Biology subjects

Burne, R. A.

Publications and source records attributed to Burne, R. A..

17 recordsLinked to original sources

bioRxiv2023

Heterogeneous lineage-specific arginine deiminase expression within dental microbiome species

Arginine catabolism by the bacterial arginine deiminase system (ADS) has anticariogenic properties through the production of ammonia, which modulates the pH of the oral environment. Given the potential protective capacity of the ADS pathway, the exploitation of ADS competent oral microbes through pre- or probiotic applications is a promising therapeutic target to prevent tooth decay. To date, most investigations of the ADS in the oral cavity and its relation to caries have focused on indirect measures of activity, or on specific bacterial groups, yet the pervasiveness and rate of expression of the ADS operon in diverse mixed microbial communities in oral health and disease remains an open question. Here we use a multivariate approach, combining ultra-deep metatranscriptomic sequencing with paired metataxonomic and in vitro citrulline quantification to characterize the microbial community and ADS operon expression in healthy and late-stage cavitated teeth. While ADS activity is higher in healthy teeth, we identify multiple bacterial lineages with upregulated ADS activity on cavitated teeth that are distinct from those found on healthy teeth using both reference-based mapping and denovo assembly methods. Our dual metataxonomic and metatranscriptomic approach demonstrates the importance of species abundance for gene expression data interpretation and that patterns of differential expression can be skewed by low abundance groups. Finally, we identify several potential candidate probiotic bacterial lineages within species that may be useful therapeutic targets for the prevention of tooth decay and propose that the development of a strain-specific, mixed-microbial probiotic may be a beneficial approach given the heterogeneity of taxa identified here across health groups. IMPORTANCETooth decay is the most common preventable chronic disease, globally affecting more than two billion people. The development of caries on teeth is primarily a consequence of acid production by cariogenic bacteria that inhabit the plaque microbiome. Other bacterial strains in the oral cavity may suppress or prevent tooth decay by producing ammonia as a byproduct of the arginine deiminase metabolic pathway, increasing the pH of the plaque biofilm. While the benefits of arginine metabolism on oral health have been extensively documented in specific bacterial groups, the prevalence and consistency of ADS activity among oral bacteria in a community context remains an open question. In the current study, we use a multi-omics approach to document the pervasiveness of expression of the ADS operon in both health and disease to better understand the conditions in which ADS activity may prevent tooth decay.

microbiology↗

bioRxiv2023

Genetic Characterization of Glyoxalase Pathway in Oral Streptococci and its Contribution to Interbacterial Competition

Substantial quantities of Reactive Electrophile Species (RES), including methylglyoxal and glyoxal, are generated by microbes and humans. To understand the impact of RES on oral microbial homeostasis, genetic analyses were performed on the glyoxalase pathway in Streptococcus mutans (SMU) and Streptococcus sanguinis (SSA). Loss of glyoxalase I (LguL), which catalyzes the rate-limiting reaction in RES degradation, reduced methylglyoxal and glyoxal tolerance to a far greater extent in SMU than in SSA, decreasing the competitiveness of SMU over SSA in planktonic cultures. MICs showed an overall greater RES tolerance by SMU than SSA; a finding consistent with the ability of methylglyoxal to induce the expression of lguL in SMU, but not in SSA. Computational analysis identified a novel paralogue of LguL in most streptococci represented by SMU.1112c in SMU. {Delta}SMU.1112c showed a minor decrease in methylglyoxal tolerance under certain conditions, but a significant growth defect on fructose; a phenotype reversed by the deletion of a fructose-1-phosphate-generating sugar: phosphotransferase system or addition of glutathione (GSH) to the medium. Further, deletion of the glucose-PTS in SMU increased RES tolerance partly through enhanced expression of the pyruvate-dehydrogenase complex. Consistent with the requirement of GSH for methylglyoxal detoxification, deletion of glutathione synthetase (gshAB) in SMU significantly reduced RES resistance. This study reveals the critical roles of RES in fitness and interbacterial competition and the effects of PTS in modulating RES metabolism. The fact that RES may impact the pathogenic potential of the oral microbiome via differential effects on beneficial and pathogenic species warrants further investigation. ImportanceAs highly reactive byproducts of sugar metabolism, very little is known regarding the contribution of methylglyoxal or related aldehyde compounds to oral health. The need to better understand the influence of these reactive electrophile species (RES) to microbial physiology and ecology is made more urgent by the widespread condition of hyperglycemia in humans, which is associated with elevated RES levels. Our study showed a significantly greater ability of a major caries pathobiont, Streptococcus mutans, to tolerate methylglyoxal and glyoxal than many commensal oral streptococci. Genetic analysis of methylglyoxal degradation in the pathobiont and commensals identified significant differences in genetic structure and gene regulation patterns that could contribute to differential fitness by constituents of the dental microbiota and ecologic shift in the presence of RES.

