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1.  The Redox-Sensing Regulator Rex Modulates Central Carbon Metabolism, Stress Tolerance Response and Biofilm Formation by Streptococcus mutans 
PLoS ONE  2012;7(9):e44766.
The Rex repressor has been implicated in regulation of central carbon and energy metabolism in Gram-positive bacteria. We have previously shown that Streptococcus mutans, the primary causative agent of dental caries, alters its transcriptome upon Rex-deficiency and renders S. mutans to have increased susceptibility to oxidative stress, aberrations in glucan production, and poor biofilm formation. In this study, we showed that rex in S. mutans is co-transcribed as an operon with downstream guaA, encoding a putative glutamine amidotransferase. Electrophoretic mobility shift assays showed that recombinant Rex bound promoters of target genes avidly and specifically, including those down-regulated in response to Rex-deficiency, and that the ability of recombinant Rex to bind to selected promoters was modulated by NADH and NAD+. Results suggest that Rex in S. mutans can function as an activator in response to intracellular NADH/NAD+ level, although the exact binding site for activator Rex remains unclear. Consistent with a role in oxidative stress tolerance, hydrogen peroxide challenge assays showed that the Rex-deficient mutant, TW239, and the Rex/GuaA double mutant, JB314, were more susceptible to hydrogen peroxide killing than the wildtype, UA159. Relative to UA159, JB314 displayed major defects in biofilm formation, with a decrease of more than 50-fold in biomass after 48-hours. Collectively, these results further suggest that Rex in S. mutans regulates fermentation pathways, oxidative stress tolerance, and biofilm formation in response to intracellular NADH/NAD+ level. Current effort is being directed to further investigation of the role of GuaA in S. mutans cellular physiology.
doi:10.1371/journal.pone.0044766
PMCID: PMC3441419  PMID: 23028612
2.  A Rex Family Transcriptional Repressor Influences H2O2 Accumulation by Enterococcus faecalis 
Journal of Bacteriology  2013;195(8):1815-1824.
Rex factors are bacterial transcription factors thought to respond to the cellular NAD+/NADH ratio in order to modulate gene expression by differentially binding DNA. To date, Rex factors have been implicated in regulating genes of central metabolism, oxidative stress response, and biofilm formation. The genome of Enterococcus faecalis, a low-GC Gram-positive opportunistic pathogen, encodes EF2638, a putative Rex factor. To study the role of E. faecalis Rex, we purified EF2638 and evaluated its DNA binding activity in vitro. EF2638 was able to bind putative promoter segments of several E. faecalis genes in an NADH-responsive manner, indicating that it represents an authentic Rex factor. Transcriptome analysis of a ΔEF2638 mutant revealed that genes likely to be involved in anaerobic metabolism were upregulated during aerobic growth, and the mutant exhibited an altered NAD+/NADH ratio. The ΔEF2638 mutant also exhibited a growth defect when grown with aeration on several carbon sources, suggesting an impaired ability to cope with oxidative stress. Inclusion of catalase in the medium alleviated the growth defect. H2O2 measurements revealed that the mutant accumulates significantly more H2O2 than wild-type E. faecalis. In summary, EF2638 represents an authentic Rex factor in E. faecalis that influences the production or detoxification of H2O2 in addition to its more familiar role as a regulator of anaerobic gene expression.
doi:10.1128/JB.02135-12
PMCID: PMC3624565  PMID: 23417491
3.  Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange. 
Infection and Immunity  1991;59(7):2316-2323.
Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.
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PMCID: PMC258013  PMID: 1828790
4.  A Pleiotropic Regulator, Frp, Affects Exopolysaccharide Synthesis, Biofilm Formation, and Competence Development in Streptococcus mutans  
Infection and Immunity  2006;74(8):4581-4589.
