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1.  Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans 
Applied and Environmental Microbiology  2016;82(15):4821-4834.
ABSTRACT
The capacity to internalize and catabolize carbohydrates is essential for dental caries pathogens to persist and cause disease. The expression of many virulence-related attributes by Streptococcus mutans, an organism strongly associated with human dental caries, is influenced by the peptide signaling pathways that control genetic competence. Here, we demonstrate a relationship between the efficiency of competence signaling and carbohydrate source. A significant increase in the activity of the promoters for comX, comS, and comYA after exposure to competence-stimulating peptide (CSP) was observed in cells growing on fructose, maltose, sucrose, or trehalose as the primary carbohydrate source, compared to cells growing on glucose. However, only cells grown in the presence of trehalose or sucrose displayed a significant increase in transformation frequency. Notably, even low concentrations of these carbohydrates in the presence of excess glucose could enhance the expression of comX, encoding a sigma factor needed for competence, and the effects on competence were dependent on the cognate sugar:phosphotransferase permease for each carbohydrate. Using green fluorescent protein (GFP) reporter fusions, we observed that growth in fructose or trehalose resulted in a greater proportion of the population activating expression of comX and comS, encoding the precursor of comX-inducing peptide (XIP), after addition of CSP, than growth in glucose. Thus, the source of carbohydrate significantly impacts the stochastic behaviors that regulate subpopulation responses to CSP, which can induce competence in S. mutans.
IMPORTANCE The signaling pathways that regulate development of genetic competence in Streptococcus mutans are intimately intertwined with the pathogenic potential of the organism, impacting biofilm formation, stress tolerance, and expression of known virulence determinants. Induction of the gene for the master regulator of competence, ComX, by competence-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of comX and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase com gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability.
doi:10.1128/AEM.01205-16
PMCID: PMC4984281  PMID: 27260355
2.  The Streptococcus mutans Cid and Lrg systems modulate virulence traits in response to multiple environmental signals 
Microbiology  2010;156(Pt 10):3136-3147.
The tight control of autolysis by Streptococcus mutans is critical for proper virulence gene expression and biofilm formation. A pair of dicistronic operons, SMU.575/574 (lrgAB) and SMU.1701/1700 (designated cidAB), encode putative membrane proteins that share structural features with the bacteriophage-encoded holin family of proteins, which modulate host cell lysis during lytic infection. Analysis of S. mutans lrg and cid mutants revealed a role for these operons in autolysis, biofilm formation, glucosyltransferase expression and oxidative stress tolerance. Expression of lrgAB was repressed during early exponential phase and was induced over 1000-fold as cells entered late exponential phase, whereas cidAB expression declined from early to late exponential phase. A two-component system encoded immediately upstream of lrgAB (LytST) was required for activation of lrgAB expression, but not for cid expression. In addition to availability of oxygen, glucose levels were revealed to affect lrg and cid transcription differentially and significantly, probably through CcpA (carbon catabolite protein A). Collectively, these findings demonstrate that the Cid/Lrg system can affect several virulence traits of S. mutans, and its expression is controlled by two major environmental signals, oxygen and glucose. Moreover, cid/lrg expression is tightly regulated by LytST and CcpA.
doi:10.1099/mic.0.039586-0
PMCID: PMC3068699  PMID: 20671018
3.  Bidirectional signaling in the competence regulatory pathway of Streptococcus mutans 
FEMS Microbiology Letters  2015;362(19):fnv159.
Streptococcus mutans expresses comX (also known as sigX), which encodes a sigma factor that is required for development of genetic competence, in response to the peptide signals XIP and CSP and environmental factors. XIP (sigX inducing peptide) is derived from ComS and activates comX unimodally in chemically defined media via the ComRS system. CSP (competence stimulating peptide) activates comX bimodally in peptide-rich media through the ComDE two-component system. However, CSP-ComDE activation of comX is indirect and involves ComRS. Therefore, the bimodality of CSP-dependent activation of comX may arise from either ComRS or ComDE. Here we study, at the single-cell level, how genes in the CSP signaling pathway respond to CSP, XIP and media. Our data indicate that activation of comX stimulates expression of comE. In addition, activation of comE requires intact comR and comS genes. Therefore, not only does CSP-ComDE stimulate the ComRS pathway to activate comX expression, but ComRS activation of comX also stimulates expression of the CSP-ComDE pathway and its regulon. The results demonstrate the mutual interconnection of the signaling pathways that control bacteriocin expression (ComDE) and genetic competence (ComRS), both of which are linked to lytic and virulence behaviors.
