Analysis of the Role of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola 1448a in Interaction with the Plant

In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway.Type III secretion systems (T3SS) are complex and specialized machineries that inject effector proteins directly into the host cell cytosol (2). In Pseudomonas syringae, T3SS-mediated secretion is essential for disease in compatible hosts and for eliciting the hypersensitive response (HR) in incompatible hosts (1). P. syringae pathovars secrete a variable number of type III effectors that form their so-called secretomes and are expressed within the plant under the control of the alternative sigma factor HrpL (47). Understanding how the T3SS determines pathogenicity requires the functional characterization of the complete type III effector inventory. However, this characterization has been partially hindered by the fact that mutation of individual effectors, usually the most straightforward approach, rarely causes virulence attenuation (14). Thus, reports showing the contribution of the type III effector to virulence in P. syringae pathovars have resorted to ectopic expression in homolog-lacking related strains (40), plasmid-cured derivatives (21), double mutants (6, 28), or polymutants (3, 26). In relation to this, we have previously established the use of the competitive index (CI) in mixed infections (13, 42) as a more sensitive virulence assay for P. syringae pathovars than traditional assays (31). Using CIs, we demonstrated for the first time the individual contribution of AvrPto, an otherwise thoroughly characterized type III effector from Pseudomonas syringae pv. tomato (9, 17, 18, 27, 36, 39, 40, 46), to pathogen growth within its natural host (31). Therefore, analysis of effector mutants by use of the CI may provide the means to establish the quantitative contribution of the members of P. syringae T3SS secretomes to virulence. In addition, genetic analysis of the effects of combinations of effector mutations on virulence has already proven a useful approach to establishing the contribution of the members of the P. syringae pv. tomato DC3000 secretome to virulence by revealing a functional overlap (6, 26, 28). Thus, generation of knockouts in all individual effector genes of a given secretome, achieved in such a manner as to allow for easy combination of these strains into double or multiple mutant strains, is a desirable task, albeit a cumbersome one, considering the size of most secretomes.The secretome of the fully sequenced wild-type (wt) representative of the Pseudomonas syringae pv. phaseolicola 1448a strain (Pph1448a) has previously been analyzed, using a differential fluorescence induction screen (7) and bioinformatics (44), to identify effector genes. Our laboratory contributed to establishing this secretome through the development and application of a very sensitive assay for T3SS-mediated translocation based on CI assays (30). This assay represents an improvement over the sensitivity of the commonly used AvrRpt2 reporter assay. When fused to a T3SS-secreted protein, AvrRpt2^81-255 is translocated inside the host cell, eliciting a hypersensitive response (HR), dependent on the resistance protein RPS2 (32). By using CIs to measure the bacterial growth reduction associated with the AvrRpt2-RPS-mediated defense response, we detected translocation for two out of four Pph1448a effector candidates previously discarded by other assays, HopAJ1 and HopAK1 (30), and demonstrated translocation for two out of five previously untested candidates, HopAH2 and A0129. However, although in planta expression has been shown to take place in an HrpL-dependent manner for HopAJ1 and HopAK1 (7), it has not been established for HopAH2 and A0129. Effector nomenclature guidelines recommend that the abbreviation for the pathovars as well as the name of the strain should be included within the effector name (29). For simplicity, we include this indication only when effectors from other pathovars are mentioned. In summary, to date, 22 effectors in Pph1448a have been experimentally validated (7, 30, 44), one HrpL-regulated candidate has given inconsistent translocation results (AvrE1) (7), two translocated candidates have not been analyzed for expression in planta (HopAH2 and A0129) (30), one bioinformatically identified candidate has not been experimentally tested (AvrB4-2) (23), and six additional candidates have been proposed but experimentally ruled out (PSPPH3757, HopAN1, HopAJ2, HopW1-2, HopV1, and HopJ1) (7, 30).In this work, we analyzed the translocation and/or expression of these and other candidate effectors to close the type III effector inventory of Pph1448a. Our results indicate that the Pph1448a complete type III secretome is formed by 27 validated effectors, including a new one, HopAY1, which does not belong to any of the previously described effector families. The work includes the development of a simplified process for quick generation of single and double knockout mutants and its application to constructing a collection of single mutants for almost all members of the Pph1448a type III secretome. Additionally, we generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the four single and two double mutant strains as well as the double mutants expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to the virulence of Pph1448a. The tools and approach used in this work set the groundwork for dissecting the contribution of the entire Pph1448a type III secretome to virulence.     

