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Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4'-phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74-fold (p .05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di-adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p .05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a1.5-fold (p .05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-β amyloid structure.

Cell surface-localized P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans mediates sucrose-independent adhesion to tooth surfaces. Previous studies showed that P1's C-terminal segment (C123, AgII) is also liberated as a separate polypeptide, contributes to cellular adhesion, interacts specifically with intact P1 on the cell surface, and forms amyloid fibrils. Identifying how C123 specifically interacts with P1 at the atomic level is essential for understanding related virulence properties of S. mutans. However, with sizes of ~ 51 and ~ 185 kDa, respectively, C123 and full-length P1 are too large to achieve high-resolution data for full structural analysis by NMR. Here, we report on biologically relevant interactions of the individual C3 domain with A3VP1, a polypeptide that represents the apical head of P1 as it is projected on the cell surface. Also evaluated are C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15 N-enriched C3 demonstrate its specific binding to A3VP1. Based on resolved C3 assignments, two binding sites, proximal and distal, are identified. Complementary NMR titration of A3VP1 with a C3/C12 complex suggests that binding of A3VP1 occurs on the distal C3 binding site, while the proximal site is occupied by C12. The MAb 6-8C binding interface to C3 overlaps with that of A3VP1 at the distal site. Together, these results identify a specific C3-A3VP1 interaction that serves as a foundation for understanding the interaction of C123 with P1 on the bacterial surface and the related biological processes that stem from this interaction. DATABASE: BMRB submission code: 27935.

The cnm gene, coding for the glycosylated collagen- and laminin-binding surface adhesin Cnm, is found in the genomes of approximately 20% of Streptococcus mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans, such as P1 and WapA, have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the β-sheet-rich N-terminal collagen-binding domain (CBD) of Cnm has a propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids in either its native glycosylated or recombinant nonglycosylated form and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro, ex vivo, and in vivo assays to characterize the amylogenic properties of Cnm. In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates, whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, amyloid formation by Cnm might act as a negative regulatory mechanism to modulate collagen-binding activity within S. mutans biofilms and warrants further investigation. IMPORTANCE Streptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor of S. mutans. Expression of Cnm by S. mutans is hypothesized to contribute to niche expansion, allowing colonization of multiple sites in the body, including collagen-rich surfaces such as dentin and heart valves. Here, we suggest that Cnm function might be modulated by its aggregation status. As a monomer, its primary function is to promote attachment to collagenous substrates via its collagen-binding domain (CBD). However, in later stages of biofilm maturation, the same CBD of Cnm could self-assemble into amyloid fibrils, losing the ability to bind to collagen and likely becoming a component of the biofilm matrix. Our findings shed light on the role of functional amyloids in S. mutans pathobiology and ecology.

Protein glycosylation has been described as the most abundant and complex post-translational modification occurring in nature. Recent studies have enhanced our view of how this modification occurs in bacteria highlighting the role of protein glycosylation in various processes such as biofilm formation, virulence and host-microbe interactions. We recently showed that the collagen- and laminin-binding adhesin Cnm of the dental pathogen Streptococcus mutans is post-translationally modified by the PgfS glycosyltransferase. Following this initial identification of Cnm as a glycoprotein, we have now identified additional genes (pgfM1, pgfE and pgfM2) that are also involved in the posttranslational modification of Cnm. Similar to the previously characterized ΔpgfS strain, inactivation of pgfM1, pgfE or pgfM2 directly impacts Cnm by altering its migration pattern, proteolytic stability and function. In addition, we identified the wall-associated protein A (WapA) as an additional substrate of Pgf-dependent modification. We conclude that the pgS-pgfM1-pgfE-pgfM2 operon encodes for a protein machinery that can modify, likely through the addition of glycans, both core and non-core gene products in S. mutans.

