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Streptococcus sanguinis is a primary colonizer of teeth and is associated with oral health. When it enters the bloodstream, however, this bacterium may cause the serious illness infective endocarditis. The genes required for survival and proliferation in blood have not been identified. The products of these genes could provide a rich source of targets for endocarditis‐specific antibiotics possessing greater efficacy for endocarditis, and also little or no activity against those bacteria that remain in the mouth. We previously created a comprehensive library of S. sanguinis mutants lacking every nonessential gene. We have now screened each member of this library for growth in human serum and discovered 178 mutants with significant abundance changes. The main biological functions disrupted in these mutants, including purine metabolism, were highlighted via network analysis. The components of an ECF‐family transporter were required for growth in serum and were shown for the first time in any bacterium to be essential for endocarditis virulence. We also identified two mutants whose growth was reduced in serum but not in saliva. This strategy promises to enable selective targeting of bacteria based on their location in the body, in this instance, treating or preventing endocarditis while leaving the oral microbiome intact., The fitness of S. sanguinis mutants in human serum was screened by ORF‐seq. The biological functions of these fitness genes were analyzed. The virulence factors in a rabbit endocarditis model and potential selective drug targets in human serum were explored.

Experimental techniques for identification of essential genes (EGs) in prokaryotes are usually expensive, time-consuming and sometimes unrealistic. Emerging in silico methods provide alternative methods for EG prediction, but often possess limitations including heavy computational requirements and lack of biological explanation. Here we propose a new computational algorithm for EG prediction in prokaryotes with an online database (ePath) for quick access to the EG prediction results of over 4,000 prokaryotes (https://www.pubapps.vcu.edu/epath/). In ePath, gene essentiality is linked to biological functions annotated by KEGG Ortholog (KO). Two new scoring systems, namely, E_score and P_score, are proposed for each KO as the EG evaluation criteria. E_score represents appearance and essentiality of a given KO in existing experimental results of gene essentiality, while P_score denotes gene essentiality based on the principle that a gene is essential if it plays a role in genetic information processing, cell envelope maintenance or energy production. The new EG prediction algorithm shows prediction accuracy ranging from 75% to 91% based on validation from five new experimental studies on EG identification. Our overall goal with ePath is to provide a comprehensive and reliable reference for gene essentiality annotation, facilitating the study of those prokaryotes without experimentally derived gene essentiality information.

Streptococcus sanguinis is an early colonizer of the tooth surface and competes with oral pathogens such as Streptococcus mutans to maintain oral health. However, little is known about its mechanism of biofilm formation. Here, we show that mutation of the ciaR gene, encoding the response regulator of the CiaRH two-component system in S. sanguinis SK36, produced a fragile biofilm. Cell aggregation, gtfP gene expression and water-insoluble glucan production were all reduced, which suggested polysaccharide production was decreased in ΔciaR. RNA sequencing and qRT-PCR revealed that arginine biosynthesis genes (argR, argB, argC, argG, argH and argJ) and two arginine/histidine permease genes (SSA_1568 and SSA_1569) were upregulated in ΔciaR. In contrast to ΔciaR, most of strains constructed to contain deletions in each of these genes produced more biofilm and water-insoluble glucan than SK36. A ΔciaRΔargB double mutant was completely restored for the gtfP gene expression, glucan production and biofilm formation ability that was lost in ΔciaR, indicating that argB was essential for ciaR to regulate biofilm formation. We conclude that by promoting the expression of arginine biosynthetic genes, especially argB gene, the ciaR mutation reduced polysaccharide production, resulting in the formation of a fragile biofilm in Streptococcus sanguinis.

Biofilm accounts for 65-80 % of microbial infections in humans. Considerable evidence links biofilm formation by oral microbiota to oral disease and consequently systemic infections. Streptococcus sanguinis, a Gram-positive bacterium, is one of the most abundant species of the oral microbiota and it contributes to biofilm development in the oral cavity. Due to its altered biofilm formation, we investigated a biofilm mutant, ΔSSA_0351, that is deficient in type I signal peptidase (SPase) in this study. Although the growth curve of the ΔSSA_0351 mutant showed no significant difference from that of the wild-type strain SK36, biofilm assays using both microtitre plate assay and confocal laser scanning microscopy (CLSM) confirmed a sharp reduction in biofilm formation in the mutant compared to the wild-type strain and the paralogous mutant ΔSSA_0849. Scanning electron microscopy (SEM) revealed remarkable differences in the cell surface morphologies and chain length of the ΔSSA_0351 mutant compared with those of the wild-type strain. Transcriptomic and proteomic assays using RNA sequencing and mass spectrometry, respectively, were conducted on the ΔSSA_0351 mutant to evaluate the functional impact of SPase on biofilm formation. Subsequently, bioinformatics analysis revealed a number of proteins that were differentially regulated in the ΔSSA_0351 mutant, narrowing down the list of SPase substrates involved in biofilm formation to lactate dehydrogenase (SSA_1221) and a short-chain dehydrogenase (SSA_0291). With further experimentation, this list defined the link between SSA_0351-encoded SPase, cell wall biosynthesis and biofilm formation.

