Cyclic-di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa: the pilY1 Gene and Its Impact on Surface-Associated Behaviors

The intracellular signaling molecule cyclic-di-GMP (c-di-GMP) has been shown to influence surface-associated behaviors of Pseudomonas aeruginosa, including biofilm formation and swarming motility. Previously, we reported a role for the bifA gene in the inverse regulation of biofilm formation and swarming motility. The bifA gene encodes a c-di-GMP-degrading phosphodiesterase (PDE), and the ΔbifA mutant exhibits increased cellular pools of c-di-GMP, forms hyperbiofilms, and is unable to swarm. In this study, we isolated suppressors of the ΔbifA swarming defect. Strains with mutations in the pilY1 gene, but not in the pilin subunit pilA gene, show robust suppression of the swarming defect of the ΔbifA mutant, as well as its hyperbiofilm phenotype. Despite the ability of the pilY1 mutation to suppress all the c-di-GMP-related phenotypes, the global pools of c-di-GMP are not detectably altered in the ΔbifA ΔpilY1 mutant relative to the ΔbifA single mutant. We also show that enhanced expression of the pilY1 gene inhibits swarming motility, and we identify residues in the putative VWA domain of PilY1 that are important for this phenotype. Furthermore, swarming repression by PilY1 specifically requires the diguanylate cyclase (DGC) SadC, and epistasis analysis indicates that PilY1 functions upstream of SadC. Our data indicate that PilY1 participates in multiple surface behaviors of P. aeruginosa, and we propose that PilY1 may act via regulation of SadC DGC activity but independently of altering global c-di-GMP levels.Pseudomonas aeruginosa forms surface-attached communities known as biofilms, and this microbe is also capable of surface-associated motility, including twitching and swarming. The mechanism by which cells regulate and coordinate these various surface-associated behaviors, or how these microbes transition from one surface behavior to another, has yet to be elucidated. Given that P. aeruginosa is capable of such diverse surface-associated lifestyles, this Gram-negative organism serves as a useful model to address questions regarding the regulation of surface-associated behaviors.Recent studies indicate that biofilm formation and swarming motility by P. aeruginosa are inversely regulated via a common pathway (12, 27, 37). Important factors that influence early biofilm formation by P. aeruginosa strain PA14 include control of flagellar motility and the robust production of the Pel exopolysaccharide (EPS). Swarming occurs when cells move across a hydrated, viscous semisolid surface, and like biofilm formation, flagellar function is important for this surface-associated motility. Additionally, swarming requires production of rhamnolipid surfactant acting as a surface-wetting agent (25, 58). In contrast to biofilm formation, swarming motility is enhanced in strains which are defective for the production of Pel EPS (12).The inverse regulation of biofilm formation and swarming motility is reminiscent of the regulation of sessile and motile behaviors that occurs in a wide range of bacterial species via the intracellular signaling molecule cyclic-di-GMP (c-di-GMP) (17, 24, 50, 51, 56). High levels of this signaling molecule promote sessile behaviors and inhibit motility, whereas low levels of c-di-GMP favor motile behaviors (8, 9, 22, 56). Recently, we reported that the BifA phosphodiesterase, which catalyzes the breakdown of c-di-GMP, inversely regulates biofilm formation and swarming motility (27). In addition, Merritt et al. reported that SadC, a diguanylate cyclase (DGC) which synthesizes c-di-GMP, participates with BifA to modulate cellular c-di-GMP levels and thus regulate biofilm formation and swarming motility (37).Consistent with a role for BifA as a c-di-GMP phosphodiesterase, ΔbifA mutants exhibit increased cellular pools of c-di-GMP relative to the wild type (WT) (27). Phenotypically, ΔbifA mutants form hyperbiofilms and are unable to swarm. The hyperbiofilm phenotype of the ΔbifA mutant results largely from increased synthesis of the pel-derived polysaccharide; that is, the ΔbifAΔpel double mutant shows a marked decrease in biofilm formation compared to the ΔbifA mutant (27). Interestingly, elevated Pel polysaccharide production alone is not sufficient to explain the swarming defect of the ΔbifA mutant, as the ΔbifAΔpel double mutant recovers only minimal swarming ability (27). These data indicate that high levels of c-di-GMP inhibit swarming motility in a largely Pel-independent manner.To better understand how elevated c-di-GMP levels in the cell inhibit swarming motility, we exploited the swarming defect of the ΔbifA mutant, and using a genetic screen, we identified suppressors in the ΔbifA background that restored the ability to swarm. Here we report a role for the PilY1 protein in repression of swarming motility in the ΔbifA mutant background. Our data are consistent with a model in which PilY1 functions upstream of the c-di-GMP diguanylate cyclase SadC to regulate swarming motility by P. aeruginosa.     