Potassium (K(+)) is the most abundant cation in dental plaque fluid. Previously, we reported the link between K(+) transport via Trk2 in Streptococcus mutans and its two critical virulence attributes: acid tolerance and surface adhesion. Herein, we build further on the intimate link between K(+) levels and S. mutans biology. High (>25 mM) versus low (≤5 mM) K(+) concentrations in the growth medium affected conformational epitopes of cell surface-localized adhesin P1. At low K(+), the expression of stress response elements gcrR and codY, cell-adhesion-associated genes such as spaP and metabolism-associated genes such as bglP was induced at stationary phase (P

Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1- and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

Potassium (K + ) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K + and a variety of K + transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K + acquisition in Streptococcus mutans and the importance of K + homeostasis for its virulence attributes. The S. mutans genome harbors four putative K + transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K + cotransporter (GlnQHMP), and a channel-like K + transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K + ] less than 5 mM eliminated biofilm formation in S. mutans . The functionality of the Trk2 system was confirmed by complementing an Escherichia coli TK2420 mutant strain, which resulted in significant K + accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K + -dependent cellular response of S. mutans to environment stresses. IMPORTANCE Biofilm formation and stress tolerance are important virulence properties of caries-causing Streptococcus mutans . To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment of S. mutans . K + is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K + transporters in S. mutans . We identified the most important system for K + homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K + for the activity of biofilm-forming enzymes, which explains why such high levels of K + would favor biofilm formation.

Proper envelope biogenesis of Streptococcus mutans, a biofilm-forming and dental caries-causing oral pathogen, requires two paralogs (yidC1 and yidC2) of the universally conserved YidC/Oxa1/Alb3 family of membrane integral chaperones and insertases. The deletion of either paralog attenuates virulence in vivo, but the mechanisms of disruption remain unclear. Here, we determined whether the deletion of yidC affects cell surface properties, extracellular glucan production, and/or the structural organization of the exopolysaccharide (EPS) matrix and biophysical properties of S. mutans biofilm. Compared to the wild type, the ΔyidC2 mutant lacked staining with fluorescent vancomycin at the division septum, while the ΔyidC1 mutant resembled the wild type. Additionally, the deletion of either yidC1 or yidC2 resulted in less insoluble glucan synthesis but produced more soluble glucans, especially at early and mid-exponential-growth phases. Alteration of glucan synthesis by both mutants yielded biofilms with less dry weight and insoluble EPS. In particular, the deletion of yidC2 resulted in a significant reduction in biofilm biomass and pronounced defects in the spatial organization of the EPS matrix, thus modifying the three-dimensional (3D) biofilm architecture. The defective biofilm harbored smaller bacterial clusters with high cell density and less surrounding EPS than those of the wild type, which was stiffer in compression yet more susceptible to removal by shear. Together, our results indicate that the elimination of either yidC paralog results in changes to the cell envelope and glucan production that ultimately disrupts biofilm development and EPS matrix structure/composition, thereby altering the physical properties of the biofilms and facilitating their removal. YidC proteins, therefore, represent potential therapeutic targets for cariogenic biofilm control. IMPORTANCE YidC proteins are membrane-localized chaperone insertases that are universally conserved in all bacteria and are traditionally studied in the context of membrane protein insertion and assembly. Both YidC paralogs of the cariogenic pathogen Streptococcus mutans are required for proper envelope biogenesis and full virulence, indicating that these proteins may also contribute to optimal biofilm formation in streptococci. Here, we show that the deletion of either yidC results in changes to the structure and physical properties of the EPS matrix produced by S. mutans, ultimately impairing optimal biofilm development, diminishing its mechanical stability, and facilitating its removal. Importantly, the universal conservation of bacterial yidC orthologs, combined with our findings, provide a rationale for YidC as a possible drug target for antibiofilm therapies.

