Genomic analysis of conjugative and chromosomally integrated mobile genetic elements in oral streptococci.

This study aimed to investigate the diversity of conjugative and chromosomally integrated mobile genetic elements (cciMGEs) within six oral streptococci species. cciMGEs, including integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs), are stably maintained on the host cell chromosome; however, under certain conditions, they are able to excise, form extrachromosomal circles, and transfer via a conjugation apparatus. Many cciMGEs encode "cargo" functions that aid survival in new niches and evolve new antimicrobial resistance or virulence properties, whereas others have been shown to influence host bacterial physiology. Here, using a workflow employing preexisting bioinformatics tools, we analyzed 551 genomes for the presence of cciMGEs across six common health- and disease-associated oral streptococci. We identified 486 cciMGEs, 173 of which were ICEs and 233 of which were IMEs. The cciMGEs were diverse in size, cargo genes, and relaxase types. We identified several novel relaxase proteins and a widespread IME carrying a small multidrug resistance transporter. Additionally, we provide evidence that several of the bioinformatically predicted cciMGEs encoded within various Streptococcus mutans strains are capable of excision and circularization, a critical step for cciMGE conjugative transfer. These findings highlight the significance and potential impact of MGEs in shaping the genetic landscape, pathogenicity, and antimicrobial resistance profiles of the oral microbiota.IMPORTANCEOral streptococci are important players in the oral microbiome, influencing both health and disease states within dental bacterial communities. Evolutionary adaptation, shaped in a major part by the horizontal transfer of genes, is essential for their survival in the oral cavity and within new environments. Conjugation is a significant driver of horizontal gene transfer; however, there is limited information regarding this process in oral bacteria. This study utilizes publicly available genome sequences to identify conjugative and chromosomally integrated mobile genetic elements (cciMGEs) across several species of oral streptococci and presents the preliminary characterization of these elements. Our findings significantly enhance our understanding of the mobile genomic landscape of oral streptococci critical for human health, with valuable insights into how cciMGEs might influence the survival and pathogenesis of these bacteria in the oral microbiome.

Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in Streptococcus mutans.

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immR Smu (immunity repressor) and a metalloprotease immA Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of immR Smu and repression of immR Smu with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immR Smu mutant. Deletion of immR Smu also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRA Smu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans.

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.

Peptides encoded in the Streptococcus mutans RcrRPQ operon are essential for thermotolerance.

The MarR-like transcriptional regulator and two ABC transporters encoded by the rcrRPQ operon in the dental caries pathogen Streptococcus mutans have important regulatory roles related to oxidative stress tolerance, genetic competence and (p)ppGpp metabolism. A unique feature of the rcrRPQ operon, when compared to other bacteria, is the presence of two peptides, designated Pep1 and Pep2, encoded in alternative reading frames at the 3' end of rcrQ. Here, we show that the rcrRPQ operon, including Pep1 and 2, is essential for S. mutans to survive and maintain viability at elevated temperatures. No major changes in the levels of the heat shock proteins DnaK or GroEL that could account for the thermosensitivity of rcrRPQ mutants were observed. By introducing a single amino acid substitution into the comX gene that deletes an internally encoded peptide, XrpA, we found that XrpA is a contributing factor to the thermosensitive phenotype of a ΔrcrR strain. Overexpression of XrpA on a plasmid also caused a significant growth defect at 42 °C. Interestingly, loss of the gene for the RelA/SpoT homologue (RSH) enzyme, relA, restored growth of the ΔrcrR strain at 42 °C. During heat stress and when a stringent response was induced, levels of (p)ppGpp were elevated in the ΔrcrR strain. Deletion of relA in the ΔrcrR strain lowered the basal levels of (p)ppGpp to those observed in wild-type S. mutans. Thus, (p)ppGpp pools are dysregulated in ΔrcrR, which likely leads to aberrant control of transcriptional/translational processes and the thermosensitive phenotype. In summary, the genes and peptides encoded in the rcrRPQ operon are critical for thermotolerance, and in some strains these phenotypes are related to altered (p)ppGpp metabolism and increased production of the XrpA peptide.