Single-cell studies show that signaling is bidirectional between the two quorum-sensing systems that regulate genetic competence in Streptococcus mutans.
Graphical Abstract Figure.Single-cell studies show that signaling is bidirectional between the two quorum-sensing systems that regulate genetic competence in Streptococcus mutans.
doi:10.1093/femsle/fnv159
PMCID: PMC4809993  PMID: 26363019
transformation; single cell; bistability; fluorescence; feedback; bimodality; quorum sensing
4.  A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans 
The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H2O2 via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)–ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.
doi:10.1128/AEM.03887-15
PMCID: PMC4807514  PMID: 26826230
5.  An Essential Role for (p)ppGpp in the Integration of Stress Tolerance, Peptide Signaling, and Competence Development in Streptococcus mutans 
The microbes that inhabit the human oral cavity are subjected to constant fluctuations in their environment. To overcome these challenges and gain a competitive advantage, oral streptococci employ numerous adaptive strategies, many of which appear to be intertwined with the development of genetic competence. Here, we demonstrate that the regulatory circuits that control development of competence in Streptococcus mutans, a primary etiological agent of human dental caries, are integrated with key stress tolerance pathways by the molecular alarmone (p)ppGpp. We first observed that the growth of a strain that does not produce (p)ppGpp (ΔrelAPQ, (p)ppGpp0) is not sensitive to growth inhibition by comX inducing peptide (XIP), unlike the wild-type strain UA159, even though XIP-dependent activation of the alternative sigma factor comX by the ComRS pathway is not impaired in the (p)ppGpp0 strain. Overexpression of a (p)ppGpp synthase gene (relP) in the (p)ppGpp0 mutant restored growth inhibition by XIP. We also demonstrate that exposure to micromolar concentrations of XIP elicited changes in (p)ppGpp accumulation in UA159. Loss of the RelA/SpoT homolog (RSH) enzyme, RelA, lead to higher basal levels of (p)ppGpp accumulation, but to decreased sensitivity to XIP and to decreases in comR promoter activity and ComX protein levels. By introducing single amino acid substitutions into the RelA enzyme, the hydrolase activity of the enzyme was shown to be crucial for full com gene induction and transformation by XIP. Finally, loss of relA resulted in phenotypic changes to ΔrcrR mutants, highlighted by restoration of transformation and ComX protein production in the otherwise non-transformable ΔrcrR-NP mutant. Thus, RelA activity and its influence on (p)ppGpp pools appears to modulate competence signaling and development through RcrRPQ and the peptide effectors encoded within rcrQ. Collectively, this study provides new insights into the molecular mechanisms that integrate intercellular communication with the physiological status of the cells and the regulation of key virulence-related phenotypes in S. mutans.
doi:10.3389/fmicb.2016.01162
PMCID: PMC4963387  PMID: 27516759
competence; biofilms; comX; stringent response; (p)ppGpp; dental caries
6.  A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance 
Molecular microbiology  2015;96(3):463-482.
Streptococcus mutans displays complex regulation of genetic competence, with ComX controlling late competence gene transcription. The rcrRPQ operon has been shown to link oxidative stress tolerance, (p)ppGpp metabolism and competence in S. mutans. Importantly, an rcrR polar (ΔrcrR-P) mutant is hyper-transformable, but an rcrR non-polar (ΔrcrR-NP) mutant cannot be transformed. Transcriptome comparisons of the rcrR mutants using RNA-Seq and quantitative real-time polymerase chain reaction revealed little expression in the 5′ region of comX in ΔrcrR-NP, but high level expression in the 3′ region. Northern blotting with comX probes revealed two distinct transcripts in the ΔrcrR-P and ΔrcrR-NP strains, and 5′ Rapid Amplification of cDNA Ends mapped the 5′ terminus of the shorter transcript to nt +140 of the comX structural gene, where a unique 69-aa open reading frame, termed XrpA, was encoded in a different reading frame than ComX. Two single-nucleotide substitution mutants (comX::T162C; comX::T210A) were introduced to disrupt XrpA without affecting the sequence of ComX. When the mutations were in the ΔrcrR-NP genetic background, ComX production and transformation were restored. Overexpression of xrpA led to impaired growth in aerobic conditions and decreased transformability. These results reveal an unprecedented mechanism for competence regulation and stress tolerance by a gene product encoded within the comX gene that appears unique to S. mutans.
doi:10.1111/mmi.12948
PMCID: PMC4414889  PMID: 25620525
7.  Regulation of competence and gene expression in Streptococcus mutans by the RcrR transcriptional regulator 
Molecular oral microbiology  2014;30(2):147-159.