Exopolysaccharide synthesis, biofilm formation, and competence are important physiologic functions and virulence factors for Streptococcus mutans. In this study, we report the role of Frp, a transcriptional regulator, on the regulation of these traits crucial to pathogenesis. An Frp-deficient mutant showed decreased transcription of several genes important in virulence, including those encoding fructosyltransferase (Ftf), glucosyltransferase B (GtfB), and GtfC, by reverse transcription and quantitative real-time PCR. Expression of Ftf was decreased in the frp mutant, as assessed by Western blotting as well as by the activity assays. Frp deficiency also inhibited the production of GtfB in the presence of glucose and sucrose as well as the production of GtfC in the presence of glucose. As a consequence of the effects on GtfB and -C, sucrose-induced biofilm formation was decreased in the frp mutant. The expression of competence mediated by the competence-signaling peptide (CSP) system, as assessed by comC gene transcription, was attenuated in the frp mutant. As a result, the transformation efficiency was decreased in the frp mutant but was partially restored by adding synthetic CSP. Transcription of the frp gene was significantly increased in the frp mutant under all conditions tested, indicating that frp transcription is autoregulated. Furthermore, complementation of the frp gene in the frp mutant restored transcription of the affected genes to levels similar to those in the wild-type strain. These results suggest that Frp is a novel pleiotropic effector of multiple cellular functions and is involved in the modulation of exopolysaccharide synthesis, sucrose-dependent biofilm formation, and competence development.
doi:10.1128/IAI.00001-06
PMCID: PMC1539613  PMID: 16861645
5.  Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus 
Molecular Microbiology  2010;76(5):1142-1161.
An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox-sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence – TTGTGAAW4TTCACAA – is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD+ while NADH, which competes with NAD+ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex-deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD+ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.
doi:10.1111/j.1365-2958.2010.07105.x
PMCID: PMC2883068  PMID: 20374494
6.  Phylogenetic Analysis of Glucosyltransferases and Implications for the Coevolution of Mutans Streptococci with Their Mammalian Hosts 
PLoS ONE  2013;8(2):e56305.
Glucosyltransferases (Gtfs) catalyze the synthesis of glucans from sucrose and are produced by several species of lactic-acid bacteria. The oral bacterium Streptococcus mutans produces large amounts of glucans through the action of three Gtfs. GtfD produces water-soluble glucan (WSG), GtfB synthesizes water-insoluble glucans (WIG) and GtfC produces mainly WIG but also WSG. These enzymes, especially those synthesizing WIG, are of particular interest because of their role in the formation of dental plaque, an environment where S. mutans can thrive and produce lactic acid, promoting the formation of dental caries. We sequenced the gtfB, gtfC and gtfD genes from several mutans streptococcal strains isolated from the oral cavity of humans and searched for their homologues in strains isolated from chimpanzees and macaque monkeys. The sequence data were analyzed in conjunction with the available Gtf sequences from other bacteria in the genera Streptococcus, Lactobacillus and Leuconostoc to gain insights into the evolutionary history of this family of enzymes, with a particular emphasis on S. mutans Gtfs. Our analyses indicate that streptococcal Gtfs arose from a common ancestral progenitor gene, and that they expanded to form two clades according to the type of glucan they synthesize. We also show that the clade of streptococcal Gtfs synthesizing WIG appeared shortly after the divergence of viviparous, dentate mammals, which potentially contributed to the formation of dental plaque and the establishment of several streptococci in the oral cavity. The two S. mutans Gtfs capable of WIG synthesis, GtfB and GtfC, are likely the product of a gene duplication event. We dated this event to coincide with the divergence of the genomes of ancestral early primates. Thus, the acquisition and diversification of S. mutans Gtfs predates modern humans and is unrelated to the increase in dietary sucrose consumption.
doi:10.1371/journal.pone.0056305
PMCID: PMC3572963  PMID: 23457545
7.  Competence-Dependent Endogenous DNA Rearrangement and Uptake of Extracellular DNA Give a Natural Variant of Streptococcus mutans without Biofilm Formation▿ 
Journal of Bacteriology  2011;193(19):5147-5154.