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

Streptococcus mutans is a Gram-positive oral bacterium that is a primary etiological agent associated with human dental caries. In the oral cavity, S. mutans adheres to immobilized salivary agglutinin (SAG) contained within the salivary pellicle on the tooth surface. Binding to SAG is mediated by cell surface P1, a multifunctional adhesin that is also capable of interacting with extracellular matrix proteins. This may be of particular importance outside of the oral cavity as S. mutans has been associated with infective endocarditis and detected in atherosclerotic plaque. Despite the biomedical importance of P1, its binding mechanisms are not completely understood. In this work, we use atomic force microscopy-based single-molecule and single-cell force spectroscopy to quantify the nanoscale forces driving P1-mediated adhesion. Single-molecule experiments show that full-length P1, as well as fragments containing only the P1 globular head or C-terminal region, binds to SAG with relatively weak forces (∼50 pN). In contrast, single-cell analyses reveal that adhesion of a single S. mutans cell to SAG is mediated by strong (∼500 pN) and long-range (up to 6000 nm) forces. This is likely due to the binding of multiple P1 adhesins to self-associated gp340 glycoproteins. Such a cooperative, long-range character of the S. mutans-SAG interaction would therefore dramatically increase the strength and duration of cell adhesion. We also demonstrate, at single-molecule and single-cell levels, the interaction of P1 with fibronectin and collagen, as well as with hydrophobic, but not hydrophilic, substrates. The binding mechanism (strong forces, cooperativity, broad specificity) of P1 provides a molecular basis for its multifunctional adhesion properties. Our methodology represents a valuable approach to probe the binding forces of bacterial adhesins and offers a tractable methodology to assess anti-adhesion therapy.

In this study, we evaluated the immunogenicity, protective efficacy and peptide-based immune signatures of antibodies raised in mice after sublingual immunization with a recombinant form of the P1 (aka AgI/II, PAc) adhesin (P139-512) of Streptococcus mutans, a major etiological agent of dental caries. Sublingual administration of P139-512 in combination with the mucosal adjuvant LTK4R (a derivative of heat-labile LT toxin) induced strong and long-lasting systemic and mucosal immune responses. Incorporation of the adjuvant resulted in an enhancement of the anti-adhesive and anti-colonization activity against S. mutans as evaluated both under in vitro and in vivo conditions. Incorporation of the adjuvant to the vaccine formulation also changed the epitope specificity of the induced antibodies as determined by immunological signatures of sera collected from vaccinated mice. Use of a peptide microarray library led to the identification of peptide targets recognized by antibodies in serum samples with enhanced anti-adhesive effects. Altogether, the results presented herein showed that the sublingual administration of a P1-based subunit vaccine represents a promising approach for the prevention of dental caries caused by S. mutans. In addition, the present study disclosed the role of adjuvants on the epitope specificity and functionality of antibodies raised by subunit vaccines.

Made available in DSpace on 2018-12-11T16:56:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-01 National Institutes of Health Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. © 2014, American Society for Microbiology. Center of Excellence in Oral and Craniofacial Biology, School of Dentistry, Louisiana State University Health Sciences Center, New Orleans, LA Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY Department of Oral Biology, University of Florida School of Dentistry, Gainesville, FL Department of Comprehensive Dentistry and Biomaterials, School of Dentistry, Louisiana State University Health Sciences Center, New Orleans, LA Department of Orthodontics, University of Pennsylvania School of Dental Medicine, Philadelphia, PA Department of Microbiology, Immunology and Parasitology, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, Sao Paulo Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, Sao Paulo National Institutes of Health: DE08007 National Institutes of Health: DE19452 National Institutes of Health: DE21789 National Institutes of Health: DE22529 National Institutes of Health: EFRI-1137186

The adhesin known as Antigen I/II, P1 or PAc of the cariogenic dental pathogen Streptococcus mutans is a target of protective immunity and candidate vaccine antigen. Previously we demonstrated that immunization of mice with S. mutans complexed with anti-AgI/II monoclonal antibodies (MAbs) resulted in changes in the specificity, isotype and functionality of elicited anti-AgI/II antibodies in the serum of immunized mice compared to administration of bacteria alone. In the current study, an anti-AgI/II MAb reported in the literature to confer unexplained long term protection against S. mutans re-colonization following passive immunization in human clinical trials (MAb Guy’s 13), and expressed in tobacco plants (MAb Guy’s 13 plantibody), was evaluated for its potential immunomodulatory properties. Immunization of BALB/c mice with immune complexes of Guy’s 13 plantibody bound to S. mutans whole cells resulted in a similar change in specificity, isotype, and functionality of elicited anti-AgI/II antibodies as had been observed for other immunomodulatory MAbs. This new information, coupled with the recently solved crystal structure of the adhesin, now provides a rational explanation and plausible mechanism of action of passively administered Guy’s 13/Guy’s 13 plantibody in human clinical trials, and how long-term prevention of S. mutans carriage well past the application period of the therapeutic antibody could have been achieved.