Levels of the Secreted Vibrio cholerae Attachment Factor GbpA Are Modulated by Quorum-Sensing-Induced Proteolysis

Vibrio cholerae is the etiologic agent of cholera in humans. Intestinal colonization occurs in a stepwise fashion, initiating with attachment to the small intestinal epithelium. This attachment is followed by expression of the toxin-coregulated pilus, microcolony formation, and cholera toxin (CT) production. We have recently characterized a secreted attachment factor, GlcNAc binding protein A (GbpA), which functions in attachment to environmental chitin sources as well as to intestinal substrates. Studies have been initiated to define the regulatory network involved in GbpA induction. At low cell density, GbpA was detected in the culture supernatant of all wild-type (WT) strains examined. In contrast, at high cell density, GbpA was undetectable in strains that produce HapR, the central regulator of the cell density-dependent quorum-sensing system of V. cholerae. HapR represses the expression of genes encoding regulators involved in V. cholerae virulence and activates the expression of genes encoding the secreted proteases HapA and PrtV. We show here that GbpA is degraded by HapA and PrtV in a time-dependent fashion. Consistent with this, ΔhapA ΔprtV strains attach to chitin beads more efficiently than either the WT or a ΔhapA ΔprtV ΔgbpA strain. These results suggest a model in which GbpA levels fluctuate in concert with the bacterial production of proteases in response to quorum-sensing signals. This could provide a mechanism for GbpA-mediated attachment to, and detachment from, surfaces in response to environmental cues.Vibrio cholerae has adapted to lifestyles in dual environments, allowing survival in aquatic locations, as well as the ability to colonize the epithelium of the human small intestine. This intestinal colonization by V. cholerae is a prerequisite for the disease cholera in humans. Intestinal colonization proceeds in a stepwise manner, initiating with attachment to the epithelial cell layer by multiple attachment factors (26). This stable attachment localizes the bacterium in an environment conducive for activation of subsequent virulence factors, including the toxin-coregulated pilus, a type IVb pilus that mediates cell-cell interactions and microcolony formation (27). Cholera toxin (CT) is produced and extracellularly secreted by bacteria within the microcolonies and enters into intestinal epithelial cells. CT causes the disruption of fluid and electrolyte balance and results in the voluminous rice water diarrhea characteristically observed with cholera patients.The ability of V. cholerae to bind to surfaces is crucial for the initial stages of colonization of both the aquatic and intestinal environments. Previous studies observing V. cholerae in the aquatic setting identified the ability of the bacteria to attach to zooplankton and phytoplankton, binding to surface structures that include chitin as a major component (7, 10, 11, 19, 21, 42). Chitin, a polymer consisting primarily of a β-1,4 linkage of GlcNAc monomers, is the most abundant aquatic carbon source and, when presented on the surfaces of zooplankton, aquatic exoskeletons, algae, and plants, provides a substrate for V. cholerae surface binding (8, 19-22). V. cholerae is able to break down chitin into carbon to use as a nutrient source via degradation by secreted chitinases (12). We have described a protein, GbpA (GlcNAc binding protein A), which facilitates the binding of V. cholerae to chitin, specifically to the chitin monomer GlcNAc, a sugar residue that is also found on the surface of epithelial cells (3, 16, 26). GbpA mediates binding to chitin, GlcNAc, and exoskeletons of Daphnia