The production of water-insoluble glucan (WIG) enables Streptococcus mutans to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous gtfB and gtfC genes. Conversely, a single hybrid gene from the endogenous recombination of gtfB and gtfC is easily generated using RecA, resulting in S. mutans UA159 WIG− (rate of ∼1.0 × 10−3). The pneumococcus recA gene is regulated as a late competence gene. comX gene mutations did not lead to the appearance of WIG− cells. The biofilm collected from the flow cell had more WIG− cells than among the planktonic cells. Among the planktonic cells, WIG− cells appeared after 16 h and increased ∼10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG− cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG+ and recipient WIG− cells, the recipient cells reverted to WIG+ and acquired an intact gtfBC region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG− cells and that both are induced by the same signal transducer, the com system. Our findings may help in understanding how S. mutans can adapt to the oral environment and may explain the evolution of S. mutans.
doi:10.1128/JB.05240-11
PMCID: PMC3187377  PMID: 21804005
8.  Comparative Analysis of Gtf Isozyme Production and Diversity in Isolates of Streptococcus mutans with Different Biofilm Growth Phenotypes 
Journal of Clinical Microbiology  2004;42(10):4586-4592.
Streptococcus mutans is the main pathogenic agent of dental caries. Glucosyltransferases (Gtfs) produced by these bacteria are important virulence factors because they catalyze the extracellular synthesis of glucans that are necessary for bacterial accumulation in the dental biofilm. The diversity of GtfB and GtfC isozymes was analyzed in 44 genotypes of S. mutans that showed a range of abilities to form biofilms in vitro. Several approaches were used to characterize these isozymes, including restriction fragment length polymorphism analysis of the gtfB and gtfC genes, zymographic analysis of the identified GtfB and GtfC genotypes, and quantitation of isozyme production in immunoblot experiments with specific monoclonal antibodies. A high diversity of gtf genes, patterns of enzymatic activity, and isozyme production was identified among the isolates tested. GtfC and, to a lesser extent, GtfB were produced in significantly higher amounts by strains that had high biofilm-forming ability than by strains with low biofilm-forming ability. Biofilm formation was independent of the GtfB and GtfC genotype. Atypical strains that showed an apparent single Gtf isozyme of intermediate size between GtfB and GtfC were also identified. The results indicate that various expression levels of GtfB and GtfC isozymes are associated with the ability of distinct S. mutans genotypes to grow as biofilms, strengthening the results of previous genetic and biochemical studies performed with laboratory strains. These studies also emphasize the need to identify factors that control gtf gene expression.
doi:10.1128/JCM.42.10.4586-4592.2004
PMCID: PMC522304  PMID: 15472313
9.  Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms▿  
Journal of Bacteriology  2010;192(12):3024-3032.
Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.
doi:10.1128/JB.01649-09
PMCID: PMC2901689  PMID: 20233920
10.  Regulatory Loop between Redox Sensing of the NADH/NAD+ Ratio by Rex (YdiH) and Oxidation of NADH by NADH Dehydrogenase Ndh in Bacillus subtilis 
Journal of Bacteriology  2006;188(20):7062-7071.
NADH dehydrogenase is a key component of the respiratory chain. It catalyzes the oxidation of NADH by transferring electrons to ubiquinone and establishes a proton motive force across the cell membrane. The yjlD (renamed ndh) gene of Bacillus subtilis is predicted to encode an enzyme similar to the NADH dehydrogenase II of Escherichia coli, encoded by the ndh gene. We have shown that the yjlC-ndh operon is negatively regulated by YdiH (renamed Rex), a homolog of Rex in Streptomyces coelicolor, and a redox-sensing transcriptional regulator that responds to the NADH/NAD+ ratio. The ndh gene regulates expression of the yjlC-ndh operon, as indicated by the fact that mutation in ndh causes a higher NADH/NAD+ ratio. An in vitro study showed that Rex binds to the downstream region of the yjlC-ndh promoter and that NAD+ enhances the binding of Rex to the putative Rex-binding sites in the yjlC-ndh operon as well as in the cydABCD operon. These results indicated that Rex and Ndh together form a regulatory loop which functions to prevent a large fluctuation in the NADH/NAD+ ratio in B. subtilis.
doi:10.1128/JB.00601-06
PMCID: PMC1636230  PMID: 17015645
11.  LuxS-Based Signaling Affects Streptococcus mutans Biofilm Formation 
Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.
doi:10.1128/AEM.71.5.2372-2380.2005
PMCID: PMC1087550  PMID: 15870324
12.  A Novel PTS of Streptococcus mutans is Responsible for Transport of Carbohydrates with α-1,3 linkage 
Molecular oral microbiology  2012;28(2):114-128.