SUMMARY
An intimate linkage between the regulation of biofilm formation, stress tolerance and genetic competence exists in the dental caries pathogen Streptococcus mutans. The rcrRPQ genes encode ABC exporters (RcrPQ) and a MarR-family transcriptional repressor of the rcr operon (RcrR) play a dominant role in regulation of the development of genetic competence and connect competence with stress tolerance and (p)ppGpp production in S. mutans. Here we identify the target for efficient RcrR binding in the rcr promoter region using purified recombinant RcrR (rRcrR) protein in electrophoretic mobility shift assays and show that DNA fragments carrying mutations in the binding region were not bound as efficiently by rRcrR in vitro. Mutations in the RcrR binding site impacted expression from the rcrR promoter in vivo and elicited changes in transformation efficiency, competence gene expression, and growth inhibition by competence stimulating peptide; even when the changes in rcrRPQ transcription were minor. An additional mechanistic linkage of RcrR with competence and (p)ppGpp metabolism was identified by showing that the rRcrR protein could bind to the promoter regions of comX, comYA and relP, although the binding was not as efficient as to the rcrRPQ promoter under the conditions tested. Thus, tightly controlled autogenous regulation of the rcrRPQ operon by RcrR binding to specific target sites is essential for cellular homeostasis, and RcrR contributes to the integration of genetic competence, (p)ppGpp metabolism, and acid and oxidative stress tolerance in S. mutans through both direct and indirect mechanisms.
doi:10.1111/omi.12079
PMCID: PMC4336644  PMID: 25146832
dental caries; genetic competence; autolysis; peptide signaling; transcriptional regulator
8.  Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans 
Applied and Environmental Microbiology  2015;81(15):5015-5025.
In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.
doi:10.1128/AEM.01160-15
PMCID: PMC4495203  PMID: 25979891
9.  Sharply Tuned pH Response of Genetic Competence Regulation in Streptococcus mutans: a Microfluidic Study of the Environmental Sensitivity of comX 
Applied and Environmental Microbiology  2015;81(16):5622-5631.
Genetic competence in Streptococcus mutans is a transient state that is regulated in response to multiple environmental inputs. These include extracellular pH and the concentrations of two secreted peptides, designated CSP (competence-stimulating peptide) and XIP (comX-inducing peptide). The role of environmental cues in regulating competence can be difficult to disentangle from the effects of the organism's physiological state and its chemical modification of its environment. We used microfluidics to control the extracellular environment and study the activation of the key competence gene comX. We find that the comX promoter (PcomX) responds to XIP or CSP only when the extracellular pH lies within a narrow window, about 1 pH unit wide, near pH 7. Within this pH range, CSP elicits a strong PcomX response from a subpopulation of cells, whereas outside this range the proportion of cells expressing comX declines sharply. Likewise, PcomX is most sensitive to XIP only within a narrow pH window. While previous work suggested that comX may become refractory to CSP or XIP stimulus as cells exit early exponential phase, our microfluidic data show that extracellular pH dominates in determining sensitivity to XIP and CSP. The data are most consistent with an effect of pH on the ComR/ComS system, which has direct control over transcription of comX in S. mutans.
doi:10.1128/AEM.01421-15
PMCID: PMC4510173  PMID: 26070670
10.  Pluronics-Formulated Farnesol Promotes Efficient Killing and Demonstrates Novel Interactions with Streptococcus mutans Biofilms 
PLoS ONE  2015;10(7):e0133886.
