciens is a soilborne pathogenic gram-negative bacterium that causes crown gall disease in a wide range of plants. Using an archetypal T4SS (9), A. tumefaciens translocates oncogenic transferred DNA and effector proteins to the host and ultimately integrates transferred DNA into the host genome. Because of its unique interkingdom DNA transfer, this bacterium has been extensively studied and used to transform foreign DNA into plants and fungi (11, 24, 40, 67). In addition to the T4SS, A. tumefaciens encodes several other secretion systems, including the Sec pathway, the Tat pathway, T1SS, T5SS, and the recently identified T6SS (72). T6SS is highly conserved and widely distributed in animal- and plant-associated Proteobacteria and plays an important role in the virulence of several human and animal pathogens (14, 19, 41, 48, 56, 63, 74). However, T6SS seems to play only a minor role or even a negative role in infection or virulence of the plant-associated pathogens or symbionts studied to date (5, 37-39, 72).T6SS was initially designated IAHP (IcmF-associated homologous protein) clusters (13). Before T6SS was documented by Pukatzki et al. in Vibrio cholerae (48), mutations in this gene cluster in the plant symbiont Rhizobium leguminosarum (5) and the fish pathogen Edwardsiella tarda (51) caused defects in protein secretion. In V. cholerae, T6SS was responsible for the loss of cytotoxicity for amoebae and for secretion of two proteins lacking a signal peptide, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein (VgrG). Secretion of Hcp is the hallmark of T6SS. Interestingly, mutation of hcp blocks the secretion of VgrG proteins (VgrG-1, VgrG-2, and VgrG-3), and, conversely, vgrG-1 and vgrG-2 are both required for secretion of the Hcp and VgrG proteins from V. cholerae (47, 48). Similarly, a requirement of Hcp for VgrG secretion and a requirement of VgrG for Hcp secretion have also been shown for E. tarda (74). Because Hcp forms a hexameric ring (41) stacked in a tube-like structure in vitro (3, 35) and VgrG has a predicted trimeric phage tail spike-like structure similar to that of the T4 phage gp5-gp27 complex (47), Hcp and VgrG have been postulated to form an extracellular translocon. This model is further supported by two recent crystallography studies showing that Hcp, VgrG, and a T4 phage gp25-like protein resembled membrane penetration tails of bacteriophages (35, 45).Little is known about the topology and structure of T6SS machinery subunits and the distinction between genes encoding machinery subunits and genes encoding regulatory proteins. Posttranslational regulation via the phosphorylation of Fha1 by a serine-threonine kinase (PpkA) is required for Hcp secretion from Pseudomonas aeruginosa (42). Genetic evidence for P. aeruginosa suggested that the T6SS may utilize a ClpV-like AAA+ ATPase to provide the energy for machinery assembly or substrate translocation (41). A recent study of V. cholerae suggested that ClpV ATPase activity is responsible for remodeling the VipA/VipB tubules which are crucial for type VI substrate secretion (6). An outer membrane lipoprotein, SciN, is an essential T6SS component for mediating Hcp secretion from enteroaggregative Escherichia coli (1). A systematic study of the T6SS machinery in E. tarda revealed that 13 of 16 genes in the evp gene cluster are essential for secretion of T6S substrates (74), which suggests the core components of the T6SS. Interestingly, most of the core components conserved in T6SS are predicted soluble proteins without recognizable signal peptide and transmembrane (TM) domains.The intracellular multiplication F (IcmF) and H (IcmH) proteins are among the few core components with obvious TM domains (8). In Legionella pneumophila Dot/Icm T4SSb, IcmF and IcmH are both membrane localized and partially required for L. pneumophila replication in macrophages (58, 70, 75). IcmF and IcmH are thought to interact with each other in stabilizing the T4SS complex in L. pneumophila (58). In T6SS, IcmF is one of the essential components required for secretion of Hcp from several animal pathogens, including V. cholerae (48), Aeromonas hydrophila (63), E. tarda (74), and P. aeruginosa (41), as well as the plant pathogens A. tumefaciens (72) and Pectobacterium atrosepticum (39). In E. tarda, IcmF (EvpO) interacted with IcmH (EvpN), EvpL, and EvpA in a yeast two-hybrid assay, and its putative nucleotide-binding site (Walker A motif) was not essential for secretion of T6SS substrates (74).In this study, we characterized the topology and interactions of the IcmF and IcmH family proteins ImpLM and ImpKL, which are two essential components of the T6SS of A. tumefaciens. We adapted the nomenclature proposed by Cascales (8), using the annotated gene designation followed by the letter indicated by Shalom et al. (59). Our data indicate that ImpLM and ImpKL are both integral inner membrane proteins and interact with each other via their N-terminal domains residing in the cytoplasm. We also provide genetic evidence showing that ImpLM may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion. 相似文献