The Siderophore Pyoverdine of Pseudomonas syringae pv. tabaci 6605 Is an Intrinsic Virulence Factor in Host Tobacco Infection

To investigate the role of iron uptake mediated by the siderophore pyoverdine in the virulence of the plant pathogen Pseudomonas syringae pv. tabaci 6605, three predicted pyoverdine synthesis-related genes, pvdJ, pvdL, and fpvA, were mutated. The pvdJ, pvdL, and fpvA genes encode the pyoverdine side chain peptide synthetase III l-Thr-l-Ser component, the pyoverdine chromophore synthetase, and the TonB-dependent ferripyoverdine receptor, respectively. The ΔpvdJ and ΔpvdL mutants were unable to produce pyoverdine in mineral salts-glucose medium, which was used for the iron-depleted condition. Furthermore, the ΔpvdJ and ΔpvdL mutants showed lower abilities to produce tabtoxin, extracellular polysaccharide, and acyl homoserine lactones (AHLs), which are quorum-sensing molecules, and consequently had reduced virulence on host tobacco plants. In contrast, all of the mutants had accelerated swarming ability and increased biosurfactant production, suggesting that swarming motility and biosurfactant production might be negatively controlled by pyoverdine. Scanning electron micrographs of the surfaces of tobacco leaves inoculated with the mutant strains revealed only small amounts of extracellular polymeric matrix around these mutants, indicating disruption of the mature biofilm. Tolerance to antibiotics was drastically increased for the ΔpvdL mutant, as for the ΔpsyI mutant, which is defective in AHL production. These results demonstrated that pyoverdine synthesis and the quorum-sensing system of Pseudomonas syringae pv. tabaci 6605 are indispensable for virulence in host tobacco infection and that AHL may negatively regulate tolerance to antibiotics.Phytopathogenic bacteria employ a variety of virulence mechanisms to overcome the defense systems of plants. Pseudomonas syringae pv. tabaci 6605 is a gram-negative bacterium that causes wildfire disease on host tobacco plants. Previously, we demonstrated that flagellin, a component of the flagellar filament of this organism, is a major elicitor of the hypersensitive reaction and is posttranslationally modified by glycosylation (26, 28-30). A genetic region composed of three open reading frames (ORFs), namely, fgt1, fgt2, and orf3, was previously identified in a flagellum gene cluster. fgt1 and fgt2 encode flagellin glycosyltransferase, and orf3 shows significant homology to the 3-oxoacyl-(acyl carrier protein [ACP]) synthase III in the fatty acid elongation cycle, required for the synthesis of acyl homoserine lactones (AHLs) (11, 30, 31). Analysis of an orf3 deletion (Δorf3) mutant revealed that orf3 played no role in the glycosylation of flagellin, although the virulence of the Δorf3 mutant on tobacco plants was remarkably reduced.Many virulence factors of bacteria have been reported to be under the regulation of a cell density-dependent system called quorum sensing. AHLs are synthesized by the coupling of the homoserine lactone ring from S-adenosylmethionine and acyl chains from the acyl-ACP by PsyI in P. syringae (9, 11). P. syringae pv. tabaci 6605 secretes three types of AHLs as signal molecules: N-hexanoyl-l-homoserine lactone, N-(3-oxohexanoyl)-l-homoserine lactone, and N-octanoyl-l-homoserine lactone (31). Our previous study indicated that the Δorf3 mutant and the quorum-sensing molecule-defective ΔpsyI mutant had significantly reduced abilities to produce AHLs and to take up iron (31). Furthermore, a scanning electron micrograph revealed little extracellular polymeric substance matrix surrounding the inoculated Δorf3 and ΔpsyI mutants on the tobacco leaf surface, indicating a lack of biofilm development (31). Iron acquisition has been reported to affect biofilm formation (2), and iron uptake is also involved in biofilm development under the regulation of quorum sensing in P. syringae pv. tabaci 6605 (2, 3, 11, 31).Iron is indispensable for the growth of almost all organisms, and the ability to acquire iron is thought to be an important factor in virulence (27). Because the concentration of Fe(III) in the environment is quite low, owing to its insolubility under environmental conditions, the fluorescent Pseudomonas group produces a yellow-green Fe(III)-chelating siderophore called pyoverdine in order to acquire iron effectively (24). The genes required for pyoverdine synthesis are well characterized in the Pseudomonas aeruginosa strain PAO1 (33), and pyoverdine biosynthesis mutants of this pathogen exhibit reduced virulence (20, 33). For the regulation of iron homeostasis, it was reported that Fur (ferric uptake regulator) is a global regulator that controls the expression of siderophore-mediated iron uptake in P. syringae pv. tabaci 11528 (6).In the present study, two genes predicted to be involved in pyoverdine synthesis, encoding the pyoverdine side chain peptide synthetase III l-Thr-l-Ser component (pvdJ) and the pyoverdine chromophore synthetase (pvdL), and the TonB-dependent ferripyoverdine receptor gene (fpvA) were disrupted in order to elucidate the roles of the pyoverdine-mediated iron acquisition system in the virulence of P. syringae pv. tabaci 6605. By use of these mutants, several important virulence factors, flagellum-dependent motility, the production of tabtoxin and extracellular polysaccharide (EPS), and biofilm formation were investigated. Although the ΔpvdJ and ΔpvdL mutants had reduced ability to produce EPS, the antibiotic tolerance of these mutants was drastically increased. The correlations among the pyoverdine-mediated iron uptake system, quorum-sensing regulation, and multidrug efflux are also discussed.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

