Ftf Gene Research Articles

The inhibitory effect of ovomucoid from egg white on biofilm formation by Streptococcus mutans.

Streptococcus mutans, the main pathogen associated with tooth decay, forms cariogenic biofilms on tooth surfaces. Therefore, controlling oral biofilm helps prevent dental caries. Hen's egg is a nutrient-dense food, and egg white is a good source of protein. Ovomucoid is one of the major proteins in egg white, with a 28 kDa molecular weight. The present study aimed to investigate the inhibitory effects of ovomucoid on the biofilm formation of S. mutans by suppressing virulence factors, including bacterial adherence, cellular aggregation and exopolysaccharide (EPS) production. Crystal violet staining showed that biofilm formation by S. mutans was inhibited by ovomucoid at 0.25-1 mg mL-1 levels. Field emission scanning electron microscopy also confirmed this inhibition. In addition, ovomucoid reduced mature biofilm, water-insoluble EPS synthesis and the metabolic activity of bacterial cells in the biofilm. The bacterial adhesion and aggregation abilities of S. mutans were also decreased in the presence of ovomucoid. Ovomucoid downregulated the expression of comDE and vicR genes involved in the two-component signal transduction system and gtfA and ftf genes involved in EPS production. Ovomucoid has the potential for use as an anti-biofilm agent for dental caries treatment because of its inhibitory effects on the virulence factors of S. mutans. © 2023 Society of Chemical Industry.

Effect of epigallocatechin gallate on dental biofilm of Streptococcus mutans: An in vitro study

BackgroundStreptococcus mutans (S. mutans) plays a major role in the formation of dental caries. The aim of this study was to examine the effect of the green tea polyphenol, epigallocatechin gallate (EGCG), on biofilm formation of S. mutans.MethodsFollowing exposure to increasing concentrations of EGCG, the planktonic growth was measured by optical density and the biofilm biomass was quantified by crystal violet staining. Exopolysaccharides (EPS) production was visualized by confocal scanning laser microscopy, and the bacterial DNA content was determined by quantitative polymerase chain reaction (qPCR). Gene expression of selected genes was analyzed by real time (RT)-qPCR and membrane potential was examined by flow cytometry.ResultsWe observed that EGCG inhibited in a dose-dependent manner both the planktonic growth and the biofilm formation of S. mutans. Significant reduction of S. mutans biofilm formation, DNA content, and EPS production was observed at 2.2–4.4 mg/ml EGCG. EGCG reduced the expression of gtfB, gtfC and ftf genes involved in EPS production, and the nox and sodA genes involved in the protection against oxidative stress. Moreover, EGCG caused an immediate change in membrane potential.ConclusionsEGCG, a natural polyphenol, has a significant inhibitory effect on S. mutans dental biofilm formation and EPS production, and thus might be a potential drug in preventing dental caries.

THE CORRELATION BETWEEN TERPENOID OF SARANG SEMUT (Myrmecodia pendans) WITH mRNA FRUCTOSYLTRANFERASE EXPRESSION OF Streptococcus mutans BIOFILM ATCC 25175