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 Å resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C(1), C(2), and C(3). Each domain adopts a DE-variant IgG fold, with two β-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C(1) and C(2)) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C(1) and C(2) domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C(1) and C(2) domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal alpha-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by alpha-helical and proline-rich regions.

Oxa/YidC/Alb family proteins are chaperones involved in membrane protein insertion and assembly. Streptococcus mutans has two YidC paralogs. Elimination of yidC2 , but not yidC1 , results in stress sensitivity with decreased membrane-associated F 1 F o ATPase activity and an inability to initiate growth at low pH or high salt concentrations (A. Hasona, P. J. Crowley, C. M. Levesque, R. W. Mair, D. G. Cvitkovitch, A. S. Bleiweis, and L. J. Brady, Proc. Natl. Acad. Sci. USA 102:17466-17471, 2005). We now show that Escherichia coli YidC complements for acid tolerance, and partially for salt tolerance, in S. mutans lacking yidC2 and that S. mutans YidC1 or YidC2 complements growth in liquid medium, restores the proton motive force, and functions to assemble the F 1 F o ATPase in a previously engineered E. coli YidC depletion strain (J. C. Samuelson, M. Chen, F. Jiang, I. Moller, M. Wiedmann, A. Kuhn, G. J. Phillips, and R. E. Dalbey, Nature 406:637-641, 2000). Both YidC1 and YidC2 also promote membrane insertion of known YidC substrates in E. coli ; however, complete membrane integrity is not fully replicated, as evidenced by induction of phage shock protein A. While both function to rescue E. coli growth in broth, a different result is observed on agar plates: growth of the YidC depletion strain is largely restored by 247YidC2, a hybrid S. mutans YidC2 fused to the YidC targeting region, but not by a similar chimera, 247YidC1, nor by YidC1 or YidC2. Simultaneous expression of YidC1 and YidC2 improves complementation on plates. This study demonstrates functional redundancy between YidC orthologs in gram-negative and gram-positive organisms but also highlights differences in their activity depending on growth conditions and species background, suggesting that the complete functional spectrum of each is optimized for the specific bacteria and environment in which they reside.

Sequences contributing to epitopes recognized by a panel of monoclonal antibodies (mAbs) against the Streptococcus mutans surface protein P1 were delineated by Western blot and enzyme-linked immunosorbent assay using a battery of deletion constructs and recombinant polypeptides. mAbs that recognize complex discontinuous epitopes reconstituted by combining the alanine-rich and proline-rich repeat domains and varying degrees of flanking sequence were identified as well as mAbs that bound epitopes contained within contiguous segments of P1. Cross-reactivity with SspA and SspB from Streptococcus gordonii is also reported. This information enables insight into the structure and function of a streptococcal adhesin and its correlates of protection and furthers our understanding of the immunomodulatory and bacterial-adherence inhibition activities of anti-P1 mAbs.

Researchers now recognize the utility of surface plasmon resonance technology to evaluate interactions of microbial pathogens with host components. The surface adhesin and candidate vaccine antigen P1 of Streptococcus mutans, the main causative agent of dental caries, interacts with a high molecular weight glycoprotein called salivary agglutinin, or gp340, in the salivary pellicle. We optimized a BIAcore assay to measure P1-mediated Ca(2+) dependent binding of S. mutans whole cells to this physiological ligand immobilized on a Pioneer F1 sensor chip. Regeneration conditions allowed cells to be eluted from the sensor chip permitting multiple reuse of the agglutinin-coated surface. An isogenic P1-deficient S. mutans mutant did not bind to immobilized agglutinin demonstrating specificity of the detected interaction. Glutaraldehyde-fixation of bacterial cells showed the assay measured a whole cell-ligand interaction and was not an artifact of solubilized or leached proteins. Adherence inhibition assays demonstrated varying degrees of disruption of the S. mutans-agglutinin interaction by anti-P1 monoclonal antibodies recognizing different epitopes, whereas a polyclonal reagent demonstrated more complete inhibition. This report describes an improved method to assess salivary agglutinin-mediated adherence of S. mutans in vitro under physiological-like conditions and to evaluate the effectiveness of antibodies of differing specificities to inhibit binding.