Competence inhibition by the XrpA peptide encoded within the comX gene of Streptococcus mutans.

Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first described negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ΔxrpA strain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.

Genome-Wide Screens Reveal New Gene Products That Influence Genetic Competence in Streptococcus mutans.

A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including the increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome, with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g., comR, comS, comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, the sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes.IMPORTANCEStreptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans, while validating technologies that can rapidly advance our understanding of the physiology, biology, and genetics of S. mutans and related pathogens.

Intercellular Communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans.

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses, and ecological competitiveness. One example is activation of natural genetic competence by ComR and the com X -inducing peptide (XIP) in Streptococcus mutans Although the competence pathway can be activated by the addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signaling molecule has not been rigorously tested. Coculture model systems were developed that included a "sender" strain that overexpressed the XIP precursor (ComS) and a "responder" strain harboring a green fluorescent protein (GFP) reporter fused to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using (i) planktonic culture systems, (ii) cells suspended in an agarose matrix, or (iii) cells growing in biofilms. XIP was shown to be freely diffusible, and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting the spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with an endogenously produced peptide(s) in populations in various environments and physiologic states.IMPORTANCE The comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids, and the addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence, and evolution of a ubiquitous human pathogen and related organisms.

Construction of an arrayed CRISPRi library as a resource for essential gene function studies in Streptococcus mutans.

IMPORTANCE: The construction of arrayed mutant libraries has advanced the field of bacterial genetics by allowing researchers to more efficiently study the exact function and importance of encoded genes. In this study, we constructed an arrayed clustered regularly interspaced short palindromic repeats interference (CRISPRi) library, known as S treptococcus mutans arrayed CRISPRi (SNAP), as a resource to study >250 essential and growth-supporting genes in Streptococcus mutans. SNAP will be made available to the research community, and we anticipate that its distribution will lead to high-quality, high-throughput, and reproducible studies of essential genes.

Development and Antibacterial Properties of 4-[4-(Anilinomethyl)-3-phenylpyrazol-1-yl]benzoic Acid Derivatives as Fatty Acid Biosynthesis Inhibitors.

A number of novel pyrazole derivatives have been synthesized, and several of these compounds are potent antibacterial agents with minimum inhibitory concentrations as low as 0.5 µg/mL. Human cell lines were tolerant to these lead compounds, and they showed negligible hemolytic effects at high concentrations. These bactericidal compounds are very effective against bacterial growth in both planktonic and biofilm contexts. Various techniques were applied to show the inhibition of biofilm growth and eradication of preformed biofilms by lead compounds. Potent compounds are more effective against persisters than positive controls. In vivo studies revealed that lead compounds are effective in rescuing C. elegans from bacterial infections. Several methods were applied to determine the mode of action including membrane permeability assay and SEM micrograph studies. Furthermore, CRISPRi studies led to the determination of these compounds as fatty acid biosynthesis (FAB) inhibitors.

Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans.

CRISPR-Cas is a bacterial immune system that restricts the acquisition of mobile DNA elements. These systems provide immunity against foreign DNA by encoding CRISPR spacers that help target DNA if it re-enters the cell. In this way, CRISPR spacers are a type of molecular tape recorder of foreign DNA encountered by the host microorganism. Here, we extracted ∼8,000 CRISPR spacers from a collection of over three hundred Streptococcus mutans genomes. Phage DNA is a major target of S. mutans spacers. S. mutans strains have also generated immunity against mobile DNA elements such as plasmids and integrative and conjugative elements. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. While there was clear evidence that these systems have acquired immunity against foreign DNA, there appeared to be minimal impact on horizontal gene transfer (HGT) constraints on a species-level. There was little or no impact on genome size, GC content and 'openness' of the pangenome when comparing between S. mutans strains with low or high CRISPR spacer loads. In summary, while there is evidence of CRISPR spacer acquisition against self and foreign DNA, CRISPR-Cas does not act as a barrier on the expansion of the S. mutans accessory genome.