microbiology↗

bioRxiv2022

Glucose PTS Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci

Spontaneous mutants with defects in the primary glucose phosphotransferase (PTS) permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. The observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of pyruvate and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide (IPS) metabolism, arginine deiminase, and pathways for metabolism of acetoin, ethanolamine, ascorbate and formate; along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm co-cultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis co-cultures increased the proportions of UA159 in the biofilms. Reduction of the buffer capacity or increasing the concentration of glucose benefited UA159 when co-cultured with SK36, but not with SK36/manL; likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that PTS-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. ImportanceDental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia, and improved persistence under acidic conditions. Significant metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

microbiology↗

bioRxiv2021

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

Genetic truncations in a gene encoding a putative glucose-PTS protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. Using an engineered mutant of manL and its complemented derivative, we showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage ([~]10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. Significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. ImportanceA health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

microbiology↗

bioRxiv2020

Mutanofactin promotes bacterial adhesion and biofilm formation of cariogenic Streptococcus mutans

Cariogenic Streptococcus mutans is known as a predominant etiological agent of dental caries due to its exceptional capacity in forming biofilms. From strains of S. mutans isolated from dental plaque, we here discover a polyketide/non-ribosomal peptide biosynthetic gene cluster, muf, which directly correlates with a strong biofilm-forming capability. We then identify the muf-associated bioactive product, mutanofactin-697 that contains a novel molecular scaffold, along with its biosynthetic logic. Further mode-of-action studies reveal mutanofactin-697 binds to S. mutans cells nonspecifically, increases bacterial hydrophobicity, and promotes bacterial adhesion and subsequent biofilm formation. Our findings provide the first example of a microbial secondary metabolite promoting biofilm formation via a physicochemical approach, highlighting the significance of secondary metabolism in mediating critical processes related to the development of dental caries.

microbiology↗

bioRxiv2020

Amino Sugars Reshape Interactions between Streptococcus mutans and Streptococcus gordonii.

Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources that are continually supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each bacterium when they were cultured alone. Likewise, co-cultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different than the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernates. Differing from a previous report, growth of S. mutans alone with GlcN inhibited expression of multiple operons required for mutacin production. Co-cultivation with S. gordonii consistently increased the expression by S. mutans of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes. Conversely, S. gordonii appeared to be less affected by the presence of S. mutans, but did show increases in genes for biosynthetic processes in the co-cultures. In conclusion, amino sugars profoundly alter the interactions between a pathogenic and commensal streptococcus by reprogramming central metabolism. IMPORTANCECarbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii) in a RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

microbiology↗

bioRxiv2019

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 the 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.

microbiology↗

bioRxiv2019

Exploring novel probiotic mechanisms of Streptococcus A12 with functional genomics

Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans. A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual, and was shown to interfere potently with the growth and virulence properties of S. mutans. Here, we apply functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans. Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated as potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to the parental strain. The A12 pcfO mutant lost the ability to inhibit comX-inducing peptide (XIP) signaling by S. mutans, while mutants in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans. Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiologic factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.\n\nImportanceAdvances in defining the composition of health-associated biofilms have highlighted the important role for beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological basis underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans, using various gene products with diverse functions. A12 displayed enhanced competitiveness by: i) disrupting intercellular communication pathways of S. mutans, ii) sensing and resisting antimicrobial peptides, and iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

microbiology↗

bioRxiv2019

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 signaling peptide (CSP) induces bimodal expression of comX, with only a fraction of cells in the population becoming transformable. Curiously, bimodal comX activation in response to CSP 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 in defined media lacking small peptides CSP induces no response in comX. In addition, growth on certain sugars other than glucose increases the proportion of the population that activates comX in response to CSP, relative to growth on glucose. By investigating the connection between media and bimodal comX expression, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, which is the origin of comX bimodality. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the intracellular XIP/ComS signal. Deletion of pepO eliminates bimodality in comX, leading to a unimodal comX response to CSP in defined and complex media. We also find that CSP upregulates comR in a carbohydrate source-dependent fashion, providing an additional stimulus to the ComRS feedback system. Our data provide mechanistic insight into how CSP regulates the bistable competence circuit and explain the puzzle of growth medium-dependence in S. mutans competence regulation.