We demonstrated previously that mucosal immunization of mice withStreptococcus mutanscoated with the monoclonal antibody (MAb) 6-11A directed against the major surface adhesin protein P1 results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with animals immunized with bacteria only. We now show that the specificity of the mucosal secretory IgA response was also influenced by this MAb. Changes in antibody specificity were associated with changes in biological activity. Serum samples which differed in antibody reactivity with P1 polypeptides generated by partial digestion withN-chlorosuccinimide but not in isotype distribution or overall reactivity withS. mutansor intact P1 demonstrated a statistically significant difference in the ability to inhibit bacterial adherence to salivary-agglutinin-coated hydroxyapatite beads. Serum IgG antibodies against P1 from mice immunized with eitherS. mutansalone orS. mutanscoated with 6-11A were shown to recognize antigenic determinants dependent on the presence of the central proline-rich repeat domain, a segment necessary for the structural integrity of the molecule. However, no statistically significant differences were observed in antibody reactivity with a panel of six partial P1 polypeptides encoded by overlappingspaPsubclones, suggesting that the targets of biologically relevant antibodies involve complex epitopes not reconstituted by the recombinant products tested. Lastly, we show that binding of MAb 6-11A to P1 on the surface ofS. mutansalters P1's susceptibility to proteolytic digestion. Hence, changes in antigen processing and presentation may contribute to the immunomodulatory effects of this MAb.

Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1 39–512 ), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro . In the present study, we further investigated the immunological features of P1 39–512 in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1 39–512 preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1 39–512 -specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1 39–512 antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1 39–512 as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.

Previously, we demonstrated successful Tn917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km(r), repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S. mutans strain NG8. Tn917-lac transposition occurred at a frequency of ca. 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression. Tn917-lac mutants that expressed beta-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were isolated. Further characterization of one of the mutants with increased beta-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The beta-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose. Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium. Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. These findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medically relevant streptococcal species.

Streptococcus mutans , the principal etiologic agent of dental caries in humans, possesses a variety of virulence traits that enable it to establish itself in the oral cavity and initiate disease. A 185-kDa cell surface-localized protein known variously as antigen I/II, antigen B, PAc, and P1 has been postulated to be a virulence factor in S. mutans . We showed previously that P1 expression is necessary for in vitro adherence of S. mutans to salivary agglutinin-coated hydroxyapatite as well as for fluid-phase aggregation. Since adherence of the organism is a necessary first step toward colonization of the tooth surface, we sought to determine what effect deletion of the gene for P1, spaP , has on the colonization and subsequent cariogenicity of this organism in vivo. Germ-free Fischer rats fed a diet containing 5% sucrose were infected with either S. mutans NG8 or an NG8-derived spaP mutant strain, PC3370, which had been constructed by allelic exchange mutagenesis. At 1-week intervals for 6 weeks after infection, total organisms recovered from mandibles were enumerated. At week 6, caries lesions also were scored. A significantly lower number of enamel and dentinal carious lesions was observed for the mutant-infected rats, although there was no difference between parent and mutant in the number of organisms recovered from teeth through 6 weeks postinfection. Coinfection of animals with both parent and mutant strains resulted in an increasing predominance of the mutant strain being recovered over time, suggesting that P1 is not a necessary prerequisite for colonization. These data do, however, suggest a role for P1 in the virulence of S. mutans , as reflected by a decrease in the cariogenicity of bacteria lacking this surface protein.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Streptococcus mutans antigen I/II (AgI/II) has been widely studied as a candidate vaccine antigen against human dental caries. In this report we follow up on prior studies that indicated that anti-AgI/II immunomodulatory monoclonal antibodies (MAbs) exerted their effects by destabilizing the native protein structure and exposing cryptic epitopes. We show here that similar results can be obtained by immunizing mice with truncated polypeptides out of the context of an intra-molecular interaction that occurs within the full-length molecule and that appears to dampen the functional response against at least two important target epitopes. Putative T cell epitopes that influenced antibody specificity were identified immediately upstream of the alanine-rich repeat domain. Adherence inhibiting antibodies could be induced against two discrete domains of the protein, one corresponding to the central portion of the molecule and the other corresponding to the C-terminus.