Identification and Analysis of Essential Genes in Streptococcus mutans with Transposon Sequencing.

Transposon sequencing (Tn-seq) has greatly accelerated the rate at which gene function can be profiled in microbial organisms. This technique has been applied to the study of the dental caries pathogen Streptococcus mutans where it has been used to generate large transposon mutant libraries. Coupled with high-throughput sequencing and bioinformatics tools, culture of these transposon mutant libraries has facilitated the identification of essential and conditional essential genes. In this chapter, we describe a procedure for performing Tn-seq studies in S. mutans that covers pooled transposon mutant construction, in vitro culture, and DNA library sequencing and data analysis.

Interspecies competition in oral biofilms mediated by Streptococcus gordonii extracellular deoxyribonuclease SsnA.

Extracellular DNA (eDNA) is a key component of many microbial biofilms including dental plaque. However, the roles of extracellular deoxyribonuclease (DNase) enzymes within biofilms are poorly understood. Streptococcus gordonii is a pioneer colonizer of dental plaque. Here, we identified and characterised SsnA, a cell wall-associated protein responsible for extracellular DNase activity of S. gordonii. The SsnA-mediated extracellular DNase activity of S. gordonii was suppressed following growth in sugars. SsnA was purified as a recombinant protein and shown to be inactive below pH 6.5. SsnA inhibited biofilm formation by Streptococcus mutans in a pH-dependent manner. Further, SsnA inhibited the growth of oral microcosm biofilms in human saliva. However, inhibition was ameliorated by the addition of sucrose. Together, these data indicate that S. gordonii SsnA plays a key role in interspecies competition within oral biofilms. Acidification of the medium through sugar catabolism could be a strategy for cariogenic species such as S. mutans to prevent SsnA-mediated exclusion from biofilms.

The bare necessities: Uncovering essential and condition-critical genes with transposon sequencing.

Querying gene function in bacteria has been greatly accelerated by the advent of transposon sequencing (Tn-seq) technologies (related Tn-seq strategies are known as TraDIS, INSeq, RB-TnSeq, and HITS). Pooled populations of transposon mutants are cultured in an environment and next-generation sequencing tools are used to determine areas of the genome that are important for bacterial fitness. In this review we provide an overview of Tn-seq methodologies and discuss how Tn-seq has been applied, or could be applied, to the study of oral microbiology. These applications include studying the essential genome as a means to rationally design therapeutic agents. Tn-seq has also contributed to our understanding of well-studied biological processes in oral bacteria. Other important applications include in vivo pathogenesis studies and use of Tn-seq to probe the molecular basis of microbial interactions. We also highlight recent advancements in techniques that act in synergy with Tn-seq such as clustered regularly interspaced short palindromic repeats (CRISPR) interference and microfluidic chip platforms.

Correction: Long-term fish assemblages of the Ohio River: Altered trophic and life history strategies with hydrologic alterations and land use modifications.

[This corrects the article DOI: 10.1371/journal.pone.0211848.].

Long-term fish assemblages of the Ohio River: Altered trophic and life history strategies with hydrologic alterations and land use modifications.