magna, as well as participates in effective intestinal colonization within the infant mouse model of cholera (26). GbpA is a secreted protein that exits the cell via the type 2 secretion system by which it mediates attachment by a yet uncharacterized mechanism (26). Previous studies examining the role of GbpA in binding to surfaces have been conducted utilizing various wild-type (WT) strains of V. cholerae, specifically O395 (26) and N16961 (33). These strains both are of the O1 serogroup but are differentially classified as classical (43) and El Tor biotypes (18), respectively. The classical biotype was responsible for the first six pandemics of cholera, whereas El Tor is the cause of the current pandemic (39).Quorum sensing regulates multiple bacterial processes, including virulence, formation of biofilms, and bioluminescence (25, 35, 36). In contrast to many other bacterial quorum-sensing systems, virulence gene expression and biofilm formation in V. cholerae is expressed under conditions of low cell density and repressed at high cell density (17, 35, 48). HapR, a member of the TetR family of regulatory proteins, is a central regulator on which the three parallel inputs of the V. cholerae quorum-sensing system converge (30, 35). During low-cell-density conditions, characteristic of growth within the aquatic environment or stages of early intestinal colonization, the quorum-sensing system is not engaged. Under conditions of high cell density, bacterial numbers and secreted autoinducer molecules are increased to a level that triggers the V. cholerae quorum-sensing system.HapR regulates gene function in two ways, serving as both an activator and repressor. At high cell density, HapR functions in the capacity of a repressor of the toxin-coregulated pilus and CT virulence cascade (29, 31) as well as a repressor of vps gene expression (17), preventing biofilm formation. In addition to repressing gene expression, at high cell density HapR activates the expression of genes encoding extracellularly secreted proteases HapA and PrtV (14, 17, 23, 45-47). HapA, also referred to as hemagglutinin/protease (HA/P), was first reported as a mucinase by Burnet (6) and later characterized as a zinc- and calcium-dependent metalloprotease (4). Extracellularly secreted via the V. cholerae type 2 secretion pathway (40), HA/P has been demonstrated to cleave fibronectin, lactoferrin, and mucin (15), as well as to participate in the activation of the CT A subunit (5). Further studies have led to the suggestion that HA/P is a detachase, critical for the release of V. cholerae from the surface of intestinal cells (2, 14, 38). PrtV is a second protease encoded by a gene that is activated by HapR (47). It has been demonstrated to be essential for both V. cholerae killing of Caenorhabditis elegans, as well as protecting V. cholerae from predator grazing by various flagellates (32, 45).The data presented here indicate that HapA and PrtV participate in the targeted degradation of the attachment factor GbpA. We demonstrate that GbpA is present during the logarithmic phase of growth and conditions of low cell density but that it is not present in the supernatant of high-cell-density cultures of strains that express functional HapR. Further studies revealed that during stages of high cell density, proteases HapA and PrtV, encoded by HapR-activated genes, are responsible for GbpA degradation in the culture supernatant. These findings suggest that the attachment factor GbpA is potentially a ligand targeted for protease degradation during the epithelial detachment process. This process could aid in the release of V. cholerae back into the aquatic environment following late stages of intestinal colonization.     