The cariogenic bacterium Streptococcus mutans has two paralogues of the YidC/Oxa1/Alb3 family of membrane protein insertases/chaperones. Disruption of yidC2 results in loss of genetic competence, decreased membrane-associated ATPase activity and stress sensitivity (acid, osmotic and oxidative). Elimination of yidC1 has less severe effects, with little observable effect on growth or stress sensitivity. To examine the respective roles of YidC1 and YidC2, a conditional expression system was developed allowing simultaneous elimination of both endogenous YidCs. The function of the YidC C-terminal tails was also investigated and a chimeric YidC1 protein appended with the C terminus of YidC2 enabled YidC1 to complement a ΔyidC2 mutant for stress tolerance, ATP hydrolysis activity and extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Elimination of yidC1 or yidC2 affected levels of extracellular proteins, including GtfB, GtfC and adhesin P1 (AgI/II, PAc), which were increased without YidC1 but decreased in the absence of YidC2. Both yidC1 and yidC2 were shown to contribute to S. mutans biofilm formation and to cariogenicity in a rat model. Collectively, these results provide evidence that YidC1 and YidC2 contribute to cell surface biogenesis and protein secretion in S. mutans and that differences in stress sensitivity between the ΔyidC1 and ΔyidC2 mutants stem from a functional difference in the C-termini of these two proteins.

Two assays were utilized to study the interaction of P1, a cell surface adhesin molecule expressed by Streptococcus mutans , and a high molecular weight mucin-like glycoprotein found in human saliva known as salivary agglutinin. A spectrophotometric assay was used to monitor the calcium-dependent aggregation of S. mutans in the presence of fluid-phase salivary agglutinin as evidenced by a measurable decrease in optical density at 700 nm. An adherence assay was used to measure the binding of 3 H-radiolabelled S. mutans to immobilized salivary agglutinin coated onto hydroxyapatite beads. The role of P1 in these interactions was assessed using P1 retainer and non-retainer strain of S. mutans as well as P1 deficeint mutants. Several monoclonal antibodies which recognize different epitopes on P1 were tested for their ability to inhibit the interaction of S. mutans with salivary agglutinin in both the aggregation or adherence assays. Different monoclonal antibodies were inhibitory in each assay suggesting that the way in which P1 interacts with salivary agglutinin differs depending on whether the agglutinin is fluid-phase or immobilized. Competitive inhibition of aggregation and adherence reactions utilizing full-length and recombinant-specified P1 polypeptides was performed as well.

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

We showed previously that deliberate immunization of BALB/c mice with immune complexes (IC) of the cariogenic bacterium Streptococcus mutans and mAbs against its surface adhesin P1 results in changes in the specificity and isotype of elicited anti-P1 Abs. Depending on the mAb, changes were beneficial, neutral, or detrimental, as measured by the ability of the serum from immunized mice to inhibit bacterial adherence to human salivary agglutinin by a BIAcore surface plasmon resonance assay. The current study further defined changes in the host response that result from immunization with IC containing beneficial mAbs, and evaluated mechanisms by which beneficial immunomodulation could occur in this system. Immunomodulatory effects varied depending upon genetic background, with differing results in C57BL/6 and BALB/c mice. Desirable effects following IC immunization were observed in the absence of activating FcRs in BALB/c Fcer1g transgenic mice. mAb F(ab′)2 mediated desirable changes similar to those observed using intact IgG. Sera from IC-immunized BALB/c mice that were better able to inhibit bacterial adherence demonstrated an increase in Abs able to compete with an adherence-inhibiting anti-P1 mAb, and binding of a beneficial immumomodulatory mAb to S. mutans increased exposure of that epitope. Consistent with a mechanism involving a mAb-mediated structural alteration of P1 on the cell surface, immunization with truncated P1 derivatives lacking segments that contribute to recognition by beneficial immunomodulatory mAbs resulted in an improvement in the ability of elicited serum Abs to inhibit bacterial adherence compared with immunization with the full-length protein.