Background: Terpenoid isolate sarang semut (Myrmecodia pendans) is a natural material that has antibacterial power through the destruction of the cell membrane of Streptoccus mutans (S. mutans). Fructosiltransferase (ftf) is one of the extrapolisaccharides produced by S. mutans. The formation of Ftf is governed by the ftf gene. The aim of research was to know the correlation between terpenoid of Sarang semut administration with ftf mRNA expression of S. mutans biofilm ATCC 25175. Method: The study used mixed methods, namely descriptive explorative method to look at the isolation of ftf gene and ftf mRNA expression. This study began with making 1 day biofilm , then given terpenoid isolates of sarang semut and chlorhexidine. Next, isolation of ftf genes and ftf mRNA expression was carried out, then the differences in S. mutans ATCC 25175 mRNA ftf expression in sarang semut and chlorhexidine were given for 1 and 30 minutes using Real-Time PCR. Continued by looking at the relationship of the duration of administration of sarang semut terpenoids to the expression of S. mutans ATCC 25175 mRNA ftf biofilm. Statistical analysis used the t test with p-value <0.05 and Pearson. Results: The results showed that ftf gene with 130 bp length and ftf mRNA expression could be isolated from S. mutans biofilm ATCC 25175 which were given terpenoid isolates from sarang semut. The expression of sarang semut mRNA ftf and chlorhexidine did not have a significant difference for 1 minute (p = 0.0761), whereas in 30 minutes the ant nest had a higher mRNA expression (p = 0.0186). There is a relationship between the duration of administration of terpenoid isolates of sarang semut with S. mutans ATCC 25175 mRNA ftf expression (r2 = 0.614), with a medium correlation coefficient. Conclusion: The conclusion of the study were that sarang semut terpenoid had the same effect as chlorhexidine for 1 minute on S. mutans biofilms, whereas at 30 minutes it was different. There is a relationship between terpenoids of sarang semut isolates and S. mutans biofilm ATCC 25175 mRNA expression.

Advances in genomics and safety studies of <italic>Weissella confusa </italic>

Weissella confusa is an important species of lactic acid bacteria in the fermentation industry, primarily presents in fresh fruits, vegetables and fermented products. Nowadays, W. confusa is often used to improve the textural properties of food products in the food industry because it is rich in extracellular polysaccharides. W. confusa is resistant to bile salts and exhibits bacteriostatic effects; in addition, it can colonise the intestines of animals and humans, thereby fulfilling probiotic functions. However, previous studies have reported that W. confusa is possibly pathogenic on animals and humans, although case reports are rare. It also showed resistance to some antibiotics, especially vancomycin. The safety of this strain is still unclear and it is not included in the list of QPS (Qualified Presumption of Safety) by the EFSA (European Food Safety Authority) for the time being. With the completion of the first whole-genome sequence of W. confusa , LBAE C39-2, genome research on this species is emerging. Presently, complete genome sequences are available for 34 W. confusa . The genome sizes are between 1.44 and 2.38 Mb and the GC contents are between 43.90% and 45.08%. In this review, we first summarise the taxonomic profile and identification methods of W. confusa at the genomic level, emphasising its high similarity to W. cibaria at the phenotypic and phylogenetic levels. The identification methods include housekeeping genes, amplified fragment length polymorphism (AFTP) and specific gene clusters. Then, the important functional genes of the W. confusa genome are introduced, such as gtf genes and ftf genes, which have a close relationship to the characteristics of extracellular polysaccharides. Subsequently, heterologous expression of functional genes has also been shown through several previous studies, and bacterial plasmids play a crucial role in this process. Furthermore, some functional genes associated with the production of bacteriocin and amino acids were also found in W. confusa . We describe the current status of safety assessment for W. confusa , animal tests and in vitro toxicological experiments with further analysis of virulence and antibiotic resistance genes, which confirmed that some strains of W. confusa are nontoxic. Finally, this review summarises the current research on the genomics of W. confusa . More attention must be paid to identify more accurate specific marker genes for interspecific identification and phylogenetic analysis of evolutionary differences among W. confusa strains. In the future, genetic engineering technology, such as heterologous gene expression, should be used to improve the fermentation efficiency of W. confusa in industrial applications. On the basis of current studies on the safety of W. confusa , corresponding MIC-specific breakpoints of this species need to be designated. Further research should be established to identify additional virulence factors and resistance genes to lay a theoretical foundation for the application of W. confusa .