SUMMARY
The most common type of carbohydrate-transport system in Streptococcus mutans is the phosphoenolpyruvate (PEP)-sugar phosphotransferase system (PTS). We previously showed that fourteen PTSs exist in S. mutans UA159 (Ajdic et al., 2002). Several studies have shown that microorganisms growing in biofilms express different genes as compared to their planktonic counterparts. In this study, we showed that one PTS of S. mutans was expressed in sucrose-grown biofilms. Furthermore, the same PTS was also responsible for the transport and metabolism of disaccharide nigerose (3-O-α-D-glucopyranosyl-D-glucose). Additionally, the results indicate that the studied PTS might be involved in the transport and metabolism of carbohydrates synthesized by glucosyltransferase B (GtfB) and glucosyltransferase C (GtfC) of S. mutans. To our knowledge, this is the first report that shows PTS transport of a disaccharide (and possibly extracellular oligosaccharides) with α-1,3 linkage.
doi:10.1111/omi.12009
PMCID: PMC3593818  PMID: 23193985
S. mutans; Biofilm; Transporter; Carbohydrate; Microarray
13.  Transcriptional Regulation of Central Carbon and Energy Metabolism in Bacteria by Redox-Responsive Repressor Rex 
Journal of Bacteriology  2012;194(5):1145-1157.
Redox-sensing repressor Rex was previously implicated in the control of anaerobic respiration in response to the cellular NADH/NAD+ levels in Gram-positive bacteria. We utilized the comparative genomics approach to infer candidate Rex-binding DNA motifs and assess the Rex regulon content in 119 genomes from 11 taxonomic groups. Both DNA-binding and NAD-sensing domains are broadly conserved in Rex orthologs identified in the phyla Firmicutes, Thermotogales, Actinobacteria, Chloroflexi, Deinococcus-Thermus, and Proteobacteria. The identified DNA-binding motifs showed significant conservation in these species, with the only exception detected in Clostridia, where the Rex motif deviates in two positions from the generalized consensus, TTGTGAANNNNTTCACAA. Comparative analysis of candidate Rex sites revealed remarkable variations in functional repertoires of candidate Rex-regulated genes in various microorganisms. Most of the reconstructed regulatory interactions are lineage specific, suggesting frequent events of gain and loss of regulator binding sites in the evolution of Rex regulons. We identified more than 50 novel Rex-regulated operons encoding functions that are essential for resumption of the NADH:NAD+ balance. The novel functional role of Rex in the control of the central carbon metabolism and hydrogen production genes was validated by in vitro DNA binding assays using the TM0169 protein in the hydrogen-producing bacterium Thermotoga maritima.
doi:10.1128/JB.06412-11
PMCID: PMC3294762  PMID: 22210771
14.  Analysis of the Streptococcus downei gtfS gene, which specifies a glucosyltransferase that synthesizes soluble glucans. 
Infection and Immunity  1990;58(8):2452-2458.
The complete nucleotide sequence was determined for the Streptococcus downei (previously Streptococcus sobrinus) MFe28 gtfS gene which specifies a glucosyltransferase (GTF-S) producing water-soluble glucan. A single open reading frame which encodes a mature protein with a molecular weight of 147,408 (1,328 amino acids) and a putative signal peptide 36 or 37 amino acids in length was detected. GTF-S shares extensive sequence similarity with GTF-I (gtfI) from S. downei and GTF-I (gtfB) and GTF-SI (gtfC) from Streptococcus mutans. GTF-S contains a highly conserved enzymatic domain and C-terminal repeated sequences which appear to be involved in glucan binding. Comparison of the deduced GTF-S protein sequence with other sequenced GTF genes of mutans streptococci revealed that these C-terminal repeats occurred in all cases, although the patterns of repeated sequences varied with respect to each other and to the glucan-binding protein of S. mutans. GTF-S contains four C-terminal repeat sequences ranging from 49 to 51 amino acids in length and a partial repeat of 13 amino acids. Nuclear magnetic resonance analysis of the glucan produced by GTF-S revealed that the product consisted of more than 90% alpha-1,6-linked glucosyl residues.