Interactions between salivary agglutinin and the adhesin P1 of Streptococcus mutans contribute to bacterial aggregation and mediate sucrose-independent adherence to tooth surfaces. We have examined biofilm formation by S. mutans UA159, and derivative strains carrying mutations affecting the localization or expression of P1, in the presence of fluid-phase or adsorbed saliva or salivary agglutinin preparations. Whole saliva- and salivary agglutinin-induced aggregation of S. mutans was adversely affected by the loss of P1 and sortase (SrtA) but not by the loss of trigger factor (RopA). Fluid-phase salivary agglutinin and, to a lesser extent, immobilized agglutinin inhibited biofilm development by S. mutans in the absence of sucrose, and whole saliva was more effective at decreasing biofilm formation than salivary agglutinin. Inhibition of biofilm development by salivary agglutinin was differently influenced by particular mutations, with the P1-deficient strain displaying a greater inhibition of biofilm development than the SrtA- or RopA-deficient strains. As expected, biofilm-forming capacities of all strains in the presence of salivary preparations were markedly enhanced in the presence of sucrose, although biofilm formation by the mutants was less efficient than that by the parental strain. Aeration strongly inhibited biofilm development, and the presence of salivary components did not restore biofilm formation in aerated conditions. The results disclose a potent ability of salivary constituents to moderate biofilm formation by S. mutans through P1-dependent and P1-independent pathways.

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is because of low abundance, poor solubility, and inherent hydrophobicity. In this study, membrane preparations from the Gram-positive bacterium Streptococcus mutans were isolated from protoplasts and by mechanical grinding. Membrane proteins were extracted using a mixture of trifluroethanol and chloroform, solubilized using highly chaotropic buffer containing ASB-14 and Triton X-100 and subjected to two-dimensional gel electrophoresis.

The P1 adhesin of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 that is covalently attached to the cell wall interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼50-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we have demonstrated that solid state NMR spectroscopy can be used to 1) globally characterize cell walls isolated from a Gram-positive bacterium; 2) quantify P1 localized on the cell surface; and 3) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent adhesion and biofilm formation via NMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

We previously identified five monoclonal antibodies (MAbs) against Streptococcus mutans adhesin P1 that modulate the humoral response when bound to whole bacteria and immune complexes (ICs) are administered to BALB/c mice. The two MAbs that redirected the response towards increased efficacy recognize discontinuous epitopes involving pre-alanine-rich domain sequence; therefore, to evaluate whether epitope specificity contributes to a desirable outcome a further MAb with this characteristic was tested. A beneficial immune response was promoted. None of the three MAbs that promoted a beneficial response was opsonic, suggesting that increased uptake of ICs by phagocytes does not mediate the improvement of the IC-elicited antibodies to inhibit bacterial adherence. Finally, two of the six anti-P1 MAbs activated complement but did not partition according to desirable vs. nondesirable effects.

Streptococcus mutans is considered to be the major etiologic agent of human dental caries. Attachment of S. mutans to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in Escherichia coli , and (v) in E. coli , P1 is secreted in the absence of SecB.