Summary Streptococcus sanguinis colonizes teeth and is an important cause of infective endocarditis. Our prior work showed that the lipoprotein SsaB is critical for S. sanguinis virulence for endocarditis and belongs to the LraI family of conserved metal transporters. In this study, we demonstrated that an ssaB mutant accumulates less manganese and iron than its parent. A mutant lacking the manganese-dependent superox- ide dismutase, SodA, was significantly less virulent than wild-type in a rabbit model of endocarditis, but significantly more virulent than the ssaB mutant. Neither the ssaB nor the sodA mutation affected sen- sitivity to phagocytic killing or efficiency of heart valve colonization. Animal virulence results for all strains could be reproduced by growing bacteria in serum under physiological levels of O2. SodA activity was reduced, but not eliminated in the ssaB mutant in serum and in rabbits. Growth of the ssaB mutant in serum was restored upon addition of Mn 2+ or removal of O2. Antioxidant supplementation experiments sug- gested that superoxide and hydroxyl radicals were together responsible for the ssaB mutant's growth defect. We conclude that manganese accumulation mediated by the SsaB transport system imparts viru- lence by enabling cell growth in oxygen through SodA-dependent and independent mechanisms.

Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259-6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe(III)2-Y(•)) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor requires NrdI, and Mn(III)2-Y(•) is more active than Fe(III)2-Y(•) with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that Mn(III)2-Y(•)-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.

Summary Streptococcus sanguinis is a major component of the oral flora and an important cause of infective endocarditis. Although S. sanguinis is naturally competent, genome sequencing has suggested significant differences in the S. sanguinis competence system relative to those of other streptococci. An S. sanguinis mutant possessing an in-frame deletion in the comC gene, which encodes competence-stimulating peptide (CSP), was created. Addition of synthetic CSP induced competence in this strain. Gene expression in this strain was monitored by microarray analysis at multiple time-points from 2.5 to 30 min after CSP addition, and verified by quantitative reverse transcription–polymerase chain reaction. Over 200 genes were identified whose expression was altered at least two-fold in at least one time point, with the majority upregulated. The ‘late’ response was typical of that seen in previous studies. However, comparison of the ‘early’ response in S. sanguinis with that of other oral streptococci revealed unexpected differences with regard to the number of genes induced, the nature of those genes, and their putative upstream regulatory sequences. Streptococcus sanguinis possesses a comparatively limited early response, which may define a minimal streptococcal competence regulatory circuit.

Hydrogen peroxide (H2O2), an important substance produced by many members of the genus Streptococcus, plays important roles in virulence and antagonism within a microbial community such as oral biofilms. The spxB gene, which encodes pyruvate oxidase, is involved in H2O2 production in many streptococcal species. However, knowledge about its regulation and relation with other genes putatively involved in the same pathway is limited. In this study, three genes – ackA, spxR and tpk – were identified as contributing to H2O2 production in Streptococcus sanguinis by screening mutants for opaque colony appearance. Mutations in all three genes resulted in significant decreases in H2O2 production, with 16–31 % of that of the wild-type. H2O2 production was restored in the complemented strains. Antagonism against Streptococcus mutans by these three S. sanguinis mutants was reduced, both on plates and in liquid cultures, indicating the critical roles of these three genes for conferring the competitive advantage of S. sanguinis. Analysis by qPCR indicated that the expression of spxB was decreased in the ackA and spxR mutants and significantly increased in the tpk mutant.

Serine carboxypeptidase-like proteins (SCPLs) comprise a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. During the course of study aimed at elucidating the molecular basis of induced immunity in rice, a gene, OsBISCPL1, encoding a putative SCPL, was isolated and identified. OsBISCPL1 contains a conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus. OsBISCPL1 is expressed ubiquitously in rice, including roots, stems, leaves and spikes. Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with benzothiadiazole, salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid, and also up-regulated in incompatible interactions between rice and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexpressing plants showed an enhanced disease resistance against Pseudomonas syringae pv. tomato and Alternaria brassicicola. Expression levels of defense-related genes, e.g. PR1, PR2, PR5 and PDF1.2, were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore, the OsBISCPL1-overexpressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes. The results suggest that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress.