PMCID: PMC258840  PMID: 2142479
15.  Isolation and characterization of the Streptococcus mutans gtfC gene, coding for synthesis of both soluble and insoluble glucans. 
Infection and Immunity  1988;56(8):1999-2005.
The intact gtfC gene from Streptococcus mutans GS-5 was isolated in Escherichia coli in plasmid vector pUC18. The glucosyltransferase activity expressed by the gene synthesized both low-molecular-weight water-soluble glucan and insoluble glucan in a primer-independent manner. Purification of the enzyme by procedures that minimize proteolytic digestion yielded a purified preparation with a molecular weight of 140,000. Insertional inactivation of the gtfC gene with a streptococcal erythromycin resistance gene fragment followed by transformation of strain GS-5 suggested that the gtfC gene product was required for sucrose-dependent colonization in vitro. In addition, evidence for the presence of a third gtf gene coding for soluble glucan synthesis was obtained following the construction of mutants containing deletions of both the gtfB and gtfC genes.
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PMCID: PMC259514  PMID: 2969375
16.  Regulation of the Glucosyltransferase (gtfBC) Operon by CovR in Streptococcus mutans 
Journal of Bacteriology  2006;188(3):988-998.
Streptococcus mutans is an important etiological agent of dental caries in humans. The extracellular polysaccharides synthesized by cell-associated glucosyltransferases (encoded by gtfBC) from sucrose have been recognized as one of the important virulence factors that promote cell aggregation and adherence to teeth, leading to dental plaque formation. In this study, we have characterized the effect of CovR, a global response regulator, on glucosyltransferase expression. Inactivation of covR in strain UA159 resulted in a marked increase in the GtfB and GtfC proteins, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With the use of a transcriptional reporter system of a single chromosomal copy of the PgtfB-gusA and PgtfC-gusA fusions, we confirmed the transcriptional regulation of these promoters by CovR. By in vitro electrophoretic mobility shift assays with purified CovR protein, we showed that CovR regulates these promoters directly. DNase I footprinting analyses suggest that CovR binds to large regions on these promoters near the transcription start sites. Taken together, our results indicate that CovR negatively regulates the expression of the gtfB and gtfC genes by directly binding to the promoter region.
doi:10.1128/JB.188.3.988-998.2006
PMCID: PMC1347363  PMID: 16428403
17.  Glucan-Binding Proteins are Essential for Shaping Streptococcus mutans Biofilm Architecture 
FEMS microbiology letters  2006;268(2):158-165.
Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases (Gtfs) that catalyze the synthesis of glucan and the non-Gtf glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties quantified by the biofilm quantification software COMSTAT. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.
doi:10.1111/j.1574-6968.2006.00576.x
PMCID: PMC1804096  PMID: 17214736
18.  Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces 
Background
10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces.
Methods
Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically.
Results
500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells.
Conclusion
Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it’s not necessary to put down the oral flora completely.
doi:10.1186/1476-0711-11-21
PMCID: PMC3495742  PMID: 22839724
Biofilm; Caries; Glucosyltransferase; Streptococcus
19.  Recombination between gtfB and gtfC Is Required for Survival of a dTDP-Rhamnose Synthesis-Deficient Mutant of Streptococcus mutans in the Presence of Sucrose 
Infection and Immunity  1999;67(7):3693-3697.
The rml genes are involved in dTDP-rhamnose synthesis in Streptococcus mutans. A gene fusion between gtfB and gtfC, which both encode extracellular water-insoluble glucan-synthesizing enzymes, accompanied by inactivation of the rml genes was observed for cells grown in the presence of sucrose. The survival rates of rml mutants isolated in the absence of sucrose were drastically reduced in the presence of sucrose. The rates were consistent with the frequency of spontaneous gene fusions between gtfB and gtfC, suggesting that the spontaneous recombinant organisms were selected in the presence of sucrose. The rml mutants with a gtfB-gtfC fusion gene had markedly reduced water-insoluble glucan synthetic activity and lost the ability to colonize glass surfaces in the presence of sucrose. These results suggest that the rml mutants of S. mutans, which are defective in dTDP-rhamnose synthesis, can survive only in the absence of water-insoluble glucan synthesis.