Streptococcus mutans is the primary causative agent of dental caries, one of the most prevalent diseases in the United States. Previously published studies have shown that Pluronic-based tooth-binding micelles carrying hydrophobic antimicrobials are extremely effective at inhibiting S. mutans biofilm growth on hydroxyapatite (HA). Interestingly, these studies also demonstrated that non-binding micelles (NBM) carrying antimicrobial also had an inhibitory effect, leading to the hypothesis that the Pluronic micelles themselves may interact with the biofilm. To explore this potential interaction, three different S. mutans strains were each grown as biofilm in tissue culture plates, either untreated or supplemented with NBM alone (P85), NBM containing farnesol (P85F), or farnesol alone (F). In each tested S. mutans strain, biomass was significantly decreased (SNK test, p < 0.05) in the P85F and F biofilms relative to untreated biofilms. Furthermore, the P85F biofilms formed large towers containing dead cells that were not observed in the other treatment conditions. Tower formation appeared to be specific to formulated farnesol, as this phenomenon was not observed in S. mutans biofilms grown with NBM containing triclosan. Parallel CFU/ml determinations revealed that biofilm growth in the presence of P85F resulted in a 3-log reduction in viability, whereas F decreased viability by less than 1-log. Wild-type biofilms grown in the absence of sucrose or gtfBC mutant biofilms grown in the presence of sucrose did not form towers. However, increased cell killing with P85F was still observed, suggesting that cell killing is independent of tower formation. Finally, repeated treatment of pre-formed biofilms with P85F was able to elicit a 2-log reduction in viability, whereas parallel treatment with F alone only reduced viability by 0.5-log. Collectively, these results suggest that Pluronics-formulated farnesol induces alterations in biofilm architecture, presumably via interaction with the sucrose-dependent biofilm matrix, and may be a viable treatment option in the prevention and treatment of pathogenic plaque biofilms.
doi:10.1371/journal.pone.0133886
PMCID: PMC4519314  PMID: 26222384
11.  Discovery of Novel Peptides Regulating Competence Development in Streptococcus mutans 
Journal of Bacteriology  2014;196(21):3735-3745.
A MarR-like transcriptional repressor (RcrR) and two predicted ABC efflux pumps (RcrPQ) encoded by a single operon were recently shown to be dominant regulators of stress tolerance and development of genetic competence in the oral pathogen Streptococcus mutans. Here, we focused on polar (ΔrcrR-P) and nonpolar (ΔrcrR-NP) rcrR mutants, which are hyper- and nontransformable, respectively, to dissect the mechanisms by which these mutations impact competence. We discovered two open reading frames (ORFs) in the 3′ end of the rcrQ gene that encode peptides of 27 and 42 amino acids (aa) which are also dramatically upregulated in the ΔrcrR-NP strain. Deletion of, or start codon mutations in, the ORFs for the peptides in the ΔrcrR-NP background restored competence and sensitivity to competence-stimulating peptide (CSP) to levels seen in the ΔrcrR-P strain. Overexpression of the peptides adversely affected competence development. Importantly, overexpression of mutant derivatives of the ABC exporters that lacked the peptides also resulted in impaired competence. FLAG-tagged versions of the peptides could be detected in S. mutans, and FLAG tagging of the peptides impaired their function. The competence phenotypes associated with the various mutations, and with overexpression of the peptides and ABC transporters, were correlated with the levels of ComX protein in cells. Collectively, these studies revealed multiple novel mechanisms for regulation of competence development by the components of the rcrRPQ operon. Given their intimate role in competence and stress tolerance, the rcrRPQ-encoded peptides may prove to be useful targets for therapeutics to diminish the virulence of S. mutans.
doi:10.1128/JB.01942-14
PMCID: PMC4248802  PMID: 25135217
12.  Growth Phase and pH Influence Peptide Signaling for Competence Development in Streptococcus mutans 
Journal of Bacteriology  2014;196(2):227-236.
The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in mid- and late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.
doi:10.1128/JB.00995-13
PMCID: PMC3911236  PMID: 24163340
13.  Evolutionary and Population Genomics of the Cavity Causing Bacteria Streptococcus mutans 
Molecular Biology and Evolution  2012;30(4):881-893.