Relatedness of Vibrio cholerae O1/O139 Isolates from Patients and Their Household Contacts,Determined by Multilocus Variable-Number Tandem-Repeat Analysis

The genetic relatedness of Vibrio cholerae O1/O139 isolates obtained from 100 patients and 146 of their household contacts in Dhaka, Bangladesh, between 2002 and 2005 was assessed by multilocus variable-number tandem-repeat analysis. Isolate genotypes were analyzed at five loci containing tandem repeats. Across the population, as well as within households, isolates with identical genotypes were clustered in time. Isolates from individuals within the same household were more likely to have similar or identical genotypes than were isolates from different households, but even within a household, isolates from different individuals often had different genotypes. When household contacts were sampled regularly for 3 weeks after the illness of the household index patient, isolates with genotypes related to the index patient appeared in contacts, on average, ∼3 days after the index patient, while isolates with unrelated genotypes appeared in contacts ∼6 days after. Limited data revealed that multiple isolates from the same individual collected within days of each other or even from a single stool sample may have identical, similar, or unrelated genotypes as well. Our results demonstrate that genetically related V. cholerae strains cluster in local outbreaks but also suggest that multiple distinct strains of V. cholerae O1 may circulate simultaneously within a household.Vibrio cholerae is the etiologic agent of cholera, a secretory diarrheal disease with a high mortality rate in humans if untreated (25). Serogroups of V. cholerae, a motile, Gram-negative, curved rod, can be defined serologically by the O side chain of the lipopolysaccharide (LPS) component of the outer membrane (9). V. cholerae is found in a variety of forms in aquatic ecosystems (41, 42), and more than 200 different serogroups have been isolated, mostly from environmental sources (45). However, the vast majority of V. cholerae strains that cause the clinical disease cholera belong to serogroup O1 or O139 (37, 42). V. cholerae O1, the historical agent of epidemic and pandemic cholera and the current leading cause of cholera both globally and in Bangladesh (42), is classified into two major biotypes, classical and El Tor (44), and two major serotypes, Ogawa and Inaba (48). The current global pandemic is caused by V. cholerae O1 El Tor. A second pathogenic serogroup, O139, emerged in the Bengal region in 1992 by horizontal transfer of new LPS biosynthesis-encoding genes into the El Tor biotype (1, 4). This new serogroup continues to cocirculate with El Tor V. cholerae O1 serotypes Ogawa and Inaba as a cause of disease in humans, although it accounts for a smaller proportion of all cholera now than in its first years of circulation (16, 20). Recently, comparative genomics has revealed an extensive amount of lateral gene transfer between strains, suggesting that genomic classification may be an alternative to serogrouping for classifying pathogenic V. cholerae strains (11).Toxigenic V. cholerae may be present in environmental sources in regions of endemicity and emerge, often seasonally, to cause cholera in humans (12, 18). Once an outbreak has begun, organisms from one infected individual are more infectious for the next individual, a property termed hyperinfectivity, and these forms may be able to pass directly from human to human through fecal-oral contamination (35). However, because vibrio organisms are difficult to isolate from implicated environmental or domestic water sources (28, 29), little is known about the diversity of V. cholerae in inocula that cause human infection.Established laboratory methods for differentiating V. cholerae strains, apart from serogrouping and serotyping, include rRNA restriction fragment length polymorphism (ribotyping), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). These methods, however, have a limited capacity to differentiate between pathogenic V. cholerae strains, as clinical isolates are relatively genetically monomorphic. For instance, V. cholerae O1 comprises approximately 30 ribotypes (39); however, only a few ribotypes are common in clinical isolates, ribotypes evolve slowly, and all isolates of a given pathogenic V. cholerae serotype in a local area over a period of multiple years often belong to a single ribotype (8, 14, 17). In a broad sampling of 154 V. cholerae isolates from Bangladesh and worldwide over several decades, only 15 ribotypes were identified, and of these, many were found in nonpathogenic environmental isolates only; only five ribotypes were associated with the V. cholerae