Long-term monitoring of species assemblages provides a unique opportunity to test hypotheses regarding environmentally induced directional trajectories of freshwater species assemblages. We used 57 years of lockchamber fish rotenone and boat electrofishing survey data (1957-2014) collected by the Ohio River Valley Water Sanitation Commission (ORSANCO) to test for directional trajectories in taxonomy, trophic classifications, and life history strategies of freshwater fish assemblages in the Ohio River Basin. We found significant changes in taxonomic and trophic composition of freshwater fishes in the Ohio River Basin. Annual species richness varied from 31 to 90 species and generally increased with year. Temporal trajectories were present for taxonomic and trophic assemblages. Assemblage structure based on taxonomy was correlated with land use change (decrease in agriculture and increase in forest). Taxonomic assemblage structure was also correlated with altered hydrology variables of increased minimum discharge, decreased fall rate, and increased rise rate. Trophic composition of fish catch correlated with land use change (decrease in agriculture and increase in forest) and altered hydrology. Altered hydrology of increased minimum discharge, increased fall discharge, decreased base flows, and increased number of high pulse events was correlated with increased counts of herbivore-detritivores and decreased counts of piscivores and planktivores. We did not find directional changes in life history composition. We hypothesized a shift occurred from benthic to phytoplankton production throughout the basin that may have decreased secondary production of benthic invertebrates. This may also be responsible for lower trophic position of invertivore and piscivore fishes observed in other studies.

Genomewide Identification of Essential Genes and Fitness Determinants of Streptococcus mutans UA159.

Transposon mutagenesis coupled with next-generation DNA sequencing (Tn-seq) is a powerful tool for discovering regions of the genome that are required for the survival of bacteria in different environments. We adapted this technique to the dental caries pathogen Streptococcus mutans UA159 and identified 11% of the genome as essential, with many genes encoding products required for replication, translation, lipid metabolism, and cell wall biogenesis. Comparison of the essential genome of S. mutans UA159 with those of selected other streptococci for which such information is available revealed several metabolic pathways and genes that are required in S. mutans, but not in some Streptococcus spp. We further identified genes that are essential for sustained growth in rich or defined medium, as well as for persistence in vivo in a rodent model of oral infection. Collectively, our results provide a novel and comprehensive view of the genes required for essential processes of S. mutans, many of which could represent potential targets for therapeutics. IMPORTANCE Tooth decay (dental caries) is a common cause of pain, impaired quality of life, and tooth loss in children and adults. It begins because of a compositional change in the microorganisms that colonize the tooth surface driven by repeated and sustained carbohydrate intake. Although several bacterial species are associated with tooth decay, Streptococcus mutans is the most common cause. Therefore, it is important to identify biological processes that contribute to the survival of S. mutans in the human mouth, with the aim of disrupting the processes with antimicrobial agents. We successfully applied Tn-seq to S. mutans, discovering genes that are required for survival, growth, and persistence, both in laboratory environments and in a mouse model of tooth decay. This work highlights new avenues for the control of an important human pathogen.

Intracellular Signaling by the comRS System in Streptococcus mutans Genetic Competence.

Entry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS quorum system. ComS is the precursor of XIP, a seven-residue peptide that is imported into the cell and interacts with the cytosolic receptor ComR to form a transcriptional activator for both comX and comS Although intercellular quorum signaling by ComRS has been demonstrated, observations of bimodal expression of comX suggest that comRS may also function as an intracellular feedback loop, activating comX without export or detection of extracellular XIP. Here we used microfluidic and single-cell methods to test whether ComRS induction of comX requires extracellular XIP or ComS. We found that individual comS-overexpressing cells activate their own comX, independently of the rate at which their growth medium is replaced. However, in the absence of lysis they do not activate comS-deficient mutants growing in coculture. We also found that induction of comR and comS genes introduced into Escherichia coli cells leads to activation of a comX reporter. Therefore, ComRS control of comX does not require either the import or extracellular accumulation of ComS or XIP or specific processing of ComS to XIP. We also found that endogenously and exogenously produced ComS and XIP have inequivalent effects on comX activation. These data are fully consistent with identification of intracellular positive feedback in comS transcription as the origin of bimodal comX expression in S. mutansIMPORTANCE The ComRS system can function as a quorum sensing trigger for genetic competence in S. mutans The signal peptide XIP, which is derived from the precursor ComS, enters the cell and interacts with the Rgg-type cytosolic receptor ComR to activate comX, which encodes the alternative sigma factor for the late competence genes. Previous studies have demonstrated intercellular signaling via ComRS, although release of the ComS or XIP peptide to the extracellular medium appears to require lysis of the producing cells. Here we tested the complementary hypothesis that ComRS can drive comX through a purely intracellular mechanism that does not depend on extracellular accumulation or import of ComS or XIP. By combining single-cell, coculture, and microfluidic approaches, we demonstrated that endogenously produced ComS can enable ComRS to activate comX without requiring processing, export, or import. These data provide insight into intracellular mechanisms that generate noise and heterogeneity in S. mutans competence.