Streptococcus mutans is widely recognized as one of the key etiological agents of human dental caries. Despite its role in this important disease, our present knowledge of gene content variability across the species and its relationship to adaptation is minimal. Estimates of its demographic history are not available. In this study, we generated genome sequences of 57 S. mutans isolates, as well as representative strains of the most closely related species to S. mutans (S. ratti, S. macaccae, and S. criceti), to identify the overall structure and potential adaptive features of the dispensable and core components of the genome. We also performed population genetic analyses on the core genome of the species aimed at understanding the demographic history, and impact of selection shaping its genetic variation. The maximum gene content divergence among strains was approximately 23%, with the majority of strains diverging by 5–15%. The core genome consisted of 1,490 genes and the pan-genome approximately 3,296. Maximum likelihood analysis of the synonymous site frequency spectrum (SFS) suggested that the S. mutans population started expanding exponentially approximately 10,000 years ago (95% confidence interval [CI]: 3,268–14,344 years ago), coincidental with the onset of human agriculture. Analysis of the replacement SFS indicated that a majority of these substitutions are under strong negative selection, and the remainder evolved neutrally. A set of 14 genes was identified as being under positive selection, most of which were involved in either sugar metabolism or acid tolerance. Analysis of the core genome suggested that among 73 genes present in all isolates of S. mutans but absent in other species of the mutans taxonomic group, the majority can be associated with metabolic processes that could have contributed to the successful adaptation of S. mutans to its new niche, the human mouth, and with the dietary changes that accompanied the origin of agriculture.
doi:10.1093/molbev/mss278
PMCID: PMC3603310  PMID: 23228887
Streptococcus mutans; demographic inference; cavities; bacterial evolution; pan and core genome; infectious disease
14.  Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX 
Molecular microbiology  2012;86(2):258-272.
Summary
Streptococcus mutans regulates genetic competence through a complex network that receives inputs from a number of environmental stimuli, including two signaling peptides designated as CSP and XIP. The response of the downstream competence genes to these inputs shows evidence of stochasticity and bistability and has been difficult to interpret. We have used microfluidic, single-cell methods to study how combinations of extracellular signals shape the response of comX, an alternative sigma factor governing expression of the late competence genes. We find that the composition of the medium determines which extracellular signal (XIP or CSP) can elicit a response from comX and whether that response is unimodal or bimodal across a population of cells. In a chemically defined medium, exogenous CSP does not induce comX, whereas exogenous XIP elicits a comX response from all cells. In complex medium, exogenous XIP does not induce comX, whereas CSP elicits a bimodal comX response from the population. Interestingly, bimodal behavior required an intact copy of comS, which encodes the precursor of XIP. The comS-dependent capability for both unimodal and bimodal response suggests that a constituent – most likely peptides – of complex medium interacts with a positive feedback loop in the competence regulatory network.
doi:10.1111/j.1365-2958.2012.08187.x
PMCID: PMC3468698  PMID: 22845615
single-cell; bistability; quorum sensing; gene regulation; feedback; transformation
15.  Transcriptional Organization and Physiological Contributions of the relQ Operon of Streptococcus mutans 
Journal of Bacteriology  2012;194(8):1968-1978.
The molecular alarmone (p)ppGpp functions as a global regulator of gene expression in bacteria. In Streptococcus mutans, (p)ppGpp synthesis is catalyzed by three gene products: RelA, RelP, and RelQ. RelA is responsible for (p)ppGpp production during a stringent response, and RelP is the primary source of (p)ppGpp during exponential growth, but the role of RelQ has not been thoroughly investigated. In this study, we analyzed the four-gene relQ operon to establish how these gene products may affect homeostasis and stress tolerance in the dental caries pathogen S. mutans. Northern blotting and reverse transcriptase PCR demonstrated that relQ is cotranscribed with the downstream genes ppnK (NAD kinase), rluE (pseudouridine synthase), and pta (phosphotransacetylase). In addition, a promoter located within the rluE gene was shown to drive transcription of pta. Inactivation of relQ, ppnK, or rluE did not significantly affect growth of or stress tolerance by S. mutans, whereas strains lacking pta were more sensitive to acid and oxidative stresses. Interestingly, introduction of an rluE deletion into the pta mutant reversed the deleterious effects of the pta mutation on growth and stress tolerance. Accumulation of (p)ppGpp was also decreased in a pta mutant strain, whereas inactivation of relQ caused enhanced (p)ppGpp synthesis in exponential-phase cells. The results reveal an important role for the relQ operon in the expression of traits that are essential for persistence and pathogenesis by S. mutans and provide evidence for a molecular connection of acetate and (p)ppGpp metabolism with tolerance of acid and oxidative stresses.
doi:10.1128/JB.00037-12
PMCID: PMC3318469  PMID: 22343297
16.  Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance 
BMC Microbiology  2012;12:187.
Background
The S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans.
