Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.Flagellins from many polarly flagellated bacteria are glycosylated (reviewed in reference 22). The best-characterized examples are the flagellins from Campylobacter spp. that are decorated with as many as 19 O-linked glycans that can contribute ∼10% to the weight of flagellin (38). The genes encoding the enzymes for biosynthesis of the glycans found on Campylobacter flagellins and the respective glycosyltransferases are located adjacent to the flagellin structural genes in one of the more hypervariable regions of the Campylobacter genome (3, 16, 28, 37). Most strains appear to carry the genes for synthesis of two distinct nine-carbon sugars that decorate flagellin: pseudaminic acid (PseAc) and an acetamidino form of legionaminic acid (LegAm) (23). In contrast, Campylobacter jejuni strain 81-176 contains only the pathway for synthesis of PseAc (9) and derivatives of PseAc that include an acetylated form (PseAcOAc), an acetamidino form (PseAm), and a form of PseAm with a glutamic acid moiety attached (PseAmOGln) (25, 34, 38). The flagellins of C. jejuni strain NCTC 11168 have recently been shown to be glycosylated with PseAc and LegAm, as well as two novel derivatives of PseAc, a di-O-methylglyceric acid and a related acetamidino form (24). Thus, although all of the flagellar glycans appear to be based on either PseAc and/or LegAm, there are variations among strains that contribute to serospecificity and reflect the heterogeneity of the flagellin glycosylation loci (23, 24).The function of the glycosyl modifications to flagellar structure and to the biology of campylobacters is not fully understood. Although most polarly flagellated bacteria appear to glycosylate flagellin, mutation of the genes involved in glycosylation does not generally result in loss of motility (22). However, flagella from C. jejuni, Campylobacter coli, and Helicobacter pylori, all members of the epsilon division of Proteobacteria, are unable to assemble a filament in the absence of a functional glycosylation system (7, 33). Also, changes in the glycans on campylobacter flagellins have been shown to affect autoagglutination (AAG) and microcolony formation on intestinal epithelial cells in vitro (5, 9). Thus, a mutant of C. jejuni 81-176 that was unable to synthesize PseAm assembled a flagellar filament, but the sites on the flagellin subunits that were normally glycosylated with PseAm were instead glycosylated with PseAc. This mutant was reduced in AAG, adherence, and invasion of INT407 cells and was also attenuated in a ferret diarrheal disease model (9). C. coli VC167 has both PseAc and LegAm pathways. Mutants that were defective in either pathway could still assemble flagellar filaments composed of subunits that were modified with the alternate sugar, but these mutants showed defects in AAG (7). A VC167 double mutant, defective in both PseAc and LegAm synthesis, was nonflagellated (7). Collectively, these data suggest that some glycosylation is required for either secretion of flagellin or for interactions between subunits within the filament.Flagellar biogenesis in C. jejuni is a complex process that is highly controlled by the alternate sigma factors σ²⁸ and σ⁵⁴, a two-component regulatory system composed of the sensor kinase FlgS and the σ⁵⁴-response regulator FlgR, and the flagellar export apparatus (15, 39). Both flgR and flgS genes undergo slip strand mismatch repair in C. jejuni strain 81-176, resulting in an on/off-phase variation of flagellar expression (13, 14). The major flagellin gene, flaA, and some other late flagellar genes are regulated by σ²⁸; the genes encoding the minor flagellin, flaB, and the hook and rod structures are regulated by σ⁵⁴. Here, we examine several aspects of glycosylation to flagellar function in C. jejuni 81-176. We demonstrate that some components of the flagellar glycosylation machinery are localized to the poles of the cell, but independently of the signal recognition particle-like flagellar protein, FlhF, and that flagellin glycosylation occurs independently of the flagellar regulon. We also show that the glycans on some amino acids appear to play a structural role in subunit interactions in the filament, while others affect interactions with adjacent filaments that result in AAG.     

Proteomic and Genomic Characterization of Highly Infectious Clostridium difficile 630 Spores