Glycoside hydrolase family 68 gene of halophilic archaeon Halalkalicoccus jeotgali B3T codes for an inulosucrase enzyme

Fructose polysaccharides and oligosaccharides are gaining strong importance due to their beneficial effects on human health and their applications in the food, feed and pharmaceutical industries. Fructan synthesis has been characterised in a wide variety of bacteria and plants. Metagenomic DNA sequence data suggests that several archaeal strains also contain fructosyltransferase (ftf) genes in their genome sequences (www.cazy.org), and thus may be able to synthesise fructans. The archaeal ftf genes are predicted to encode glycoside hydrolase family 68 enzymes. These genes are of strong interest in view of their potential role in the synthesis of novel prebiotic compounds. In the present studies, we have expressed putative ftf gene of an archaeon Halalkalicoccus jeotgali B3T in E. coli. The recombinant enzyme produced water-insoluble polysaccharide using sucrose as a substrate. Analysis by 13C and 1H NMR spectroscopy revealed the polysaccharide to be an inulin-type fructan polymer, depicting that the GH68 gene of H. jeotgali B3T codes for an inulosucrase enzyme. This is the first report on heterologous expression of an archaeal inulosucrase gene, which also provides conclusive evidence of the functional identity of this gene.

Lactobacillus rhamnosus biosurfactant inhibits biofilm formation and gene expression of caries-inducing Streptococcus mutans

It is cleared that some probiotic strains inhibit biofilm formation of oral bacteria, but its mechanisms are not clearly understood yet. It is proposed that one of the mechanisms can be biosurfactant production, a structurally diverse group of surface-active compounds synthesized by microorganisms. Hence, this study focused on the evaluation of the anti-biofilm and antiadhesive activities of the L. rhamnosus derived-biosurfactant against Streptococcus mutans and its effect on gtfB/C and ftf genes expression level. In this in vitro study Lactobacillus rhamnosus ATCC7469 overnight culture was used for biosurfactant production. The biosurfactant effect on the surface tension reduction was confirmed by drop collapse method. Chemical bonds in the biosurfactant were identified by Fourier transform infrared (FTIR). Anti-biofilm and antiadhesive activities of the biosurfactant were determined on glass slides and in 96-well culture plates, respectively. The effect of the biosurfactant on gtfB/C and ftf genes expression level was also investigated after biofilm formation, total RNA extraction, and reverse transcription by quantitative real-time reverse transcriptase polymerase chain reaction (PCR) assay (quantitative PCR). The data were assessed by one-way analysis of variance in the Tukey-Kramer postdeviation test for all pairs. P < 0.05 was considered statistically significant. The FTIR results of biosurfactant showed that it was protein rich. It also showed anti-biofilm formation activity on the glass slide and antiadhesive activity till 40% on microtiter plate wells. It also showed a significant reduction (P < 0.05) in gtfB/C and ftf genes expression level. L. rhamnosus-derived biosurfactant exhibits a significant inhibitory effect on biofilm formation ability of S. mutans due to downregulation of biofilm formation associated genes, gtfB/C and ftf. L. rhamnosus-derived biosurfactant with substantial antiadhesive activity is suitable candidates for use in new generations of microbial antiadhesive agents.

Effects of biosurfactant produced by Lactobacillus casei on gtfB, gtfC, and ftf gene expression level in S. mutans by real-time RT-PCR.

Background:The Streptococci are the pioneer strains in plaque formation and Streptococcus mutans are the main etiological agent of dental plaque and caries. In general, biofilm formation is a step-wise process, which begins by adhesion of planktonic cells to the surfaces. Evidences show that expression of glucosyltransferase B and C (gtfB and gtfC) and fructosyltransferase (ftf) genes play critical role in initial adhesion of S. mutans to the tooth surface which results in formation of dental plaques and consequently caries and other periodontal disease.Materials and Methods:The aim of this study was to determine the effect of biosurfactants produced by a probiotic strain; Lactobacillus casei (ATCC39392) on gene expression profile of gftB/C and tft of S. mutans (ATCC35668) using quantitative real-time PCR.Results:The application of the prepared biosurfactant caused dramatic down regulation of all the three genes under study. The reduction in gene expression was statistically highly significant (for gtfB, P > 0.0002; for gtfC, P > 0.0063, and for ftf, P > 0.0057).Conclusion:Considerable downregulation of all three genes in the presence of the prepared biosurfactant comparing to untreated controls is indicative of successful inhibition of influential genes in bacterial adhesion phenomena. In view of the importance of glucosyltransferase gene products for S.mutans attachment to the tooth surface which is the initial important step in biofilm production and dental caries, further research in this field may lead to an applicable alternative for successful with least adverse side effects in dental caries prevention.

Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (β-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.

Characterization of a Large Amount of Water-insoluble Polysaccharide Synthesis Streptococcus salivarius

The cariogenicity of a large amount of insoluble polysaccharide synthesis isolate from the human oral cavity on Mitis Salivarius agar was investigated. The isolate was identified as Streptococcus salivarius subsp. salivarius by genetical and biochemical analysis and was designated the NUM 6069 strain. NUM 6069 produced a large amount of water-insoluble polysaccharide from sucrose, and the plaque formation of the NUM 6069 strain was also strong ; however, no adherent or sticky plaques were observed. Acid production of NUM 6069 from glucose was rapid. The time taken to critical pH in the strain was faster than in the S. salivarius subsp. salivarius type strain JCM5707T and was similar to that in S. mutans. Expression of mRNA of the NUM 6069 ftf gene increased 42-fold compared to that of JCM5707T. All gtf genes, gtfJ, gtfK, gtfL and gtfM of NUM 6069 increased 2- to 6-fold compared to those of JCM5707T. In in vivo experiments, the caries scores and bacterial colonization of the NUM 6069 infected group were obviously decreased compared to those of the S. mutans infected group. These results reconfirm that sticky water-insoluble polysaccharides are important in the induction of dental caries.

Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two differentWeissella confusastrains from soya

Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and fructansucrases/fructosyltransferase (FTF) enzymes from genomic DNA of 'rare' Indonesian exopolysaccharide-producing LAB. From a total of 63 exopolysaccharide-producing LAB isolates obtained from foods, beverages and environmental samples, 18 isolates showing the most slimy and mucoid colony morphologies on sucrose were chosen for further study. By comparing bacterial growth on De Man, Rogosa and Sharpe (MRS)-sucrose with that on MRS-raffinose, and using the results of a previous PCR screening study with degenerate primer pairs targeting the conserved catalytic domain of GTFs, various strains were identified as producers of fructan (13), of glucan only (five) or as potential producers of both glucan and fructan (nine). Here, we report the characteristics of three gtf genes and one ftf gene obtained from Weissella confusa strains MBF8-1 and MBF8-2. Strain MBF8-1 harbored two putative gtf genes with high sequence similarity to GTFB of Lactobacillus reuteri 121 and GTF180 of L. reuteri 180, respectively. Strain MBF8-2 possessed single gtf and ftf genes with high sequence similarity to GTFKg3 of Lactobacillus fermentum Kg3 and DSRWC of Weissella cibaria, and FTF levansucrase of L. reuteri 121, respectively.

Characterization of Glycansucrase Activities from Leuconostoc Mesenteroides LM17 and URE 13 Strains

ABSTRACT14 original strains from traditional Bulgarian fermented food have been studied in order to characterize new orginal glycosyltransferase activities In order to characterize the whole spectrum of functional glycosyltransferases in the studied strains, the EPS produced in liquid media where first purified and hydrolysed and the global enzymatic activities measured by DNS assay. Activities present in the culural supernatant and cell associated were determined using SD_SPAGE and in situ Periodic Acid Schiffs staining after incubation with sucrose and raffinose. Among the studied strains Lm17 and URE 13 strains shown interesting activities. Three polymers bands were detected during in situ EPS production in presence of sucrose associated respectively to ∼300 kD (only in URE 13) and 180 kDa and 120 kDa (in both Lm 17 and URE13) proteins The 180KDa band could correspond to a glucansucrase activity of these strains. Analysis of zymogram incubated in raffinose as substrate and hydrolysis of the EPS produced in liquid culture are in good correlation with the hypothesis of a fructosyltransferase activity that could be attributed to a 120 kDa extracellular and cell-associated protein‥ GTF specific motifs (YG repeats from the Glucan Binding Domain) from Lm 17 and URE 13 strains were amplified by PCR and sequenced. They share a high percentage of identity to each other, and to the YG-repeats from ATCC 8293 and NRRL B-512F. A conserved motif from the catalytic core of FTF genes from Lm 17 and URE 13 strains was also shown to be very conserved between these two strains and with sequences of levansucrase from Ln. mesenteroides NRRL B-512F.

Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase)

In the present study, we have coupled detailed acceptor and donor substrate studies of the fructosyltransferase (FTF, levansucrase) (EC 2.4.1.162) from Bacillus subtilis NCIMB 11871, with a structural model of the substrate enzyme complex in order to investigate in detail the roles of the active site amino acids in the catalytic action of the enzyme and the scope and limitation of substrates. Therefore we have isolated the ftf gene, expressed in Escherichia coli, yielding a levansucrase. Consequently, detailed acceptor property effects in the fructosylation by systematic variation of glycoside acceptors with respect to the positions (2, 3, 4 and 6) of the hydroxyl groups from equatorial to axial have been studied for preparative scale production of new oligosaccharides. Such investigations provided mechanistic insights of the FTF reaction. The configuration and the presence of the C-2 and C-3 hydroxyl groups of the glucopyranoside derivatives either as substrates or acceptors have been identified to be rate limiting for the trans-fructosylation process. The rates are rationalized on the basis of the coordination of d-glycopyranoside residues in 4 C 1 conformation with a network of amino acids by Arg360, Tyr411, Glu342, Trp85, Asp247 and Arg246 stabilization of both acceptors and substrates. In addition we also describe the first FTF reaction, which catalyzes the β-(1→2)-fructosyl transfer to 2-OH of l-sugars ( l-glucose, l-rhamnose, l-galactose, l-fucose, l-xylose) presumably in a 1 C 4 conformation. In those conformations, the l-glycopyranosides are stabilized by the same hydrogen network. Structures of the acceptor products were determined by NMR and mass spectrometry analysis.

Development of a technique for multiple site-directed mutagenesis of the ftf gene of Streptococcus salivarius containing palindromic sequences.

Attempts at site-directed mutagenesis of the fructosyltransferase (ftf) gene of Streptococcus salivarius ATCC 25975 using standard protocols were unsuccessful and resulted in a series of deletions. These deletions appeared to commence at points within the ftf gene where there were palindromic sequences which were capable of forming closed loop structures that acted as terminators under the conditions of mutagenesis. To overcome this problem, two modified mutagenic techniques were developed. They made use of T4 DNA polymerase in conjunction with either T7 DNA polymerase at 37 degrees C or Vent DNA polymerase from Thermococcus litoralis at an elevated temperature. These methods eliminated the need for a single-stranded DNA template and allowed polymerisation through palindromic sequences to rapidly produce multiple site-directed mutations.

Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate.

Streptococcus mutans produces a number of extracellular sucrose-metabolizing enzymes that contribute to the ability of the organism to cause dental caries, including three glucosyltransferases, the products of the gtfB, gtfC and gtfD genes, and a fructosyltransferase, encoded by the ftf gene. To better understand the regulation of the expression of these genes under environmental conditions that more closely mimic those in dental plaque, two strains of S. mutans harbouring fusions of the gtfBC (SMS102) and ftf (SMS101) promoters to a chloramphenicol acetyltransferase (CAT) gene were examined in biofilms formed in vitro. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium. CAT specific activity in biofilm cells was measured at quasi-steady state or following additions of 25 mM sucrose or glucose, with or without pH control. After approximately 10 generations of biofilm growth, the ftf and gtfBC genes of S. mutans were found to be expressed at levels different from those reported for planktonic cells growing under otherwise similar conditions. The expression of these genes was induced by the addition of sucrose to the quasi-steady-state cultures. Expression of the gtfBC genes was influenced by environmental pH, since CAT specific activities in quasi-steady-state biofilms of strain SMS102 grown without pH control were twice those produced by cells grown with pH control. Moreover, addition of glucose to quasi-steady-state biofilms resulted in increased expression of the gtfBC-cat fusion, although the magnitude of the induction was less than that seen with sucrose. The effect of pH on ftf expression was negligible. A modest and transient induction of ftf was observed in biofilms pulsed with excess glucose and the kinetics and level of induction of ftf by excess carbohydrate were dependent on the pH of the biofilms. This study demonstrates that the type and amount of carbohydrate and the environmental pH have a major influence on transcription of the gtfBC and ftf genes when the organisms are growing in biofilms, and provides evidence for previously undisclosed regulatory circuits for exopolysaccharide gene expression in S. mutans.