O1 El Tor biotype that currently predominates as the cause of clinical disease, while pathogenic isolates of serogroup O139 were indistinguishable from each other by ribotype (19).PFGE, in which restriction endonuclease digestion of genomic DNA generates mutation-sensitive banding patterns, is often more sensitive than ribotyping in detecting strain variation (7, 34, 51) and detects extensive genetic variation within nonpathogenic V. cholerae serogroups (3, 46). However, PFGE types change slowly and are useful primarily for distinguishing between strains in different pandemics or between different continental branches of those pandemics. In an analysis of 180 mostly western-hemisphere isolates (7), PFGE differences had developed from a prior pandemic strain over the 30 years since its arrival in Latin America, but a new strain that had been causing disease for 2 years still had only a single PFGE type across the 64 isolates analyzed. Similarly, in a Japanese study (2), although 19 PFGE types were identified among O1 isolates, the majority of the domestic isolates, along with several imported isolates, belonged to a single PFGE type.Further differentiation between V. cholerae isolates is achievable by MLST, which characterizes isolates by internal DNA sequences in selected housekeeping genes (32). Nevertheless, epidemic strains also cluster tightly in this typing scheme (5, 32) and the method has been useful primarily for determining relationships between nontoxigenic strains (36) or for linking regional outbreaks (which typically appear monoclonal by these methods) with the pandemic strain responsible (5, 33).Although these methods have distinguished major pandemic clones from other nonpathogenic human and environmental isolates of V. cholerae, the near clonality of pathogenic O1 and O139 strains means that established methods may not provide sufficiently robust differentiation of these genetically similar pathogenic strains to answer important epidemiological questions. Therefore, there is a need for other methods that can distinguish among clinical O1 and O139 isolates and track the epidemiology of outbreaks in a restricted geographic area on a shorter time scale.Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is one method that may be useful for differentiating between pathogenic V. cholerae O1 and O139 strains that would be indistinguishable by other techniques (15). This method examines short repeating DNA segments at various locations in the genome that can vary in number at each location and uses the number of repeats at each varying locus as a fingerprint to distinguish between isolates.Escherichia coli is the paradigm organism for demonstrating the value of the MLVA method. Noller et al. (38) showed that E. coli O157 isolates that were indistinguishable by MLST could be distinguished to some extent by PFGE but that MLVA distinguished between isolates that had the same PFGE type and did so in a manner consistent with the known epidemiology of the isolates (38a). In addition, machine-scored VNTR assays have been demonstrated to be robust and portable and to discriminate clearly between isolates by using relatively few loci, therefore limiting the effect of compounding genotyping errors (6).For V. cholerae, five VNTR loci have been identified (15), and the initial application of MLVA at those loci has demonstrated distinct populations of clinical isolates of V. cholerae in different geographic regions within Bangladesh and India (23, 47). Predominant isolates in each of two rural Bangladeshi regions varied gradually over a time scale of months to years (47), and isolates collected from India over a 15-year period varied widely, with individual MLVA types clustering in time and place—some with widespread dissemination and others with limited local occurrence only (23). MLVA has also been used to classify hybrid and altered V. cholerae variants and to demonstrate their genetic distance from the pandemic El Tor strain (10). Use of the MLVA method for epidemiologic study of cholera requires that V. cholerae VNTR alleles remain reasonably stable during bacterial replication in patients or in laboratory culture after isolation. Some degree of stability of two of the five loci used in V. cholerae MLVA has been demonstrated previously by serial passage in vitro through four overnight cultures (15). In this study, we used MLVA to examine V. cholerae O1 and O139 isolates obtained from infected patients and their household contacts—including multiple isolates from the same individual and isolates from multiple individuals within the same household—in a large city where cholera is endemic.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