Clostridium difficile, a major cause of antibiotic-associated diarrhea, produces highly resistant spores that contaminate hospital environments and facilitate efficient disease transmission. We purified C. difficile spores using a novel method and show that they exhibit significant resistance to harsh physical or chemical treatments and are also highly infectious, with <7 environmental spores per cm² reproducibly establishing a persistent infection in exposed mice. Mass spectrometric analysis identified ∼336 spore-associated polypeptides, with a significant proportion linked to translation, sporulation/germination, and protein stabilization/degradation. In addition, proteins from several distinct metabolic pathways associated with energy production were identified. Comparison of the C. difficile spore proteome to those of other clostridial species defined 88 proteins as the clostridial spore “core” and 29 proteins as C. difficile spore specific, including proteins that could contribute to spore-host interactions. Thus, our results provide the first molecular definition of C. difficile spores, opening up new opportunities for the development of diagnostic and therapeutic approaches.Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that can asymptomatically colonize the intestinal tracts of humans and other mammals (3, 30, 39). Antibiotic treatment can result in C. difficile overgrowth and can lead to clinical disease, ranging from diarrhea to life-threatening pseudomembranous colitis, particularly in immunocompromised hosts (2, 4, 7). In recent years, C. difficile has emerged as the major cause of nosocomial antibiotic-induced diarrhea, and it is frequently associated with outbreaks (21, 22). A contributing factor is that C. difficile can be highly infectious and difficult to contain, especially when susceptible patients are present in the same hospital setting (13).Person-to-person transmission of C. difficile is associated with the excretion of highly resistant spores in the feces of infected patients, creating an environmental reservoir that can confound many infection control measures (29, 44). Bacterial spores, which are metabolically dormant cells that are formed following asymmetric cell division, normally have thick concentric external layers, the spore coat and cortex, that protect the internal cytoplasm (15, 42). Upon germination, spores lose their protective external layers and resume vegetative growth (24, 27, 36). Bacillus spores and the spores of most Clostridium species germinate in response to amino acids, carbohydrates, or potassium ions (24, 36). In contrast, C. difficile spores show an increased level of germination in response to cholate derivatives found in bile (40, 41). Thus, spores are well adapted for survival and dispersal under a wide range of environmental conditions but will germinate in the presence of specific molecular signals (24, 36).While the spores of a number of Bacillus species, such as Bacillus subtilis and Bacillus anthracis, and those of other Clostridium species, such as Clostridium perfringens (15, 20), have been well characterized, research on C. difficile spores has been relatively limited. A greater understanding of C. difficile spore biology could be exploited to rationalize disinfection regimes, molecular diagnostics, and the development of targeted treatments such as vaccines. Here we describe a novel method to isolate highly purified C. difficile spores that maintain their resistance and infectious characteristics, thus providing a unique opportunity to study C. difficile spores in the absence of vegetative cells. A thorough proteomic and genomic analysis of the spore provides novel insight into the unique composition and predictive biological properties of C. difficile spores that should underpin future research into this high-profile but poorly understood pathogen.     

Inhibiting the Initiation of Clostridium difficile Spore Germination using Analogs of Chenodeoxycholic Acid,a Bile Acid