Results
Real-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (H2O2) resulted in increased intracellular ROS in the lytS mutant.
Conclusions
Overall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans.
doi:10.1186/1471-2180-12-187
PMCID: PMC3507848  PMID: 22937869
Stress; Oxygen; Competence; Cid/Lrg system; Streptococcus mutans
17.  Implication of Mitochondrial Cytoprotection in Human Islet Isolation and Transplantation 
Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and long-term insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes.
doi:10.1155/2012/395974
PMCID: PMC3352213  PMID: 22611495
18.  A Transcriptional Regulator and ABC Transporters Link Stress Tolerance, (p)ppGpp, and Genetic Competence in Streptococcus mutans▿ †  
Journal of Bacteriology  2010;193(4):862-874.
Streptococcus mutans, a primary agent of dental caries, has three (p)ppGpp synthases: RelA, which is required for a mupirocin-induced stringent response; RelP, which produces (p)ppGpp during exponential growth and is regulated by the RelRS two-component system; and RelQ. Transcription of relPRS and a gene cluster (SMu0835 to SMu0837) located immediately upstream was activated in cells grown with aeration and during a stringent response, respectively. Bioinformatic analysis predicted that SMu0836 and SMu0837 encode ABC exporters, which we designated rcrPQ (rel competence-related) genes, respectively. SMu0835 (rcrR) encodes a MarR family transcriptional regulator. Reverse transcriptase PCR (RT-PCR) and quantitative RT-PCR analysis showed that RcrR functions as an autogenous negative regulator of the expression of the rcrRPQ operon. A mutant in which a polar insertion replaced the SMu836 gene (Δ836polar) grew more slowly and had final yields that were lower than those of the wild-type strain. Likewise, the Δ836polar strain had an impaired capacity to form biofilms, grew poorly at pH 5.5, and was more sensitive to oxidative stressors. Optimal expression of rcrPQ required RelP and vice versa. Replacement of rcrR with a nonpolar antibiotic resistance marker (Δ835np), which leads to overexpression of rcrPQ, yielded a strain that was not transformable with exogenous DNA. Transcriptional analysis revealed that the expression of comYA and comX was dramatically altered in the Δ835np and Δ836polar mutants. Collectively, the data support the suggestion that the rcrRPQ gene products play a critical role in physiologic homeostasis and stress tolerance by linking (p)ppGpp metabolism, acid and oxidative stress tolerance, and genetic competence.
doi:10.1128/JB.01257-10
PMCID: PMC3028664  PMID: 21148727
19.  Opportunities for Disrupting Cariogenic Biofilms 
Advances in dental research  2009;21(1):17-20.
Bacteria adhere to a surface and, through cell division and coordinated expression of gene products, to develop into a structurally-complex population of adherent cells. This process, known as biofilm formation, requires that intrinsic and extrinsic signals are transduced into appropriate gene expression patterns as biofilms mature. Mutational analysis has begun to reveal the complexity of systems used by Streptococcus mutans to ensure proper biofilm formation. These studies have revealed new and unique targets for the design of broadly-effective anti-caries strategies.
doi:10.1177/0895937409335593
PMCID: PMC2853230  PMID: 19710079
20.  Biofilm formation and virulence expression by Streptococcus mutans are altered when grown in dual-species model 
BMC Microbiology  2010;10:111.
Background
Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.
Results
As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.
Conclusions
These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.
doi:10.1186/1471-2180-10-111
PMCID: PMC2867949  PMID: 20398271
21.  Changes in Biochemical and Phenotypic Properties of Streptococcus mutans during Growth with Aeration▿ † 
Oxygen has a potent influence on the expression of genes and the activity of physiological and biochemical pathways in bacteria. We have found that oxygen significantly altered virulence-related phenotypic properties of Streptococcus mutans, the primary etiological agent of human dental caries. Transport of glucose, fructose, or mannose by the sugar:phosphotransferase system was significantly enhanced by growth under aerobic conditions, whereas aeration caused an extended lag phase and slower growth of S. mutans in medium containing glucose, fructose, or mannose as the carbohydrate source. Aeration resulted in a decrease in the glycolytic rate and enhanced the production of intracellular storage polysaccharides. Although aeration decreased the acid tolerance of S. mutans, aerobically grown cells had higher F-ATPase activity. Aeration altered biofilm architecture but did not change the ability of S. mutans to interact with salivary agglutinin. Growth in air resulted in enhanced cell-associated glucosyltransferase (Gtf) activity at the expense of cell-free Gtf activity. These results demonstrate that S. mutans can dramatically alter its pathogenic potential in response to exposure to oxygen, suggesting that the phenotype of the organism may be highly variable in the human oral cavity depending on the maturity of the dental plaque biofilm.
doi:10.1128/AEM.02367-08
PMCID: PMC2675223  PMID: 19251884
22.  Characteristics of Biofilm Formation by Streptococcus mutans in the Presence of Saliva▿  
Infection and Immunity  2008;76(9):4259-4268.