Streptococcus sanguinis is one of the pioneers in the bacterial colonization of teeth and is one of the most abundant species in the oral biofilm called dental plaque. S. sanguinis is also the most common viridans group streptococcal species implicated in infective endocarditis. To investigate the association of biofilm and endocarditis, we established a biofilm assay and examined biofilm formation with a signature-tagged mutagenesis library of S. sanguinis . Four genes that have not previously been associated with biofilm formation in any other bacterium, purB, purL, thrB , and pyrE , were putatively identified as contributing to in vitro biofilm formation in S. sanguinis . By examining 800 mutants for attenuation in the rabbit endocarditis model and for reduction in biofilm formation in vitro, we found some mutants that were both biofilm defective and attenuated for endocarditis. However, we also identified mutants with only reduced biofilm formation or with only attenuation in the endocarditis model. This result indicates that the ability to form biofilms in vitro is not associated with endocarditis virulence in vivo in S. sanguinis .

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B 12 biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD + . The oxidation of NADH to NAD + was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H 2 O 2 ; however, the intracellular level of H 2 O 2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence.

Species-specific antimicrobial therapy has the potential to combat the increasing threat of antibiotic resistance and alteration of the human microbiome. We therefore set out to demonstrate the beginning of a pathogen-selective drug discovery method using the periodontal pathogen Porphyromonas gingivalis as a model. Through our knowledge of metabolic networks and essential genes we identified a “druggable” essential target, meso-diaminopimelate dehydrogenase, which is found in a limited number of species. We adopted a high-throughput virtual screen method on the ZINC chemical library to select a group of potential small-molecule inhibitors. Meso-diaminopimelate dehydrogenase from P. gingivalis was first expressed and purified in Escherichia coli then characterized for enzymatic inhibitor screening studies. Several inhibitors with similar structural scaffolds containing a sulfonamide core and aromatic substituents showed dose-dependent inhibition. These compounds were further assayed showing reasonable whole-cell activity and the inhibition mechanism was determined. We conclude that the establishment of this target and screening strategy provides a model for the future development of new antimicrobials.

Essential genes in pathogens are important for the development of antibacterial drugs. In this report, we described a protocol to identify essential genes in the Streptococcus sanguinis SK36 strain using genome-wide deletion mutation. A fusion PCR-based method is used to construct gene deletion fragments, which contain kanamycin resistance cassettes with two flanking arms of DNA upstream and downstream of the target gene. The linear fused PCR amplicons were transformed into S. sanguinis SK36 cells. No kanamycin-resistant transformants suggested the gene essentiality because the deletion of the essential gene renders a lethal phenotype of the transformants. The putative essential genes were further confirmed by independent transformations up to five attempts. The false nonessential genes were also identified by removing double-band mutants.

This work examined the accumulation of diterpenoid tanshinones and related secondary metabolism pathways in hairy root cultures of Salvia miltiorrhiza Bunge (Lamiacae) induced by a biotic elicitor, the carbohydrate fraction of yeast extract (YE, 100 m gm L 1 ), and an abiotic elicitor, Ag + (30 mM). The activity of 3-hydroxy-3-methylglutaryl CoA redutase (HMGR) was only stimulated by Ag + , and 1-deoxyD-xylulose 5-phosphate synthase (DXS) was stimulated by both, but more strongly by YE. While the non-MVA pathway inhibitor fosmidomycin inhibited the tanshinone accumulation induced by both elicitors, the MVA-pathway inhibitor mevinolin only suppressed the Ag-induced tanshinone accumulation. The results suggest that the tanshinone accumulation induced by the two elicitors was mainly synthesized via the non-MVA pathway (DXS activity), but could depend on crosstalk between the MVA and non-MVA pathways. Furthermore, the pretreatment of hairy roots with Ag + for 24–48 h potentiated the YE-induced tanshinone production and DXS activity.

The non-protein amino acid beta-aminobutyric acid (BABA) is a proven inducer of plant defense against pathogens. This work examines its effect on the production of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures, both separately and in combination with a yeast elicitor (YE, the carbohydrate fraction of yeast extract). In the absence of YE, BABA at 0.1, 1 and 2 mM caused a dose-dependent enhancement of tanshinone accumulation, with up to a 4.5-fold increase (from 0.24 to 1.09 mg/g DW) in total content of three major tanshinones (cryptotanshinone, tanshinone I and tanshinone IIA) in the hairy roots. The combination of BABA with YE treatment further enhanced tanshinone production, but only when the BABA treatment was applied to the culture a few days before the YE treatment. Compared with methyl jasmonate, BABA was more effective in enhancing tanshinone production. A 3-day pretreatment with 1 mM BABA followed by YE-treatment, increased the total tanshinone content of roots by 9.4 times to 2.26 mg/g cells, and the volumetric tanshinone yield of culture by 6.3 times (from 3.2 to 20.1 mg/l). The results suggest that BABA can strongly potentiate elicitor-induced secondary metabolism in plant tissue cultures.

