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.     

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).     

Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection

Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.Herpesviruses are well known to interfere with major histocompatibility complex class I (MHC-I) molecules in order to ensure evasion from immune recognition. A majority of evidence so far indicates that they target MHC-I maturation events and MHC-I trafficking in the secretory pathway (33), although evidence exists suggesting that herpesviruses could also interfere with MHC-I functions in endosomal pathways (8). Murine cytomegalovirus (MCMV), a member of the herpesvirus family, dedicates a substantial part of its genome to encoding nonessential genes for the modulation of cellular functions (40), including MHC-I trafficking in the secretory pathway (24, 27, 45, 48, 49, 52). All known immune evasion functions encoded by MCMV are based on a direct interaction of viral gene products with MHC-I complexes in the secretory pathway. The egress of nascent MHC-I complexes to the cell surface of MCMV-infected cells is abolished as a consequence of their retention in the endoplasmic reticulum (ER)-cis-Golgi intermediate compartment (ERGIC) by the m152 gene product (10, 19, 24, 52, 56) as well as redirection of those that escape into the Golgi compartment toward late endosomes (LEs) for degradation by the m06 MCMV gene product (45). These effects are opposed by gp34, a product of the MCMV m04 gene, which associates with MHC-I complexes and reaches the cell surface (24, 27).The loss of MHC-I from the cell surface is an expected consequence of the activity of m152 and m06, which act in the secretory pathway. The level of cell surface MHC-I is substantially reduced at later times of infection (10, 19, 24, 48, 52), and cells stably transfected with either the m152 or m06 gene do not display MHC-I at the cell surface (20, 24). If the loss of MHC-I from the cell surface is a consequence of the prevented egress from the secretory pathway, then the cell surface loss should follow the kinetics of the constitutive internalization of MHC-I complexes in the endosomal pathway. Given that the constitutive internalization is the net result of cell surface supply from the secretory pathway, endocytic uptake, and endocytic recycling, it is a slow process that occurs in normal fibroblasts at a rate of ∼6 to 8% per hour (36). Therefore, the effect of MCMV immunoevasins on cell surface MHC-I should be expected at later times of infection. However, several reports demonstrated that the level of MHC-I surface expression was already reduced in the early phase of infection (10, 45, 48, 52). Thus, it would be reasonable to expect that MCMV contributes with a function that causes the accelerated retrieval of cell surface-resident MHC-I complexes.In this report we demonstrate that MCMV perturbs endosomal trafficking very early in infection by acting on distal parts of early endosome (EE) route and affecting the trafficking of both clathrin-dependent and clathrin-independent cargoes. Clathrin-dependent cargo does not share primary endocytic carriers with MHC-I proteins (12, 14), which enter the cell via the nonclathrin Arf6-associated endocytic carriers (12, 14, 41, 42, 53), but they meet in the proximal part of the common early endocytic route and redirect to distal endocytic carriers around the cell center (12, 14). The perturbation of the distal part of the EE route has dramatic consequences on MHC-I, since it supplements the viral mechanisms that act in the secretory pathway. The net result of this perturbation is a complete loss of MHC-I molecules from the cell surface.     

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.     

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

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

Expression of a Synthesized Gene Encoding Cationic Peptide Cecropin B in Transgenic Tomato Plants Protects against Bacterial Diseases

The cationic lytic peptide cecropin B (CB), isolated from the giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. In this study, the effects of chemically synthesized CB on plant pathogens were investigated. The S₅₀s (the peptide concentrations causing 50% survival of a pathogenic bacterium) of CB against two major pathogens of the tomato, Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, were 529.6 μg/ml and 0.29 μg/ml, respectively. The CB gene was then fused to the secretory signal peptide (sp) sequence from the barley α-amylase gene, and the new construct, pBI121-spCB, was used for the transformation of tomato plants. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato plant demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato plants (∼0.05 μg in 50 mg of leaves) were far lower than the S₅₀ determined in vitro. CB transgenic tomatoes could therefore be a new mode of bioprotection against these two plant diseases with significant agricultural applications.Bacterial plant diseases are a source of great losses in the annual yields of most crops (5). The agrochemical methods and conventional breeding commonly used to control these bacterially induced diseases have many drawbacks. Indiscriminate use of agrochemicals has a negative impact on human, as well as animal, health and contributes to environmental pollution. Conventional plant-breeding strategies have limited scope due to the paucity of genes with these traits in the usable gene pools and their time-consuming nature. Consequently, genetic engineering and transformation technology offer better tools to test the efficacies of genes for crop improvement and to provide a better understanding of their mechanisms. One advance is the possibility of creating transgenic plants that overexpress recombinant DNA or novel genes with resistance to pathogens (36). In particular, strengthening the biological defenses of a crop by the production of antibacterial proteins with other origins (not from plants) offers a novel strategy to increase the resistance of crops to diseases (35, 39, 41). These antimicrobial peptides (AMPs) include such peptides as cecropins (2, 15, 20, 23-24, 27, 31, 42, 50), magainins (1, 9, 14, 29, 47), sarcotoxin IA (35, 40), and tachyplesin I (3). The genes encoding these small AMPs in plants have been used in practice to enhance their resistance to bacterial and fungal pathogens (8, 22, 40). The expression of AMPs in vivo (mostly cecropins and a synthetic analog of cecropin and magainin) with either specific or broad-spectrum disease resistance in tobacco (14, 24, 27), potato (17, 42), rice (46), banana (9), and hybrid poplar (32) have been reported. The transgenic plants showed considerably greater resistance to certain pathogens than the wild types (4, 13, 24, 27, 42, 46, 50). However, detailed studies of transgenic tomatoes expressing natural cecropin have not yet been reported.The tomato (Solanum lycopersicum) is one of the most commonly consumed vegetables worldwide. The annual yield of tomatoes, however, is severely affected by two common bacterial diseases, bacterial wilt and bacterial spot, which are caused by infection with the Gram-negative bacteria Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, respectively. Currently available pesticides are ineffective against R. solanacearum, and thus bacterial wilt is a serious problem.Cecropins, one of the natural lytic peptides found in the giant silk moth, Hyalophora cecropia (25), are synthesized in lipid bodies as proteins consisting of 31 to 39 amino acid residues. They adopt an α-helical structure on interaction with bacterial membranes, resulting in the formation of ion channels (12). At low concentrations (0.1 μM to 5 μM), cecropins exhibit lytic antibacterial activity against a number of Gram-negative and some Gram-positive bacteria, but not against eukaryotic cells (11, 26, 33), thus making them potentially powerful tools for engineering bacterial resistance in crops. Moreover, cecropin B (CB) shows the strongest activity against Gram-negative bacteria within the cecropin family and therefore has been considered an excellent candidate for transformation into plants to improve their resistance against bacterial diseases.The introduction of genes encoding cecropins and their analogs into tobacco has been reported to have contradictory results regarding resistance against pathogens (20). However, subsequent investigations of these tobacco plants showed that the expression of CB in the plants did not result in accumulation of detectable levels of CB, presumably due to degradation of the peptide by host peptidases (20, 34). Therefore, protection of CB from cellular degradation is considered to be vital for the exploitation of its antibacterial activity in transgenic plants. The secretory sequences of several genes are helpful, because they cooperate with the desired genes to enhance extracellular secretion (24, 40, 46). In the present study, a natural CB gene was successfully transferred into tomatoes. The transgenic plants showed significant resistance to the tomato diseases bacterial wilt and bacterial spot, as well as with a chemically synthesized CB peptide.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Host-Interactive Genes in Amerindian Helicobacter pylori Diverge from Their Old World Homologs and Mediate Inflammatory Responses