Inhibition of Selenium Metabolism in the Oral Pathogen Treponema denticola

In this report we provide evidence that the antimicrobial action of stannous salts and a gold drug, auranofin, against Treponema denticola is mediated through inhibition of the metabolism of selenium for synthesis of selenoproteins.The biological use of selenium as a catalyst, incorporated into proteins as selenocysteine, is broad. It plays an essential role in energy metabolism, redox balance, and reproduction in a variety of organisms, from bacterial pathogens to eukaryotic parasites to humans. The results of several epidemiological studies indicate that higher levels of selenium in the mammalian diet can have a negative effect on dental health (2, 17-19, 39). Although the impact of selenium is attributed to its influence on the physical properties of the enamel surface (10), the role of selenium in supporting the oral microbial community has not been studied.The oral cavity is a highly complex microbiome, with a large proportion of its residents uncharacterized due to their fastidious nature and resistance to traditional culture methods (11). Analysis of whole saliva indicates that bacterial metabolism influences the amino acid composition and indicates a role for amino acid fermentation (38). Curtis et al. demonstrated the occurrence of Stickland reactions in dental plaque (9). These reactions were first described in clostridia (35-37). They involve the coupled fermentation of amino acids in which one amino acid is oxidized (Stickland donor) and another (Stickland acceptor) is reduced (29). Treponema denticola, an established resident of the oral cavity, performs Stickland reactions via the selenoprotein glycine reductase (32). Glycine reductase is composed of a multiprotein complex that contains two separate selenoproteins, termed selenoprotein A and selenoprotein B (1, 7, 8, 15, 16). This complex of proteins converts glycine to acetyl phosphate by using inorganic phosphate and the reducing potential from thioredoxin. For the organisms that use this complex, this is a vital source of ATP. Thus far, the requirement for selenocysteine at the active site of this enzyme complex is universally conserved, even though all other selenoproteins that have been identified using computational techniques have a putative cysteine homologue (24).Treponema denticola is considered one of the primary pathogens responsible for periodontitis, a chronic inflammatory disease that is the major cause of adult tooth loss (11, 27, 33). It is the best-studied oral spirochete, commonly found with other spirochetes within the periodontal pocket. It expresses a variety of virulence factors and is capable of adhering to and penetrating endothelial cell monolayers (31). Its health impact may reach beyond the oral cavity. A recent study linked periodontitis with peripheral arterial disease and detected T. denticola, along with other periodontal pathogens, in atherosclerotic plaque (3). Sequence analysis indicates the presence of several selenoproteins in addition to glycine reductase within the genome of T. denticola (24). This organism exhibits a strict growth requirement for selenium (32).A significant literature exists that clearly demonstrates the antimicrobial activity of fluoride compounds against microorganisms associated with dental decay and periodontitis. Both sodium fluoride and stannous fluoride, as well as stannous ions alone, inhibit the growth of T. denticola (21). The inhibitory effect of stannous salts on T. denticola''s growth is unexplained. It should be noted that toothpastes containing stannous fluoride are more effective in reducing gingivitis and plaque (28, 30).Tin, as well as several other trace elements, modulates the effects of acute selenium toxicity (20). Conversely, selenium affects the activity of tin in animal models (4-6). In this study, we examine the possibility that stannous ions interfere with selenium metabolism in T. denticola.     

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.     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).     

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.     

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).     

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.