biophysics↗

bioRxiv2019

Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans

N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) enhance the competitiveness of the laboratory strain DL1 of Streptococcus gordonii against the caries pathogen Streptococcus mutans. Here we examine how amino sugars affect the interaction of five low-passage clinical isolates of abundant commensal streptococci with S. mutans utilizing a dual-species biofilm model. Compared to glucose, growth on GlcN or GlcNAc significantly reduced the viability of S. mutans in co-cultures with most commensals, shifting the proportions of species. Consistent with these results, production of H2O2 was increased in most commensals when growing on amino sugars, and inhibition of S. mutans by Streptococcus cristatus, Streptococcus oralis, or S. gordonii was enhanced by amino sugars on agar plates. All commensals except S. oralis had higher arginine deiminase activities when grown on GlcN, and in some cases GlcNAc. In ex vivo biofilms formed using pooled cell-containing saliva (CCS), the proportions of S. mutans were drastically diminished when GlcNAc was the primary carbohydrate. Increased production of H2O2 could account in large part for the inhibitory effects of CCS biofilms. Surprisingly, amino sugars appeared to improve mutacin production by S. mutans on agar plates, suggesting that the commensals have mechanisms to actively subvert antagonism by S. mutans in co-cultures. Collectively, these findings demonstrate that amino sugars can enhance the beneficial properties of low-passage commensal oral streptococci and highlight their potential for moderating the cariogenicity of oral biofilms. SIGNIFICANCEDental caries is driven by dysbiosis of oral biofilms in which dominance by acid-producing and acid-tolerant bacteria results in loss of tooth mineral. Our previous work demonstrated the beneficial effects of amino sugars, GlcNAc and GlcN, in promoting the antagonistic properties of a health-associated oral bacterium, Streptococcus gordonii, in competition with the major caries pathogen Streptococcus mutans. Here we investigated 5 low-passage clinical isolates of the most common streptococcal species to establish how amino sugars may influence the ecology and virulence of oral biofilms. Using multiple in vitro models, including a human saliva-derived microcosm biofilm, experiments showed significant enhancement by at least one amino sugar in the ability of most of these bacteria to suppress the caries pathogen. Therefore, our findings demonstrated the mechanism of action by which amino sugars may affect human oral biofilms to promote health.

microbiology↗

bioRxiv2019

Characterization of Spontaneously Arising Chlorhexidine-Tolerant Variants in Streptococcus mutans

Broad spectrum antimicrobials, both in dental products and within the clinic, have been used in the suppression of cariogenic bacteria such as Streptococcus mutans for over 40 years. One such antimicrobial is chlorhexidine (CHX), and serves as a standard in dental research against which other antimicrobial therapies are compared against for their efficacy. However, very little is known about the mode of action for CHX against Streptococci and whether tolerance can be developed from repeated exposures. Here, we begin to answer such questions by passaging S. mutans with increasing concentrations of CHX and isolating spontaneously-arising tolerant variants (CTVs) from separate lineages. We find that these CTVs display a higher minimal inhibitory concentration (MIC) against CHX than the wild-type strain and have altered virulence properties such as acid tolerance and biofilm formation. We record higher MICs for the variants against both daptomycin and bacitracin, but find increased sensitivity to triclosan and sodium fluoride. Measurements of antagonistic capabilities against other health-associated oral streptococci show decreased bacteriocin production compared to wild-type and increased sensitivity to hydrogen peroxide. Finally whole genome sequencing of the CTVs show common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a glycolipid synthesis enzyme, altering LTA accumulation and potentially lipid profile of the cell wall. Together, these findings confirm that streptococci may develop tolerance to antimicrobial agents such as CHX but in the case of S. mutans, increased tolerance may come at a fitness cost for survival within oral biofilms that keeps variants suppressed within the population.

microbiology↗

bioRxiv2018

Species Designations Belie Phenotypic and Genotypic Heterogeneity in Oral Streptococci