A new arginine-based dental adhesive system: formulation, mechanical and anti-caries properties.

Secondary caries at the margins of composite restorations has been attributed to adhesive failure and consequent accumulation of cariogenic biofilms. OBJECTIVES: To develop and evaluate an etch-and-rinse adhesive system containing arginine for sustainable release and recharge without affecting its mechanical properties. Arginine metabolism by oral bacteria generates ammonia, which neutralizes glycolytic acids and creates a neutral environmental pH that is less favorable to the growth of caries pathogens, thus reducing the caries risk at the tooth-composite interface. METHODS: Experimental adhesives were formulated with methacrylate monomers and arginine at 5%, 7%, and 10% or no arginine (control). Adhesives were tested for: (i) mechanical properties of true stress (FS and UTS), modulus of elasticity (E), degree of conversion (DC), Knoop hardness number (KHN) and dentin microtensile bond strength (µ-TBS), (ii) arginine release and recharge, and (iii) antibacterial activities. Data was analyzed by t-test, one-way ANOVA and Tukey's tests. RESULTS: FS and UTS results showed no statistically significant differences between the 7% arginine-adhesive and control, while the results for E, DC, KHN and µ-TBS showed no difference among all groups. The 7% arginine-adhesive showed a high release rate of arginine (75.0µmol/cm2) at 2h, and a more sustainable, controlled release rate (up to 0.2µmol/cm2) at 30days. CONCLUSIONS: Incorporation of 7% arginine did not affect the physical and mechanical properties of the adhesive. Arginine was released from the adhesive at a rate and concentration that exhibited antibacterial effects, regardless of shifts in biofilm conditions such as sugar availability and pH. CLINICAL SIGNIFICANCE: Secondary caries is recognized as the main reason for failure of dental restorations. The development of an arginine-based adhesive system has the potential to dramatically reduce the incidence and severity of secondary caries in adhesive restorations in a very economical fashion.

Growth of Streptococcus mutans in Biofilms Alters Peptide Signaling at the Sub-population Level.

Streptococcus mutans activates multiple cellular processes in response to the formation of a complex between comX-inducing peptide (XIP) and the ComR transcriptional regulator. Bulk phase and microfluidic experiments previously revealed that ComR-dependent activation of comX is altered by pH and by carbohydrate source. Biofilm formation is a major factor in bacterial survival and virulence in the oral cavity. Here, we sought to determine the response of S. mutans biofilm cells to XIP during different stages of biofilm maturation. Using flow cytometry and confocal microscopy, we showed that exogenous addition of XIP to early biofilms resulted in robust comX activation. However, as the biofilms matured, increasing amounts of XIP were required to activate comX expression. Single-cell analysis demonstrated that the entire population was responding to XIP with activation of comX in early biofilms, but only a sub-population was responding in mature biofilms. The sub-population response of mature biofilms was retained when the cells were dispersed and then treated with XIP. The proportion and intensity of the bi-modal response of mature biofilm cells was altered in mutants lacking the Type II toxins MazF and RelE, or in a strain lacking the (p)ppGpp synthase/hydrolase RelA. Thus, competence signaling is markedly altered in cells growing in mature biofilms, and pathways that control cell death and growth/survival decisions modulate activation of comX expression in these sessile populations.