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

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

An IcmF Family Protein,ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL,and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

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 ImpL_M, 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 ImpL_M 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 ImpL_M 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. impL_M mutants with substitutions or deletions in the Walker A motif failed to complement the impL_M deletion mutant for Hcp secretion, which provided evidence that ImpL_M 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 ImpL_M and another essential T6SS component, ImpK_L. Topology and biochemical fractionation analyses suggested that ImpK_L 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 ImpL_M-ImpK_L interaction domains suggested that ImpL_M interacts with ImpK_L via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL_M interacts with ImpK_L, 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 ImpL_M and ImpK_L, 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 ImpL_M and ImpK_L 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 ImpL_M may function as a nucleoside triphosphate (NTP)-binding protein or nucleoside triphosphatase to mediate T6S machinery assembly and/or substrate secretion.     

Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration

HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified α- and β-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of α-, β-, and γ-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in theregulation of cell motility by modulating microtubule stability.HtrA1 (for high temperature requirement) belongs to a family of serine proteases and is so named because of its essential role in thermal tolerance in Escherichia coli, which requires HtrA (also known as DegP) for survival at elevated temperatures (14). This survival is attributed to the ability of HtrA proteins to switch from chaperones to proteases that reduce the amount of unfolded and aggregated protein upon heat stress (46). Human, as well as bacterial, HtrA proteins contain trypsin and PDZ (PSD95, Dlg, ZO1) domains that display a high degree of sequence conservation from bacteria to human (14). Of the four human HtrA proteins, HtrA1, HtrA3, and HtrA4 also contain a signal peptide, insulin-like growth factor binding protein (IGFBP), and Kazal-type trypsin inhibitor domains, while HtrA2 lacks these domains. Although HtrA1 contains signal peptide, an intracellular form of HtrA1 has been reported as well (15, 17). The mitochondrial protein HtrA2 is well characterized and has been shown to be involved in apoptosis (27, 37, 39, 47, 52, 53) and neurodegenerative disease (35). However, HtrA1 is the first in the family to be implicated as a tumor suppressor in ovarian cancer and melanoma (3, 5, 13). In addition, HtrA1 is implicated in various pathogenic and developmental processes, including osteoarthritis, Alzheimer''s disease, neuronal maturation and development, age-related macular degeneration, and tumor progression (11, 23, 24, 33, 36, 50, 56). Specific to its role in tumor progression, HtrA1 is downregulated in various cancers, and its downregulation is associated with resistance to chemotherapy and a metastatic phenotype (4, 11, 19). Recently, we developed a mixture-based peptide library to determine the specificities of cleavage site motifs for HtrA1 serine protease. The results identified tubulins as potential substrates of HtrA1. Furthermore, we showed that exogenously expressed HtrA1 disrupts microtubules (MTs) and targets tubulins for degradation (data not shown). These results suggest a potential role for HtrA1 as an MT-associated protein (MAP) and its potential to regulate MT and tubulin stability and MT-associated cellular functions.MTs are highly dynamic noncovalent polymers of α- and β-tubulins that undergo cyclical shrinking (catastrophe) and growing (rescue) (18, 31, 43). The dynamic instability of MTs is central to their diverse biological functions, including the coordination of cell division (40, 55), morphogenesis (25), cell polarity (42), and motility (48). MT instability is, in part, modulated by MAPs (2, 29). Many tumor suppressors, such as adenomatous polyposis coli (APC) (20), RASSF1A (45), and Dlg (6), associate with MTs and impose tumor suppressor activities by regulating their functions related to cell division, polarity, and motility. Deregulation of these processes, as a consequence of loss of function of these tumor suppressors, contributes to unchecked proliferation; cytoarchitecture disruption; and the ability to migrate, invade, and metastasize distant organs (6, 7, 26). Therefore, the regulation of MT stability and dynamics or the lack of it has dire consequences for normal cell functions.Given the fact that HtrA1 is downregulated in various cancers, particularly in metastatic cancer, it is possible that HtrA1 may regulate certain aspects of cancer, namely, the motility of cancer cells, by modulating MT stability and dynamics. Therefore, to better characterize the interaction between HtrA1 and MTs and to gain mechanistic insights into the functional consequences of HtrA1 downregulation in cancer, we investigated the biochemical interaction between HtrA1 and tubulin, the domain within HtrA1 required for localization to MTs, and the effect on cell migration. Here, we describe the identification of HtrA1 as an MT-associated serine protease and a novel role of HtrA1 in the regulation of cell motility.     

The Cyclic-di-GMP Phosphodiesterase BinA Negatively Regulates Cellulose-Containing Biofilms in Vibrio fischeri

Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the ΔbinA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of the syp regulator sypG induced binA expression. Thus, this work reveals a mechanism by which V. fischeri inhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.Bacterial biofilms play important roles in the environment and in interactions with eukaryotic hosts (for reviews, see references 17 and 32). Exopolysaccharides are a major component of biofilms (23), and many bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Salmonella spp., have the ability to produce multiple different exopolysaccharides (23). For some of these bacteria, it has been demonstrated that the different polysaccharides contribute to biofilm formation in different settings. For example, several strains of Salmonella require an exopolysaccharide called O-antigen capsule to form biofilms on human gallstones but not to form biofilms on glass or plastic (11). Conversely, the exopolysaccharides cellulose and colanic acid are required for optimal biofilm formation by Salmonella spp. on glass and plastic but are not required for biofilm formation on human gallstones (11, 35). For some bacteria, a particular exopolysaccharide promotes attachment to one surface but seems to interfere with attachment to other surfaces. One example of this is E. coli O157:H7, which requires the exopolysaccharides poly-β-1,6-N-acetylglucosamine (PGA), colanic acid, and cellulose for optimal binding to alfalfa sprouts and plastic (27). In contrast, these polysaccharides are not required for binding by E. coli O157:H7 cells to human intestinal epithelial (Caco-2) cells and binding was actually enhanced in cellulose and PGA mutants, suggesting that while these polysaccharides are important for attachment to sprouts and plastic, they interfere with attachment to Caco-2 cells (27).The marine bacterium Vibrio fischeri is known to produce at least two different exopolysaccharides that play roles in biofilm formation, the symbiosis polysaccharide (Syp) and cellulose (12, 59, 60). The Syp polysaccharide is critical for the formation of a biofilm-like aggregate at the initiation of symbiosis with the Hawaiian bobtail squid Euprymna scolopes (59). The natural condition(s) under which V. fischeri cells use cellulose in biofilm formation is not yet known. However, in other bacteria, cellulose contributes to the ability to attach to a variety of surfaces, including plant roots, other plant cells, mammalian epithelial cells, glass, and plastic (27, 28, 31, 35, 37, 45).Although V. fischeri biofilm formation appears to be important for interaction with its symbiotic host and is likely also to be important in the marine environment outside the host, wild-type V. fischeri cells do not produce substantial biofilms under a variety of standard laboratory conditions. V. fischeri''s ability to form biofilms in culture, however, is greatly enhanced when the syp biosynthetic gene cluster (Fig. ​(Fig.1)1) is induced by overexpression of the response regulator SypG, the sensor kinase RscS, or the sensor kinase SypF (12, 59, 60).Open in a separate windowFIG. 1.The binA gene and its predicted protein. (A) The binA gene (VFA1038) is located downstream from and oriented in the same direction as the syp gene locus. Individual genes are indicated by block arrows, and the four known or putative promoters within the syp locus are indicated by line arrows. (B) The BinA protein (630 amino acids [aa]) is predicted to have three domains, GAF (∼Q20 to L151), GGDEF (∼H205 to A338), and EAL (∼L374 to D611), as indicated. Only the EAL domain is well conserved.In culture, V. fischeri also forms biofilms when cellulose production is induced. Cellulose contributes to the biofilms formed when either SypF or the putative response regulator VpsR is overexpressed (12). Additionally, overexpression of the diguanylate cyclase MifA induces biofilm formation and increases binding to two dyes that are cellulose indicators, Congo red and calcofluor (34, 37, 54).The product of diguanylate cyclase activity, cyclic diguanylate (c-di-GMP), is an intracellular signaling molecule that plays an important role in regulating biofilm formation and motility (reviewed in references 10, 22, 38, 39, 48, and 56). C-di-GMP is produced from two GTP molecules by diguanylate cyclases with conserved GGDEF domains and is depleted by hydrolysis to linear pGpG by phosphodiesterases (PDEs) with EAL or HD-GYP domains. There is evidence of increased cellulose production in response to increased c-di-GMP levels in many bacteria (36, 41). In general, high levels of c-di-GMP enhance biofilm formation and low levels of c-di-GMP enhance motility (39).Here we report that V. fischeri biofilm formation also increases in the absence of BinA, a GGDEF/EAL domain protein encoded by a gene that is adjacent to the syp cluster. We also report the discovery of genes involved in biofilm formation by binA mutants and the identification of amino acids that are critical to BinA activity. Finally, we suggest a mechanism by which the production of two different polysaccharides may be coordinated by BinA.     