Health-associated oral Streptococcus species are promising probiotic candidates to protect against dental caries. Ammonia production through the arginine deiminase system (ADS), which can increase the pH of oral biofilms, and direct antagonism of caries-associated bacterial species are desirable properties for oral probiotic strains. ADS and antagonistic activities can vary dramatically among individuals, but the genetic basis for these differences is unknown. We sequenced whole genomes of a diverse set of clinical oral Streptococcus isolates and examined the genetic basis of variability in ADS and antagonistic activities. A total of 113 isolates were included and represented ten species: S. australis, A12-like, S. cristatus, S. gordonii, S. intermedius, S. mitis, S. oralis including S. oralis subsp. dentisani, S. parasanguinis, S. salivarius, and S. sanguinis. Mean ADS activity and antagonism on Streptococcus mutans UA159 were measured for each isolate, and each isolate was whole genome-shotgun sequenced on an Illumina MiSeq. Phylogenies were built of genes known to be involved in ADS activity and antagonism. Several approaches to correlate the pan-genome with phenotypes were performed. Phylogenies of genes previously identified in ADS activity and antagonism grouped isolates by species but not by phenotype. GWAS identified additional genes potentially involved in ADS activity or antagonism across all the isolates we sequenced as well as within several species. Phenotypic heterogeneity in oral streptococci is not necessarily reflected by genotype and is not species-specific. Probiotic strains must be carefully selected based on characterization of each strain, and not based on inclusion within a certain species.\n\nImportanceRepresentative type strains are commonly used to characterize bacterial species, yet species are phenotypically and genotypically heterogeneous. Conclusions about strain physiology and activity based on a single strain therefore may be inappropriate and misleading. When selecting strains for probiotic use, the assumption that all strains within a species share the same desired probiotic characteristics share those characteristics may result in selection of a strain that lacks the desired traits, and therefore makes a minimally effective or ineffective probiotic. Health-associated oral streptococci are promising candidates for anti-caries probiotics, but strains need to be carefully selected based on observed phenotypes. We characterized the genotype and anti-caries phenotypes of strains from ten species of oral Streptococci and demonstrate poor correlation between genotype and phenotype across all species.

microbiology↗

bioRxiv2018

Essential Roles of the sppRA Fructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression by Streptococcus mutans

The dental caries pathogen Streptococcus mutans can ferment a variety of sugars to produce organic acids. Exposure of S. mutans to certain non-metabolizable carbohydrates such as xylitol impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress in S. mutans was demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here we studied an operon in S. mutans, sppRA, which was highly expressed in the fruK mutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg2+ and Mn2+, but inhibited by NaF. SppR, a DeoR-family regulator, repressed the expression of the sppRA operon to minimum levels in the absence of the fructose-derived metabolites, F-1-P and likely also F-6-P. Accumulation of F-1-P, as a result of growth on fructose, not only induced sppA expression, it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression of sppA, via a plasmid or by deleting sppR, greatly alleviated fructose-induced stress in a fruK mutant, enhanced resistance to xylitol, and reversed effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show that S. mutans is capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.\n\nImportanceStreptococcus mutans is a major etiologic agent for dental caries, primarily due to its ability to form biofilms on tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon in S. mutans that regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.

microbiology↗

bioRxiv2018

Preferred hexoses influence long-term memory and induction of lactose catabolism by Streptococcus mutans