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O and H2O2 and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US. © 2004 Wiley Periodicals, Inc.

Streptococcus sanguinis is a Gram-positive bacterium that is indigenous to the oral cavity. S. sanguinis, a primary colonizer of the oral cavity, serves as a tether for the attachment of other oral pathogens. The colonization of microbes on the tooth surface forms dental plaque, which can lead to the onset of periodontal disease. We examined a comprehensive mutant library to identify genes related to cellular chain length and morphology using phase-contrast microscopy. A number of hypothetical genes related to the cellular chain length were identified in this study. Genes related to the cellular chain length were analysed along with clusters of orthologous groups (COG) for gene functions. It was discovered that the highest proportion of COG functions related to cellular chain length was ‘cell division and chromosome separation’. However, different COG functions were also found to be related with altered cellular chain length. This suggested that different genes related with multiple mechanisms contribute to the cellular chain length in S. sanguinis SK36.

We examined the subgingival bacterial biodiversity in untreated chronic periodontitis patients by sequencing 16S rRNA genes. The primary purpose of the study was to compare the oral microbiome in deep (diseased) and shallow (healthy) sites. A secondary purpose was to evaluate the influences of smoking, race and dental caries on this relationship. A total of 88 subjects from two clinics were recruited. Paired subgingival plaque samples were taken from each subject, one from a probing site depth >5 mm (deep site) and the other from a probing site depth ≤3mm (shallow site). A universal primer set was designed to amplify the V4-V6 region for oral microbial 16S rRNA sequences. Differences in genera and species attributable to deep and shallow sites were determined by statistical analysis using a two-part model and false discovery rate. Fifty-one of 170 genera and 200 of 746 species were found significantly different in abundances between shallow and deep sites. Besides previously identified periodontal disease-associated bacterial species, additional species were found markedly changed in diseased sites. Cluster analysis revealed that the microbiome difference between deep and shallow sites was influenced by patient-level effects such as clinic location, race and smoking. The differences between clinic locations may be influenced by racial distribution, in that all of the African Americans subjects were seen at the same clinic. Our results suggested that there were influences from the microbiome for caries and periodontal disease and these influences are independent.

Streptococci resident in the oral cavity have been linked to infective endocarditis (IE). While other viridans streptococci are commonly studied in relation to IE, less research has been focused on Streptococcus pneumoniae. We established for the first time an animal model of S. pneumoniae IE, and examined the virulence of the TIGR4 strain in this model. We hypothesized that two-component systems (TCS) may mediate S. pneumoniae TIGR4 strain virulence in IE and examined TCS response regulator (RR) mutants of TIGR4 in vivo with the IE model. Thirteen of the 14 RR protein genes were mutagenized, excluding only the essential gene SP_1227. The requirement of the 13 RRs for S. pneumoniae competitiveness in the IE model was assessed in vivo through use of quantitative real-time PCR (qPCR) and competitive index assays. Using real-time PCR, several RR mutants were detected at significantly lower levels in infected heart valves compared with a control strain suggesting the respective RRs are candidate virulence factors for IE. The virulence reduction of the ΔciaR mutant was further confirmed by competitive index assay. Our data suggest that CiaR is a virulence factor of S. pneumoniae strain TIGR4 for IE.

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

A clear perception of gene essentiality in bacterial pathogens is pivotal for identifying drug targets to combat emergence of new pathogens and antibiotic-resistant bacteria, for synthetic biology, and for understanding the origins of life. We have constructed a comprehensive set of deletion mutants and systematically identified a clearly defined set of essential genes for Streptococcus sanguinis. Our results were confirmed by growing S. sanguinis in minimal medium and by double-knockout of paralogous or isozyme genes. Careful examination revealed that these essential genes were associated with only three basic categories of biological functions: maintenance of the cell envelope, energy production, and processing of genetic information. Our finding was subsequently validated in two other pathogenic streptococcal species, Streptococcus pneumoniae and Streptococcus mutans and in two other gram-positive pathogens, Bacillus subtilis and Staphylococcus aureus. Our analysis has thus led to a simplified model that permits reliable prediction of gene essentiality.

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase ( lgt ) and signal peptidase II ( lspA ). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt , lspA , or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB , encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [ 3 H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.

Background: Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings: We applied bioinformatics for antigen prediction and pooled antigens for immunization. Fortyseven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions: The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.