PMCID: PMC116568  PMID: 10377163
20.  Transdominant repressors for human T-cell leukemia virus type I rex and human immunodeficiency virus type 1 rev function. 
Journal of Virology  1991;65(1):81-88.
Human T-cell leukemia virus type I (HTLV-I) encodes a 27-kDa trans-acting gene product (Rex) which is involved in the regulated expression of transcripts coding for the viral structural proteins. We used oligonucleotide-directed mutagenesis to generate a series of mutant HTLV-I rex genes. Transient expression experiments demonstrated that 3 of 28 mutant proteins are functionally inactive on the homologous HTLV-I rex response element, whereas an additional 2 mutant proteins are functionally inactive on the heterologous human immunodeficiency virus type 1 rev response element. One of these mutants is able to suppress the function of the wild-type HTLV-I Rex protein in trans on the homologous rex response element sequence. Furthermore, all of these mutants are able to inhibit Rex function on the heterologous rev response element sequence. Intriguingly, only three of these mutants are able to inhibit the human immunodeficiency virus type 1 Rev protein in a dominant-negative manner.
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PMCID: PMC240491  PMID: 1985219
21.  Molecular cloning and characterization of the glucosyltransferase C gene (gtfC) from Streptococcus mutans LM7. 
Infection and Immunity  1987;55(9):2176-2182.
A glucosyltransferase (GTF) gene, designated gtfC, was cloned from Streptococcus mutans LM7. Its gene product was detected by screening a bacteriophage lambda library with rabbit antiserum raised against S. mutans LM7 extracellular proteins. DNA isolated from the immunopositive recombinant phage revealed two S. mutans chromosomal EcoRI fragment inserts, 8.1 and 4.7 kilobase pairs in size. Escherichia coli minicell analyses revealed the approximate position and direction of transcription of the gtfC gene. The gene product was determined to be a polypeptide of about 150 kilodaltons which synthesized a water-soluble glucan. Restriction endonuclease mapping and DNA hybridization indicated a repeated region of DNA corresponding to a portion of the coding region of gtfC immediately downstream from the intact gtfC locus on the chromosome. A 300-base-pair gtfC-specific probe showed that the gene and the putative duplicated sequence were present in S. mutans serotypes c, e, and f, but not in other related oral streptococci which had GTF activity. In addition, the gtfC determinant displayed homology to sequences corresponding to the carboxy-terminal coding region of a gene (gtfB) encoding a GTF activity that synthesized water-insoluble glucans. These data suggest that at least one class of GTF genes may be present in multiple copies in S. mutans and, further, that GTF genes may contain conserved sequences internal to their coding regions.
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PMCID: PMC260675  PMID: 3040591
22.  Influence of Apigenin on gtf Gene Expression in Streptococcus mutans UA159 
Apigenin, a potent inhibitor of glucosyltransferase activity, affects the accumulation of Streptococcus mutans biofilms in vitro by reducing the formation of insoluble glucans and enhancing the soluble glucan content of the polysaccharide matrix. In the present study, we investigated the influence of apigenin on gtfB, gtfC, and gtfD expression in S. mutans UA159. Apigenin (0.1 mM) significantly decreased the expression of gtfB and gtfC mRNA (P < 0.05); in contrast, it increased the expression of gtfD in S. mutans growing in the planktonic state. The protein levels of GTF B, GTF C, and GTF D in culture supernatants were also affected; less GTF B and C were detected, whereas the level of GTF D was significantly elevated (P < 0.05). A similar profile of gtf expression was obtained with biofilms, although an elevated concentration (1 mM) of apigenin was required to elicit the effects. The influence of apigenin on gtf gene expression was independent of any effect on GTF activity, did not involve inhibition of growth or effects on pH, and was not affected by addition of sucrose. The data show that apigenin modulates the genetic expression of virulence factors in S. mutans.
doi:10.1128/AAC.50.2.542-546.2006
PMCID: PMC1366919  PMID: 16436708
23.  Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. 