Bacteria prioritize sugar metabolism via carbohydrate catabolite repression, which regulates global gene expression to optimize the catabolism of preferred substrates. Here, we report an unusual long-term memory effect in certain Streptococcus mutans strains that alters adaptation to growth on lactose after prior exposure to glucose or fructose. In strain GS-5, cells that were first cultured on fructose then transferred to lactose displayed an exceptionally long lag (>11 h) and slower growth, compared to cells first cultured on glucose or cellobiose, which displayed a reduction in lag phase by as much as 10 h. Mutants lacking the cellobiose-PTS or phospho-{beta}-glucosidase lost the accelerated growth on lactose associated with prior culturing on glucose. The memory effects of glucose or fructose on lactose catabolism were not as profound in strain UA159, but the lag phase was considerably shorter in mutants lacking the glucose-PTS EIIMan. Interestingly, when S. mutans was cultivated on lactose, significant quantities of free glucose accumulated in the medium, with higher levels found in the cultures of strains lacking EIIMan, glucokinase, or both. Free glucose was also detected in cultures that were utilizing cellobiose or trehalose, albeit at lower levels. Such release of hexoses by S. mutans is likely of biological significance as it was found that cells required small amounts of glucose or other preferred carbohydrates to initiate efficient growth on lactose. These findings suggest that S. mutans modulates the induction of lactose utilization based on its prior exposure to glucose or fructose, which can be liberated from common disaccharides.\n\nIMPORTANCEUnderstanding the molecular mechanisms employed by oral bacteria to control sugar metabolism is key to developing novel therapies for management of dental caries and other oral diseases. Lactose is a naturally occurring disaccharide that is abundant in dairy products and commonly ingested by humans. However, for the dental caries pathogen Streptococcus mutans, relatively little is known about the molecular mechanisms that regulate expression of genes required for lactose uptake and catabolism. Two peculiarities of lactose utilization by S. mutans are explored here: a) S. mutans excretes glucose that it cleaves from lactose and b) prior exposure to certain carbohydrates can result in a long-term inability to use lactose. The study begins to shed light on how S. mutans may bet-hedge to optimize its persistence and virulence in the human oral cavity.

microbiology↗

bioRxiv2018

Intracellular signaling through the comRS system in Streptococcus mutans genetic competence

AbstractEntry 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 signaling system, which consists of the cytosolic receptor ComR and the 7-residue signal peptide XIP, which is derived from ComS. Extracellular XIP imported by an oligopeptide permease interacts with ComR to form a transcriptional activator for both comX and comS. Therefore, extracellular XIP can function as an exogenous signal to trigger S. mutans competence. However, the mechanisms that process ComS and export it as XIP are not fully known in S. mutans. The observation that comX is expressed bimodally under some environmental conditions suggests that ComR may also interact with endogenously produced XIP or ComS, creating an intracellular positive feedback loop in comS transcription. Here we use single cell and microfluidic methods to compare the effects of the native comS gene and extracellular XIP on comX expression. We find that deletion of comS reduces the response of comX to extracellular XIP. We also find that comS-overexpressing cells autoactivate their comX even when their growth medium is rapidly exchanged, although this autoactivation requires an intact copy of comS under control of its own promoter. However comS-overexpressing cells do not activate comS-deficient mutants growing in coculture. These data show that individual cells can activate comX without exporting or importing the XIP or ComS signal, and that endogenously and exogenously produced ComS/XIP have inequivalent effects on comX behavior. These data are fully consistent with a model in which intracellular positive feedback in comS transcription plays a role in ComRS signaling, and is responsible for the bimodal expression of comX.\n\nAuthor SummaryHeterogeneous gene expression in genetically identical populations plays an important role in bacterial persistence and survival under changing environmental conditions. In the oral pathogen Streptococcus mutans, the physiological state of genetic competence can exhibit bimodality, with only some cells becoming competent. S. mutans controls its entry into competence by using the ComRS signaling system to activate comX, a gene encoding the master competence regulator ComX. The ComRS system is understood as a quorum sensing system, in which the extracellular accumulation of the small signal peptide XIP, derived from ComS, induces comX expression. We coupled observation of bacteria that fluoresce when comX is active with mathematical analysis and chemical binding assays to show that activation of comX does not necessarily require extracellular XIP or ComS, and that comX-active cells do not necessarily export XIP. Our experiments and mathematical modeling indicate that a positive feedback loop in comS transcription allows a cell to activate comX in response to its own XIP or ComS in the absence of extracellular XIP, or to amplify its comX response to extracellular XIP if present. Such positive feedback loops are often the cause of bimodal gene expression like that seen in S. mutans competence.

microbiology↗

bioRxiv2018

Threshold regulation and stochasticity from the MecA/ClpCP proteolytic system in Streptococcus mutans competence

Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell-to-cell heterogeneity in MecA levels creates variability in that threshold. Therefore MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.

microbiology↗

bioRxiv2017

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 comX-inducing peptide (XIP) in Streptococcus mutans. Although the competence pathway can be activated by addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signal molecule has not been rigorously tested. Co-culture model systems were developed that included a \"sender\" strain that overexpressed the XIP precursor (ComS) and a \"responder\" strain harboring a GFP reporter fusion 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 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 endogenously-produced peptide(s) in populations in various environments and physiologic states.\n\nIMPORTANCEThe 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 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.

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