Genome-Derived Criteria for Assigning Environmental narG and nosZ Sequences to Operational Taxonomic Units of Nitrate Reducers

Ninety percent of cultured bacterial nitrate reducers with a 16S rRNA gene similarity of ≥97% had a narG or nosZ similarity of ≥67% or ≥80%, respectively, suggesting that 67% and 80% could be used as standardized, conservative threshold similarity values for narG and nosZ, respectively (i.e., any two sequences that are less similar than the threshold similarity value have a very high probability of belonging to different species), for estimating species-level operational taxonomic units. Genus-level tree topologies of narG and nosZ were generally similar to those of the corresponding 16S rRNA genes. Although some genomes contained multiple copies of narG, recent horizontal gene transfer of narG was not apparent.Nitrate reducers (i.e., both dissimilatory nitrate reducers and denitrifiers) reduce nitrate to nitrite, which can then be reduced to ammonium by dissimilatory nitrate reducers or sequentially reduced to nitric oxide, nitrous oxide, and dinitrogen by denitrifiers (29). narG codes for the alpha subunit of the dissimilatory nitrate reductase, which reduces nitrate to nitrite and is thus common to both dissimilatory nitrate reducers and denitrifiers (29). nosZ codes for nitrous oxide reductase, which reduces nitrous oxide to dinitrogen and is common to denitrifiers but not dissimilatory nitrate reducers (29). Both narG and nosZ are commonly used as gene markers for community level analysis of nitrate reducers (2, 8, 9, 16, 18, 19, 20, 25). However, standardized criteria for assigning environmental narG and nosZ sequences to operational taxonomic units (OTUs) are required so that diverse data sets on nitrate-reducing communities can be normalized. The widespread ability of bacteria and archaea to denitrify (29) complicates the development of such criteria for genes involved in denitrification. Some closely related narG and closely related nosZ genes occur in distantly related taxa, and narG or nosZ phylogenies do not always reflect 16S rRNA phylogenies (17). However, nosZ-based phylogenies in general have a high degree of congruency with 16S rRNA gene-based phylogenies (3, 10, 30), and recent horizontal gene transfer of nosZ seems unlikely (10), indicating that denitrifier structural genes might be used for estimating the species-level novelty, as well as species-level diversity, of denitrifiers in environmental samples. The limited amount of data on horizontal gene transfer of narG (4, 24) identifies a need to extend such an approach to this gene. The limited number of studies that have compared 16S rRNA with narG or nosZ phylogenies accentuates the need for a more thorough analysis of the phylogenetic relatedness of these three genes (3, 4, 7). Thus, the main objectives of this study were to (i) resolve criteria for standardizing OTU assignment of environmental narG and nosZ sequences, (ii) determine whether those criteria can be used as indicators of novel species, and (iii) investigate the impact of horizontal gene transfer on narG.     

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).     

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.     

The dlt Operon of Bacillus cereus Is Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Δdlt_Bc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Δdlt_Bc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Δdlt_Bc). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.Gram-positive bacteria are generally enclosed by cell walls consisting of macromolecular assemblies of cross-linked peptidoglycan (murein), polyanionic teichoic acids (TAs), and surface proteins (69). TAs are polymers of repeating glycerophosphate residues. They may be covalently anchored to either peptidoglycan (wall-associated TAs) or the cytoplasmic membrane via glycolipids (lipoteichoic acids [LTAs]). TAs may be involved in controlling cell shape, autolytic enzyme activity, and cation homeostasis (69). They make a significant contribution to the overall negative charge of the bacterial cell wall, attracting negatively charged compounds, including the cationic antimicrobial peptides (AMPs) of the innate humoral immune systems of higher organisms (69).Many of the gram-positive bacterial species pathogenic to humans display resistance to cationic AMPs because of a decrease in the net negative charge of bacterial cell envelopes (75). Modifications to the TAs at the bacterial surface involving the incorporation of positively charged residues, such as d-alanine, prevent cationic AMPs from reaching their target, thereby protecting the organism against these compounds. This process involves the Dlt proteins encoded by the dltABCD operon present in most of the genome sequences established to date for gram-positive bacteria (44, 58, 74). d-Alanine is incorporated into LTAs through a two-step reaction involving a d-alanine-d-alanyl carrier protein ligase (Dcl) and a d-alanyl carrier protein (Dcp), encoded by the dltA and dltC genes, respectively (18, 44, 45, 70). The dltB and dltD genes encode other proteins required for the d-alanylation of LTAs. DltD is involved in selection of the Dcp carrier protein for ligation with d-alanine (19), whereas DltB is thought to be involved in d-alanyl-Dcp secretion (69). d-Alanine may be transferred from d-alanylated LTAs to wall-associated TAs by transacylation. For many human gram-positive bacterial pathogens, dlt operon inactivation has been shown to affect bacterial resistance to cationic AMPs and virulence. Indeed, Listeria monocytogenes, Bacillus anthracis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Lactobacillus reuteri, and group B streptococci harboring mutations in dlt genes all have a higher negative charge on the cell surface and are more susceptible to cationic AMPs of various origins (1, 34, 56, 58, 59, 77, 78, 89). The inactivation of dlt genes in these pathogenic bacterial species also decreases interactions with phagocytic and nonphagocytic cells (1, 13, 34, 78).The impact of Dlt proteins on cationic AMP resistance and virulence in insect bacterial pathogens has never before been studied, despite the major role of cationic AMPs in insect humoral immunity (9, 61). Insect bacterial pathogens also termed entomopathogenic bacteria are able to multiply in the insect hemocoel from small inocula (<10,000 viable cells) and produce fatal septicemia (8, 57). Entomopathogenic bacteria entering the hemolymph are targeted by an array of immune system mediators of both cellular and humoral reactions. The cellular response results in bacterial phagocytosis or encapsulation by circulating hemocytes, whereas the humoral response generates cationic AMPs (61). These peptides are small, inducible molecules produced in large amounts in hemolymph by the fat body (9, 26). They participate to the insect antimicrobial defense in a systemic response. Many AMP have been reported to cause damage in microbial membranes, which may ultimately lead to bacterial cell lysis (94).We investigated the role of the dlt operon in cationic AMP resistance and virulence in Bacillus cereus, a human opportunistic and insect facultative bacterial pathogen. B. cereus sensu stricto is a spore-forming gram-positive bacterium. The B. cereus sensu lato group of bacteria also includes the closely related insect pathogen Bacillus thuringiensis and the human pathogen B. anthracis. Genomic data have shown that B. thuringiensis and B. cereus have almost identical chromosomal genetic backgrounds (54, 55) but that B. thuringiensis carries a plasmid encoding entomopathogenic cytoplasmic crystalline δ-endotoxins (Cry proteins) specifically active against insect larvae upon ingestion (22, 23, 83). B. cereus can cause opportunistic food-borne gastroenteritis and local/systemic infections in immunocompromised humans (85). Both B. thuringiensis (with and without Cry toxins) and B. cereus strains are highly pathogenic when injected directly into the hemocoels of insect larvae, in which they cause lethal septicemia (46, 82, 86, 96). The occurrence, structure, and glycosylation of LTAs were studied for different Bacillus species, including B. cereus strains containing LTAs (built up of polyglycerol phosphate chains and hydrophobic anchors) and d-alanine (11, 50, 51, 62). Therefore, the presence of a dlt operon in the B. cereus 14579 genome suggests that the LTAs may be alanylated.We report here that the dlt operon of B. cereus is required for resistance to cationic AMPs of bacterial or insect origin. The dlt operon is required for full B. cereus virulence following bacterial injection into two lepidopteran insects, the caterpillar Spodoptera littoralis and the wax moth Galleria mellonella. We also detected the dlt operon in three gram-negative bacterial genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01).     