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is pathogenic for both humans and animals (33, 44). Infections caused by C. difficile range from mild diarrhea to more life-threatening conditions, such as pseudomembranous colitis (33). In the classic case, prior antibiotic treatment that disrupts the normally protective colonic flora makes patients susceptible to C. difficile infection (CDI) (35, 53). Other antibiotics, such as vancomycin and metronidazole, are the most commonly used treatments for CDI (54). However, because these antibiotics also disrupt the colonic flora, 10 to 40% of patients whose symptoms have been ameliorated suffer from relapsing CDI (15, 24). The annual treatment-associated cost for CDI in the United States is estimated to be between $750 million and $3.2 billion (8, 9, 16, 31). Moreover, the number of fatal cases of CDI has been increasing rapidly (14, 39). Thus, there is an urgent need to find alternative therapies for CDI.C. difficile infection likely is initiated by infection with the spore form of C. difficile (12). C. difficile elicits disease through the actions of two secreted toxins, TcdA and TcdB (48). TcdB was recently shown to be critical for pathogenesis in an animal model of disease (18). Since the toxins are produced by vegetative cells, not by spores (17), germination and outgrowth are prerequisites for pathogenesis.Spore germination is triggered by the interaction of small molecules, called germinants, with receptors within the spore inner membrane. These germinants vary by bacterial species and can include ions, amino acids, sugars, nucleotides, surfactants, or combinations thereof (43). The recognition of germinants triggers irreversible germination, leading to Ca²⁺-dipicolinic acid release, the uptake of water, the degradation of the cortex, and, eventually, the outgrowth of the vegetative bacterium (43). The germination receptors that C. difficile uses to sense the environment have not been identified. Based on homology searches, C. difficile germination receptors must be very different from known germination receptors (42), but they appear to be proteinaceous (13).Taurocholate, a primary bile acid, has been used for approximately 30 years by researchers and clinical microbiologists to increase colony formation by C. difficile spores from patient and environmental samples (3, 49, 51, 52). This suggested that C. difficile spores interact with bile acids along the gastrointestinal (GI) tract and that spores use a host-derived signal to initiate germination.The liver synthesizes the two major primary bile acids, cholate and chenodeoxycholate (40). These compounds are modified by conjugation with either taurine (to give taurocholate or taurochenodeoxycholate) or glycine (producing glycocholate or glycochenodeoxycholate). Upon secretion into the digestive tract, bile aids in the absorption of fat and cholesterol; much of the secreted bile is actively absorbed and recycled back to the liver for reutilization (40). Though efficient, enterohepatic recirculation is not complete; bile enters the cecum of the large intestine at a concentration of approximately 2 mM (30).In the cecum, bile is modified by the normal, benign colonic flora. First, bile salt hydrolases found on the surfaces of many bacterial species remove the conjugated amino acid, producing the deconjugated primary bile acids cholate and chenodeoxycholate (40). These deconjugated primary bile acids are further metabolized by only a few species of intestinal bacteria, including Clostridium scindens. C. scindens actively transports unconjugated primary bile acids into the cytoplasm, where they are 7α-dehydroxylated, converting cholate to deoxycholate and chenodeoxycholate to lithocholate (21, 40). The disruption of the colonic flora by antibiotic treatment abolishes 7α-dehydroxylation activity (41).Building upon the work on Wilson and others (51, 52), we demonstrated that taurocholate and glycine, acting together, trigger the loss of the birefringence of C. difficile spores (45). All cholate derivatives (taurocholate, glycocholate, cholate, and deoxycholate) stimulate the germination of C. difficile spores (45). Recently it was shown that taurocholate binding is prerequisite to glycine binding (37). At physiologically relevant concentrations, chenodeoxycholate inhibits taurocholate-mediated germination (46) and also inhibits C. difficile vegetative growth, as does deoxycholate (45). In fact, C. difficile spores use the relative concentrations of the various bile acids as cues for germination within the host (10).Since chenodeoxycholate is absorbed by the colonic epithelium and metabolized to lithocholate by the colonic flora (25, 40), the use of chenodeoxycholate as a therapy against C. difficile disease might be hindered by its absorption and conversion to lithocholate.Here, we further characterize the interaction of C. difficile spores with various bile acids and demonstrate that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination. Further, we identify chemical analogs of chenodeoxycholate that are more potent inhibitors of germination and that resist 7α-dehydroxylation by the colonic flora, potentially increasing their stability and effectiveness as inhibitors of C. difficile spore germination in the colonic environment.     

Application of a Short,Disordered N-Terminal Flagellin Segment,a Fully Functional Flagellar Type III Export Signal,to Expression of Secreted Proteins

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.     

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.     

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.     

Use of Purified Clostridium difficile Spores To Facilitate Evaluation of Health Care Disinfection Regimens

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 10⁶ pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is a major cause of health care-acquired infections and antibiotic-associated diarrhea (2). In recent years, several genetic variants of C. difficile have emerged as important health care pathogens (6). Perhaps most notable is the “hypervirulent” variant, commonly referred to as PCR ribotype 027/restriction endonuclease analysis (REA) group BI, that produces elevated levels of toxins TcdA and TcdB (17, 19). Other virulent ribotypes that display extensive heterogeneity among their toxin protein sequences (26) and gene activities (8) have emerged. Using whole-genome sequencing, we demonstrated that there are broad genetic differences between the entire genomes of several common variants, including ribotype/REA group variants 012/R, 017/CF, and 027/BI used in this study (12, 27, 31). In contrast, phylogeographic analysis of 027/BI isolates from Europe and the United States demonstrates that this clade is extremely clonal and implies recent transcontinental spread of hypervirulent C. difficile (12).C. difficile is distinct from many other health care pathogens because it produces highly infectious spores that are shed into the environment (25, 28). C. difficile spores can resist disinfection regimens that normally inactivate other health care pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, therefore challenging current infection control measures (2). A multifaceted approach is normally used to control C. difficile in health care facilities (32). Interventions include antimicrobial stewardship, increased clinical awareness, patient isolation (11), and enhanced environmental disinfection regimens based on hydrogen peroxide (H₂O₂) vapor (HPV) (4). While attempts to break the spore-mediated infection cycle and interrupt these efficient routes of transmission are important for infection control measures, there is little quantitative evidence indicating which interventions are most effective (7). Here we describe the exploitation of pure C. difficile spores (16) and a murine transmission model (15) in simple, practical methods to quantitatively monitor the impact of health care disinfection regimens on C. difficile viability. These methods can be used to optimize disinfection regimens targeted at C. difficile.     

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.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

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).     

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.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

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.