Acid tolerance is regarded as an important determinant for survival of cariogenic bacteria in the biofilm on teeth (7, 37, 49.) The microorganisms in dental plaque are exposed to continual cycles of acid shock resulting from the formation of acid end products during metabolism of dietary carbohydrate by the acidogenic microflora. In vivo pH measurements have revealed that the intake of carbohydrates can drop the plaque pH from 7 to 4 in less than 20 min (21, 24, 52). In addition, frequent carbohydrate intake results in a lower pH of “resting plaque,” which has been associated with increased dental caries. Early studies (41) demonstrated that high caries activity is associated with rapid plaque acidification as well as tolerance of low pH by the plaque microorganisms. Streptococcus mutans has been recognized as one of the major etiological agents in human dental caries (14, 27). In addition to its ability to colonize tooth surfaces and acidogenicity, S. mutans is acid tolerant. Glycolysis can occur at pH values as low as pH 4.4, whereas the minimum pH for growth is 4.8 (5). Svensater et al. (42) demonstrated that a 2-h exposure of exponential-phase cells to pH 5.5 induced an acid tolerance response (ATR) that increased subsequent survival at pH 3.0. The mechanisms involved in acid tolerance of S. mutans are poorly understood. Protein synthesis is required for induction of an ATR, since it could be blocked by chloramphenicol in the medium (17). Sustained mildly acidic conditions increase H+/ATPase and glycolytic activities (2, 4, 15, 16). These metabolic changes permit S. mutans to maintain a constant transmembrane pH gradient of approximately 1.0 pH unit (8, 15). Previous genetic studies in our laboratory focused on identification of genes that when inactivated by transposon Tn917 rendered the mutants acid sensitive (12). One of the mutants (AS17) affected in acid tolerance had the transposon integrated into the intergenic region between the ylxM and ffh homologs. This locus was subsequently designated sat for secretion and acid tolerance (12). The ffh gene encodes a 54-kDa subunit homolog of the eucaryotic signal recognition particle (SRP) complex, which is part of a protein translocation pathway that is highly conserved throughout the domains of life (6, 9, 29, 54). Detailed studies of Escherichia coli and Bacillus subtilis have shown that this pathway is involved in protein export and membrane biogenesis. The protein encoded by ylxM is homologous to a protein of unknown function in Bacillus subtilis that is transcribed as an operon with ffh (19). YlxM contains helix-turn-helix motifs indicative of DNA-binding proteins, therefore suggesting a function in transcriptional regulation (35). Remarkably, the transposon integration in AS17 resulted in a viable mutant. In E. coli and B. subtilis, ffh is a gene essential for viability (19, 33, 51). Our previous data (11) suggested that AS17 had a reduction in rather than a complete loss of Ffh expression. Further characterization of mutant AS17 revealed that it was incapable of an ATR (11). Furthermore, the H+/ATPase activity in membrane fractions of pH 5.0-shocked AS17 cells was comparable to that in pH 7.5-grown mutant cells, whereas it was twofold lower than the pH 5.0-induced activity in wild-type cell membranes (11). A lack of Ffh could result in an inability to rearrange the membrane in the way needed to generate an ATR, which would include the incorporation of additional H+/ATPase polypeptides. However, the phenotypic effects observed could be the result of polar effects of the insertion. In the present study, we addressed this issue by characterizing the transcriptional organization of the sat operon by Northern blotting and primer extension analysis. Subsequently, we created nonpolar kanamycin insertions in the individual genes of the sat operon. Growth experiments were performed to assess the involvement of each of the sat operon genes in acid tolerance of S. mutans. Our data show that the S. mutans sat operon consists of five open reading frames (ORFs) and confirm the functional role of ffh in acid tolerance.

A two-stage radioimmunoassay (RIA) has been developed that identifies type-specific antigens on the surfaces of group B streptococci. In addition to detecting the type-specific carbohydrate antigens Ia, Ib, II, and III, the RIA identified four unique antigens reactive with the Centers for Disease Control's c-protein typing antiserum. Analysis of hot-acid extracts by immunoelectrophoresis confirmed that two of these reactivities corresponded to the reported alpha and beta antigens of the c-protein marker. In addition, the identification of two heretofore unidentified antigens, gamma and delta, by the two-stage RIA is detailed in this report. The assay uses intact bacteria and does not require hot-acid extraction, a procedure thus enabling detection of acid-labile antigens in their native unmodified form. The semiquantitative RIA requires less typing reagent than does precipitin testing and is more objective, reproducible, and rapid. The assay described here could be applied to the detection of any cell surface antigen for which a monospecific antiserum is available.