Evidence for Nitrogen Fixation by “Dehalococcoides ethenogenes” Strain 195

Genome annotation of the chlorinated ethene-respiring “Dehalococcoides ethenogenes” strain 195 indicated the presence of a complete nitrogenase operon. Here, results from long-term growth experiments, gene expression, and ¹⁵N₂-isotope measurements confirm that strain 195 is capable of fixing atmospheric dinitrogen when a defined fixed-nitrogen source such as ammonium is unavailable.“Dehalococcoides ethenogenes” strain 195 is the first isolated bacterium that is capable of reductively dechlorinating tetrachloroethene and trichloroethene (TCE) to vinyl chloride (VC) and ethene (22). Annotation of the 1.5-Mbp genome of strain 195 has identified 17 intact reductive dehalogenase (RDase) genes (25). The variety of RDases has essentially defined the metabolic capabilities of strain 195 and other Dehalococcoides strains for respiration of chlorinated ethenes (8, 9, 15, 23, 27) and other chlorinated compounds (1, 2, 6, 21), making them important participants in bioremediation processes (19). Expression of different putative RDase genes has been examined previously in pure culture (6) and in Dehalococcoides-containing enrichment cultures (3, 4, 13, 17, 24, 28).Genome annotation of strain 195 has revealed the presence of a nitrogenase-encoding operon (nif) (DET1151-58) typical of those found in anaerobes (25). According to the published genome annotations of four strains of Dehalococcoides, strain 195 is the only one that contains a nif operon (16, 25; Joint Genome Institute, 2009, Integrated Microbial Genomes system [www.jgi.doe.gov]). A nif operon closely related to that in strain 195 has also been identified in a mixed Dehalococcoides-containing community (29); thus, the nitrogen-fixing function might be present in other unsequenced strains of Dehalococcoides.Phylogenetically, the nitrogenase structural genes of strain 195 are clustered with diverse anaerobic Bacteria, including the molybdenum (Mo)-nitrogenase in Clostridium pasteurianum, as well as Archaea, including the Mo-nitrogenase in Methanosarcina barkeri (25, 30). In the genome of strain 195, the presence of an ABC transporter for molybdenum (DET1159-61) and a nifV gene (DET1614), which encodes homocitrate synthetase used in nitrogenase FeMo-cofactor biosynthesis, suggests that the nitrogenase is of the typical molybdenum-iron type (25). While strain 195 is the only sequenced Dehalococcoides isolate that contains a nif operon, Ju et al. (14) previously identified functional nifH genes in dechlorinating organisms from diverse genera such as Sulfurospirillum multivorans, Desulfovibrio dechloracetivorans, and Desulfomonile tiedjei.Aquifers containing groundwater contaminated with chlorinated ethenes can potentially be limited in nutrients. For example, at the Wurtsmith Air Force Base, the chlorinated ethene-contaminated groundwater was found to contain less than 0.09 mM of ammonia, prompting ammonium amendment (26). Little is currently known about the potential effects of nitrogen limitation on reductive dechlorination in the environment, and the demonstration of nitrogen fixation in strain 195 was previously hindered by the use of an undefined medium (21). Here, we present results demonstrating that strain 195 is capable of fixing atmospheric dinitrogen and the physiological implications of the stress caused by nitrogen limitation.     

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).     

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.