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Murat Balaban Stephanie N. Joslin David R. Hendrixson 《Journal of bacteriology》2009,191(21):6602-6611
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. 相似文献
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Tohru Minamino Masafumi Shimada Mayuko Okabe Yumiko Saijo-Hamano Katsumi Imada May Kihara Keiichi Namba 《Journal of bacteriology》2010,192(7):1929-1936
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 (FlhAC) 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 FlhAC. 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 FlhAC consists of at least three subdomains, a flexible linker, FlhACN, and FlhACC, and that FlhACN 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 FlhAC 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 (FlhATM) and a hydrophilic C-terminal cytoplasmic region (FlhAC) (12, 15). FlhATM is responsible for the association with the MS ring (9). FlhAC 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 FlhAC 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 FlhAC is also required for substrate recognition (7). These observations suggest that an interaction between FlhAC 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 FlhAC for possible rearrangement for the export function. 相似文献
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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. 相似文献
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Manabu Konishi Masaomi Kanbe Jonathan L. McMurry Shin-Ichi Aizawa 《Journal of bacteriology》2009,191(19):6186-6191
The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a Fla− phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN- or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact.The flagellum is the ultrastructure for motility in many bacterial species (1). Flagellar assembly requires about 50 genes, among which about 20 gene products are incorporated in the complete flagellum (12). Most structural proteins and others necessary for assembly are exported through a flagellum-specific type III secretion apparatus housed within the basal body. The apparatus consists of at least six integral membrane proteins: FlhA, FlhB, FliP, FliQ, FliR, and FliO (for salmonellae and other species) (1, 12). Other proteins are also involved. FliI is the only known ATPase among flagellar proteins (2). FliI interacts with FliJ, which is of unknown function, and with a dimer of FliH, an inhibitor of FliI. The apparatus can be visualized by quick-freeze electron microscopy and has been termed the C (cytoplasmic) rod by virtue of its appearance and membrane-proximal location inside the C ring (7). The C ring is composed of three component proteins: FliG, FliM, and FliN (3). Mutations or deletions of any of these proteins cause a nonflagellate (Fla−) phenotype, strongly suggesting that the C ring is necessary for flagellar protein export (6, 22, 26). The trimer FliH2-FliI specifically binds FliN (4, 15), suggesting that FliI docks at the periphery of the C ring through interactions with FliN-bound FliH, standing ready to escort export substrates to the secretion gate that is probably composed by FlhA, FlhB, and others (15).The C ring has long been studied with respect to motor function rather than export function. It has been proposed that FliG plays a major role in torque generation in concert with MotAB complexes, leaving the other two proteins, FliM and FliN, in minor and supporting roles (10, 11). However, as mentioned above, all three components are required for flagellar protein export (6, 22, 26). Together with the C ring, FliI pushes export substrates into the gate using the energy of ATP hydrolysis. Just recently, it was shown that FliI ATPase activity is not absolutely necessary for protein export and that increasing proton motive force (PMF) or reversion mutations in FlhA and FlhB can compensate for its absence (17, 21).In order to elucidate the roles that FliG, FliM, and FliN play in export, we employed C-ring-defective mutants. Here, we show that the overproduction of FliI allows flagellar formation in C-ring-defective mutants. We closely examined flagella formed in those mutants by electron microscopy, noting percentages of flagellation in each population, analyzing partially formed structures, and measuring hook length. 相似文献
15.
Gabriele Margos Stephanie A. Vollmer Muriel Cornet Martine Garnier Volker Fingerle Bettina Wilske Antra Bormane Liliana Vitorino Margarida Collares-Pereira Michel Drancourt Klaus Kurtenbach 《Applied and environmental microbiology》2009,75(16):5410-5416
Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48). 相似文献
16.
Masafumi Koike Hiroyuki Terashima Seiji Kojima Michio Homma 《Journal of bacteriology》2010,192(1):375-378
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. 相似文献
17.
Andrea Koerdt Anja Paulick Marlen Mock Kathinka Jost Kai M. Thormann 《Journal of bacteriology》2009,191(16):5085-5093
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. 相似文献
18.
19.