The Tumor Suppressor Functions of p27kip1 Include Control of the Mesenchymal/Amoeboid Transition

In many human cancers, p27 downregulation correlates with a worse prognosis, suggesting that p27 levels could represent an important determinant in cell transformation and cancer development. Using a mouse model system based on v-src-induced transformation, we show here that p27 absence is always linked to a more aggressive phenotype. When cultured in three-dimensional contexts, v-src-transformed p27-null fibroblasts undergo a morphological switch from an elongated to a rounded cell shape, accompanied by amoeboid-like morphology and motility. Importantly, the acquisition of the amoeboid motility is associated with a greater ability to move and colonize distant sites in vivo. The reintroduction of different p27 mutants in v-src-transformed p27-null cells demonstrates that the control of cell proliferation and motility represents two distinct functions of p27, both necessary for it to fully act as a tumor suppressor. Thus, we highlight here a new p27 function in driving cell plasticity that is associated with its C-terminal portion and does not depend on the control of cyclin-dependent kinase activity.Dissemination of tumor cells is strictly linked to their ability to attach to and move within the extracellular matrix (ECM) in a three-dimensional (3D) environment. The use of 3D experimental model systems revealed that a higher complexity in cell migration and adaptation responses exists in the 3D model than in the classical 2D model (10, 16, 41, 49). A striking example is given by the fact that only in 3D could individually migrating cells use different mechanisms such as mesenchymal and amoeboid motility (16, 17). The relative slow mesenchymal migration is characterized by a fibroblast-like spindle shape and is dependent on integrin-mediated adhesion and on protease function (16). The amoeboid motility can in some cases represent a less adhesive, integrin-independent type of movement. Cells use a propulsive mechanism and are highly deformable, and rather than degrade the matrix, they are able to squeeze through it (16). As a result, the cells that use the amoeboid motility can potentially move faster than cells that use a mesenchymal strategy. Mesenchymal and amoeboid movements are also characterized by a different involvement of small GTPases of the Rho family. A high RhoA activity is associated mainly with the amoeboid motility, while the mesenchymal migration needs a high Rac activity at the leading edge to promote the extension of cellular protrusions (41, 48). Under certain circumstances, cancer cells can undergo conversion from a mesenchymal toward an amoeboid motility, an event referred as mesenchymal-amoeboid transition (MAT) (50). MAT represents a putative escape mechanism in tumor cell dissemination that could be induced by inhibition of pericellular proteolysis (50) or by increased membrane-associated RhoA activity (18, 40).Key mediators of cell motility through ECM substrates are the members of the Src family kinases. The prototype of Src family kinases, c-Src (14), is activated following cell-ECM adhesion and contributes to regulate the focal adhesion turnover and the cytoskeletal modifications necessary for normal cell adhesion and motility (52). The c-Src gene is the proto-oncogene of the transforming gene v-src of Rous sarcoma virus, and its elevated protein level and activity have been found in many human tumors (20, 28, 27, 34). Despite the accumulation of information and new molecular understanding of how Src is controlled, there is still an incomplete picture about its role in the generation of the malignant phenotype. v-Src shows higher levels of the kinase activity and transforming ability than c-Src (14, 15, 52). It induces normal cells to acquire a variety of transformed features, including alteration of morphology and increase of invasion ability due to its role in focal adhesion remodeling (7, 9, 13).Many data suggest that there is a close relationship between cell-ECM interaction and the proliferation and movements in both normal and tumor cells (5, 38, 43). Accordingly, Src activation may influence not only cell motility but also cell cycle progression by targeting the cell cycle inhibitor p27^kip1 to proteasomal degradation (22, 39). Recent evidences indicated that p27^kip1 (hereafter called p27) can also regulate cell migration, even though its role still remains controversial since it has been reported to either block or stimulate cell movements (1, 4, 11, 19, 21, 23, 29, 45).Based on these notions, we tested the possible contribution of p27 to the growth and motility phenotypes induced by v-src transformation, with special regard to those cellular invasive features that can be observed in 3D environments. By studying in vitro and in vivo the behavior of wild-type (WT) and p27-null fibroblasts transformed with v-src, we highlight a new role for p27 in the regulation of cellular plasticity that can ultimately drive tumor cell shape, motility, and invasion.     

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).