Infection and Immunity  1993;61(9):3811-3817.
The role of each of the Streptococcus mutans gtf genes coding for glucan synthesis in cariogenesis was evaluated by using strain UA130 in the specific-pathogen-free (SPF) rat model system. Mutants defective in either or both of the genes required for insoluble glucan synthesis, the gtfB and gtfC genes, exhibited markedly reduced levels of smooth-surface carious lesions relative to that of the parental organism. Likewise, the mutant defective in the gtfD gene coding for the glucosyltransferase-S enzyme synthesizing water-soluble glucans also produced significantly fewer smooth-surface lesions than strain UA130. None of these mutations markedly altered the rate of sulcal caries induction relative to that of the parental organism. In addition, a mutant of strain UA130 defective in the gtfA gene was reexamined in the SPF rat model. In contrast to previous results from a gnotobiotic rat system, these mutants also induced significantly fewer smooth-surface carious lesions compared with that by strain UA130. These results suggest that all four genes are important for smooth-surface caries formation. Furthermore, these results are discussed relative to the differences in the diets utilized in the SPF and gnotobiotic rat model systems for assessing the virulence factors of S. mutans.
Images
PMCID: PMC281081  PMID: 8359902
24.  Streptococcus Mutans Glucan-Binding Protein-A Affects Streptococcus Gordonii Biofilm Architecture 
FEMS microbiology letters  2006;267(1):80-88.
The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. Since S. mutans synthesizes multiple glucosyltransferases (GTF) and non-GTF glucan-binding proteins (GBPs), it’s possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished GTF activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily α-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512 that produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to α-1,6-linkages within glucan. The magnitude of the GbpA effect appears dependent on the quantity and linkage of available glucan.
doi:10.1111/j.1574-6968.2006.00557.x
PMCID: PMC1780135  PMID: 17166223
25.  Streptococcus gordonii’s Sequenced Strain CH1 Glucosyltransferase Determines Persistent But Not Initial Colonization of Teeth of Rats 
Archives of oral biology  2007;53(2):133-140.
Objective
Extracellular glucan synthesis from sucrose by Streptococcus gordonii, a major dental plaque biofilm bacterium, is assumed important for colonization of teeth; but this hypothesis is un-tested in vivo.
Methods
To do so, we studied an isogenic glucosyltransferase (Gtf)-negative mutant (strain AMS12, gtfG−) of S. gordonii sequenced wild type (WT, strain Challis CH1, gtfG+), comparing their in vitro abilities to grow in the presence of glucose and sucrose and, in vivo, to colonize and persist on teeth and induce caries in rats. Weanling rats of two breeding colonies, TAN:SPFOM(OM)BR and TAN:SPFOM(OMASF)BR, eating high sucrose diet, were inoculated with either the WT (gtfG+), its isogenic gtfG− mutant, or reference strains of Streptococcus mutans. Control animals were not inoculated.
Results
In vitro, the gtfG− strain grew at least as rapidly in the presence of sucrose as its WT gtfG+ progenitor, but formed soft colonies on sucrose agar, consistent with its lack of insoluble glucan synthesis. It also had a higher growth yield due apparently to its inability to channel carbon flow into extracellular glucan. In vivo, the gtfG− mutant initially colonized as did the WT as but, unlike the WT, failed to persist on the teeth as shown over time. By comparison to three S. mutans strains, S. gordonii WT, despite its comparable ecological success on the teeth, was associated with only modest caries induction. Failure of the gtfG− mutant to persistently colonize was associated with slight diminution of caries scores by comparison with its gtfG+ WT.
Conclusions
Initial S. gordonii colonization does not depend on Gtf-G synthesis; rather, Gtf-G production determines S. gordonii’s ability to persist on the teeth of sucrose-fed rats. S. gordonii appears weakly cariogenic by comparison with S. mutans reference strains.
doi:10.1016/j.archoralbio.2007.08.011
PMCID: PMC2243221  PMID: 17961499
S. gordonii; glucosyltransferase; colonization persistence; biofilm; plaque; dental caries

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