Examination of streptococcal gene expression in the mammalian environment

Laboratory studies of bacterial gene expression have not allowed the investigation of in vivo factors crucial to pathogenesis. Recently, genetic tools have been used to identify in vivo-activated genes. These tools, however, have not allowed the measurement of specific expression levels of virulence-associated bacterial genes in vivo. This report describes a system, previously tested utilizing the fructosyltransferase (ftf) gene of Streptococcus mutans, that allows the measurement of activity of specific virulence-associated streptococcal genes while bacteria are living in the animal environment.

Development of a technique for multiple site-directed mutagenesis of the ftf gene of Streptococcus salivarius containing palindromic sequences

Attempts at site-directed mutagenesis of the fructosyltransferase ( ftf) gene of Streptococcus salivarius ATCC 25975 using standard protocols were unsuccessful and resulted in a series of deletions. These deletions appeared to commence at points within the ftf gene where there were palindromic sequences which were capable of forming closed loop structures that acted as terminators under the conditions of mutagenesis. To overcome this problem, two modified mutagenic techniques were developed. They made use of T4 DNA polymerase in conjunction with either T7 DNA polymerase at 37°C or Vent DNA polymerase from Thermococcus litoralis at an elevated temperature. These methods eliminated the need for a single-stranded DNA template and allowed polymerisation through palindromic sequences to rapidly produce multiple site-directed mutations.

Molecular and genetic analysis of multiple changes in the levels of production of virulence factors in a subcultured variant of Streptococcus mutans

We previously isolated a variant strain, Xc100L, which shows decreased production of a surface protein antigen with a molecular mass of 190 kDa (PAc), after repeated subculturing of Streptococcus mutans strain Xc [Koga T. et al. (1989) J. Gen. Microbiol. 135, 3199–3202]. In the present study, the levels of expression of the gtfB, gtfC, gtfD and ftf genes coding for polysaccharide-synthesizing enzymes in strain Xc100L were compared with those in strain Xc. Western blot analysis revealed multiple differences in the levels of production of these enzymes between these two strains. The amounts of the gtfB and gtfC gene products responsible for water-insoluble glucan synthesis in strain Xc100L were lower than those in strain Xc, whereas the amounts of the gtfD and ftf gene products responsible for water-soluble glucan synthesis and fructan synthesis, respectively, in strain Xc100L were higher than those in strain Xc. Northern blot analysis revealed that the amounts of the four enzymes and PAc produced by strain Xc100L reflected the relative amounts of mRNAs from the genes. The chloramphenicol acetyltransferase gene was fused with each of these five genes, and the transcriptional activity of each gene in strain Xc100L was quantitatively compared with that in strain Xc. The chloramphenicol acetyltransferase assay also indicated that the phenotypic differences between strain Xc and strain Xc100L were due to differences in the transcriptional activities of the virulence genes. No differences in the nucleotide sequences of the promoter regions of the gtfB, gtfC, gtfD, ftf and pac genes were found between strain Xc and strain Xc100L. It is possible that a factor(s) affecting the levels of transcription of the multiple virulence genes exists in S. mutans.