Interactions between salivary agglutinin and the adhesin P1 of Streptococcus mutans contribute to bacterial aggregation and mediate sucrose-independent adherence to tooth surfaces. We have examined biofilm formation by S. mutans UA159, and derivative strains carrying mutations affecting the localization or expression of P1, in the presence of fluid-phase or adsorbed saliva or salivary agglutinin preparations. Whole saliva- and salivary agglutinin-induced aggregation of S. mutans was adversely affected by the loss of P1 and sortase (SrtA) but not by the loss of trigger factor (RopA). Fluid-phase salivary agglutinin and, to a lesser extent, immobilized agglutinin inhibited biofilm development by S. mutans in the absence of sucrose, and whole saliva was more effective at decreasing biofilm formation than salivary agglutinin. Inhibition of biofilm development by salivary agglutinin was differently influenced by particular mutations, with the P1-deficient strain displaying a greater inhibition of biofilm development than the SrtA- or RopA-deficient strains. As expected, biofilm-forming capacities of all strains in the presence of salivary preparations were markedly enhanced in the presence of sucrose, although biofilm formation by the mutants was less efficient than that by the parental strain. Aeration strongly inhibited biofilm development, and the presence of salivary components did not restore biofilm formation in aerated conditions. The results disclose a potent ability of salivary constituents to moderate biofilm formation by S. mutans through P1-dependent and P1-independent pathways.
doi:10.1128/IAI.00422-08
PMCID: PMC2519434  PMID: 18625741
23.  Effects of Oxygen on Virulence Traits of Streptococcus mutans▿  
Journal of Bacteriology  2007;189(23):8519-8527.
Oxygen profoundly affects the composition of oral biofilms. Recently, we showed that exposure of Streptococcus mutans to oxygen strongly inhibits biofilm formation and alters cell surface biogenesis. To begin to dissect the underlying mechanisms by which oxygen affects known virulence traits of S. mutans, transcription profiling was used to show that roughly 5% of the genes of this organism are differentially expressed in response to aeration. Among the most profoundly upregulated genes were autolysis-related genes and those that encode bacteriocins, the ClpB protease chaperone subunit, pyruvate dehydrogenase, the tricarboxylic acid cycle enzymes, NADH oxidase enzymes, and certain carbohydrate transporters and catabolic pathways. Consistent with our observation that the ability of S. mutans to form biofilms was severely impaired by oxygen exposure, transcription of the gtfB gene, which encodes one of the primary enzymes involved in the production of water-insoluble, adhesive glucan exopolysaccharides, was down-regulated in cells growing aerobically. Further investigation revealed that transcription of gtfB, but not gtfC, was responsive to oxygen and that aeration causes major changes in the amount and degree of cell association of the Gtf enzymes. Moreover, inactivation of the VicK sensor kinase affected the expression and localization the GtfB and GtfC enzymes. This study provides novel insights into the complex transcriptional and posttranscriptional regulatory networks used by S. mutans to modulate virulence gene expression and exopolysaccharide production in response to changes in oxygen availability.
doi:10.1128/JB.01180-07
PMCID: PMC2168947  PMID: 17921307
24.  Effects of Oxygen on Biofilm Formation and the AtlA Autolysin of Streptococcus mutans▿  
Journal of Bacteriology  2007;189(17):6293-6302.
The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.
doi:10.1128/JB.00546-07
PMCID: PMC1951938  PMID: 17616606
25.  The atlA Operon of Streptococcus mutans: Role in Autolysin Maturation and Cell Surface Biogenesis 
Journal of Bacteriology  2006;188(19):6877-6888.
The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.
doi:10.1128/JB.00536-06
PMCID: PMC1595523  PMID: 16980491

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