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.Helicobacter pylori is a microaerophilic bacterium of the Epsilonproteobacteria that has colonized the stomach since early in human evolution (45) and diverged with ancient human migrations (24, 45, 92). Thus, several major H. pylori populations, such as hpAfrica1, hpEurope, hspEAsia, and hspAmerind, whose names indicate their original geographic associations (45, 51), have been defined. In particular, similarities between the hspAmerind and hspEAsia populations suggest that the first colonizers of the New World brought H. pylori with them (24, 28). With recent mixing of human groups, H. pylori populations are also mixing and competing, with an apparent dominance by the hpEurope population at least in Latin America (19).H. pylori usually does not cause illness, but colonization with strains bearing the cag (cytotoxin-associated gene) pathogenicity island (cag PAI) (3, 7, 25, 52, 57, 61, 63) is associated with an increased risk of noncardia gastric adenocarcinoma and peptic ulcer disease (56, 64). Nonetheless, a high prevalence of cag-positive H. pylori strains occurs concurrently with low gastric cancer rates in Africa (40) and some regions in Latin America, such as the Venezuelan savannas and Amazonas (29, 53). Moreover, clinical and epidemiological data provide evidence for an inverse relationship between H. pylori colonization and the prevalence of certain metabolic disorders, esophageal diseases, asthma and allergic disorders, and acute infectious diseases, as well as a direct relationship with improved nutritional status of rural children (3, 14, 34, 37, 49, 68). That the host interaction with an indigenous gastric microbe provides some health benefits to the host is not unexpected given the well-established role of gastrointestinal microflora in maintaining gastroenteric homeostasis (8).The most thoroughly studied H. pylori proteins that interact with human cells are CagA and VacA. CagA is an effector protein injected into gastric epithelial cells by a type IV secretion system encoded by the cag PAI (10, 12, 15, 83). VacA is initially secreted from the bacterial cell by an autotransporter mechanism (16). Both proteins have multiple effects on host cells. Inside the host cell, phosphorylation of CagA on EPIYA repeats in the phosphotyrosine (PY) region (73) induces cellular elongation known as the hummingbird phenotype (72). CagA may also induce secretion of interleukin-8 (IL-8) (11), a process commonly attributed to NF-κB, and disrupt the barrier function of the tight junctions in polarized epithelial cells, leading to a loss of adhesion (1, 5). Other motifs in the PY region promote phosphorylation-independent effects (79). In addition, cagA may be considered an oncogene (60), since transgenic expression of cagA in mice leads to gastric epithelial hyperplasia through aberrant epithelial cell signaling and gastric carcinogenesis (60, 62). In contrast, VacA is a multifunctional protein with several activities in epithelial and immune cells (16). VacA induces cell vacuolation (43), alters mitochondrial membrane permeability (27, 41, 90), and increases epithelial monolayer permeability. VacA also activates several signal transduction pathways that are important in immune and epithelial cells, including the mitogen-activated protein (MAP) kinase and p38/ATF-2-mediated signal pathways (9, 55).Genomic analysis provides insights into the evolution of H. pylori strains and their relation with their human hosts and may be useful for the development of diagnostic tools and novel therapies. To date, there are six published complete H. pylori genomes, mostly from the hpEurope population (see Table SA1 in the supplemental material). Here, we report the whole genome of a newly characterized hspAmerind strain, V225d, and assess its genetic structure in comparison to those of Old World H. pylori strains through a comprehensive multiprotein phylogenetic analysis, as well as through single-gene examination of cagA and vacA, revealing clues to the evolution and migration of this strain into the New World and the implications for human health. We also present the results of functional and genomic studies using gastric epithelial cells demonstrating that V225d can induce an inflammatory host response, an effect that was lost following passage through the mouse stomach.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.