Genetic regulation of fructosyltransferase in Streptococcus mutans

Streptococcus mutans possesses several extracellular sucrose-metabolizing enzymes which have been implicated as important virulence factors in dental caries. This study was initiated to investigate the genetic regulation of one of these enzymes, the extracellular fructosyltransferase (Ftf). Fusions were constructed with the region upstream of the S. mutans GS5 Ftf gene (ftf) and a promoterless chloramphenicol acetyltransferase (CAT) gene. The fusions were integrated at a remote site in the chromosome, and transcriptional activity in response to the addition of various carbohydrates to the growth medium was measured. A significant increase in CAT activity was observed when glucose-grown cells were shifted to sucrose-containing medium. Sucrose-induced expression was repressed immediately upon addition of phosphoenolpyruvate phosphotransferase system sugars to the growth media. Deletion analysis of the ftf upstream region revealed that an inverted repeat structure was involved in the control of ftf expression in response to carbohydrate. However, the control of the level of ftf transcription appeared to involve a region distinct from that mediating carbohydrate regulation. CAT gene fusions also were constructed with the ftf upstream region from S. mutans V403, a fructan-hyperproducing strain which synthesizes increased levels of Ftf. Sequence analysis of the upstream ftf region in this strain revealed several nucleotide sequence changes which were associated with high-level ftf expression. Comparison of the GS5 and V403 ftf expression patterns suggested the presence of a trans-acting factor(s) involved in modulation of ftf expression in response to carbohydrate. This factor(s) was either absent or altered in V403, resulting in the inability of this organism to respond to the presence of carbohydrate. The sequences of the ftf regions from three additional fructan-hyperproducing strains were determined and compared with that of V403. Only one strain displayed nucleotide changes similar to those of V403. Two additional strains did not have these changes, suggesting that several mechanisms for up-regulation of ftf expression exist.

Construction of a model secretion system for oral streptococci

A DNA fragment corresponding to the secretory domain from the Streptococcus mutans GS-5 gtfB gene, which encodes the putative 38-amino-acid signal peptide of the glucosyltransferase I (GTF-I) enzyme product, has been constructed. This fragment was fused with the alpha-amylase structural gene from alkalophilic Bacillus sp. strain 707. This hybrid gene as well as the intact amylase gene were introduced into an Escherichia coli-streptococcus shuttle vector consisting of three components: the E. coli replicon p15Aori from pACYC177, an erythromycin resistance gene from pAM beta-1, and the streptococcal replicon from pVA838. Transformation of the oral noncariogenic bacterium Streptococcus gordonii with the chimeric plasmid harboring the hybrid amylase gene resulted in strong extracellular amylase production. By contrast, transformants containing the intact amylase gene exhibited only trace amounts of amylase activity in culture fluids. Since the two signal peptide structures of the GTF-I enzyme and the Bacillus amylase are distinct from each other, these differences might result from the inability of S. gordonii to correctly process the Bacillus signal peptide. Furthermore, culture fluids from transformants of S. mutans as well as Streptococcus milleri harboring the hybrid amylase gene showed only weak amylase activity. Deletion of the gtfB, gtfC, or ftf gene from S. mutans GS-5 did not increase amylase secretion following transformation with the hybrid amylase gene. These results suggest that in contrast to S. gordonii, the inability of S. mutans and S. milleri to secrete hybrid amylase molecules could result from incorrect interaction of the secretory components of these organisms with amylase precursor molecules.

DNA probes for detection of cariogenic Streptococcus mutans.

Streptococcus mutans has been identified as the principal etiological agent in human dental caries. Therefore, the specificity and sensitivity of 3 potential gene probes derived from S. mutans GS5 have been examined as potential reagents for detecting the cariogenic bacteria. The gene probes derived from the cloned gtfB, gtfD and ftf genes were examined in Southern blots with a panel of representative oral bacteria. The gtfB and ftf genes were apparently specific for S. mutans under high-stringency hybridization conditions. However, the gtfD gene fragment did cross-hybridize with the DNA from other mutans streptococci. These results suggest that gtfB and ftf gene fragments may be used as specific probes for S. mutans.