The regulation of Streptococcus mutans glucan-binding protein A expression

The S. mutans GBP-A is hypothesized to be constitutively expressed and to contribute to the sucrose-dependent colonization of S. mutans. To investigate GBP-A expression, a reporter gene encoding chloramphenicol acetyltransferase (CAT) was placed downstream of the gbpA promoter and CAT activity was measured under conditions that would be associated with the sucrose-dependent colonization of S. mutans. Expression of GBP-A was optimal under anaerobiosis and neutral pH conditions, and correlated with optimal growth. The addition of sucrose to the growth medium did not elevate the expression of GBP-A.     

Construction of Streptococcus mutans glucosyltransferase mutants utilizing a cloned gene fragment

Abstract An internal 1.6-kb BAM HI DNA fragment of the previously cloned Streptococcus mutans GS-5 gtfB gene was utilized to construct plasmids capable of insertion in toto into the GS-5 chromosome. The resultant insertions primarily yielded mutants defective in glucosyltransferase-I activity. These mutants were also defective in sucrose-dependent colonization of smooth surfaces.     

替代失活法构建变形链球菌LuxS基因缺陷株

目的 LuxS基因是变形链球菌生物膜早期形成过程中的关键基因,构建该基因的缺陷菌。方法采用长臂同源多聚酶链反应（LFH-PCR）方法构建含红霉素耐药基因片段的LuxS基因上、下游同源序列的连接片段,转化到变形链球菌中,在红霉素的平板上筛选缺陷菌株,并采用PCR鉴定。结果对变形链球菌LuxS基因缺陷菌株进行PCR和DNA序列测定分析证实构建成功。结论成功构建出变形链球菌LuxS基因的缺陷菌株,为后期针对变形链球菌LuxS基因的相关研究奠定基础。     

变形链球菌致龋抗原基因在乳酸乳球菌的表达

目的 克隆变形链球菌葡聚糖结合蛋白B(GbpB)功能区的基因片段,并在乳酸乳球菌中表达.方法 在实验中利用了分子克隆技术构建携带GbpB基因的重组原核表达质粒pNI1,将重组质粒转化乳酸乳球菌YF02株,筛选鉴定阳性菌落,诱导表达的GbpB蛋白用SDS-PAGE进行鉴定.结果 成功克隆了GbpB功能区的基因片段,并在乳酸乳球菌中得到其融合蛋白的表达.结论 利用分子生物学技术能够成功克隆GbpB功能区基因并获得乳酸乳球菌融合蛋白的表达,为后续研究奠定了基础.     

Construction of scrA::lacZ gene fusions to investigate regulation of the sucrose PTS of Streptococcus mutans

The scrA gene coding for sucrose EnzymeII of the phosphoenolpyruvate dependent phosphotransferase system previously isolated from Streptococcus mutans was fused in vitro to the promoterless lacZ' gene to monitor the expression of the scrA gene. The scrA::lacZ gene fusion was introduced back into S. mutans GS-5IS3 by two independent transformation procedures involving either linear or plasmid DNA to produce both scrA and scrA+ mutants. These mutants should prove useful for analyzing the regulation of sucrose transport in S. mutans.     

电转化法构建变形链球菌UA159 comD缺陷菌株

目的构建变形链球菌UAl59密度感应相关的comD基因缺陷菌株,为进一步研究该基因功能做准备。方法根据同源重组原理,利用变形链球菌UAl59comD基因同源重组DNA片段,采用电击转化方法获取转化菌落,通过形态学观察、生化特性检测、PCR及测序、RT-PCR对缺陷菌进行鉴定。结果在含有红霉素（10μg/mL）的琼脂平板上出现转化菌落,鉴定结果均显示转化菌为comD缺陷菌。结论成功构建了变形链球菌UA159comD基因缺陷菌株。     

Complete nucleotide sequence of the sr gene from Streptococcus mutans OMZ 175

The nucleotide sequence encoding the SR protein of Streptococcus mutans OMZ 175 (serotype f) has been determined. The sr gene consists of 4667 bp and codes for a 171177 Da protein. Comparison of the inferred amino acid sequence with the one of PAc antigen from S. mutans MT 8148 (serotype c) indicates a 88% conservation of amino acid residues which reflects the close relatedness of both proteins. Major differences in amino acid composition are located at the C-terminal part of the sequence where only 298 amino acids of the terminal 420 are conserved.     

长臂同源多聚酶链反应法构建变形链球菌comD基因同源重组DNA片段

目的构建变形链球菌UAl59密度感应相关的comD基因同源重组DNA片段,为利用同源重组原理构建基因功能丧失菌株做准备。方法通过NCBI基因数据库获取变形链球菌的DNA序列,利用聚合酶链反应技术分别扩增变形链球菌UA159comD基因上、下游片段及抗红霉素基因片段,再通过长臂同源多聚酶链反应将这3个片段连接起来,形成同源重组DNA片段。结果经过PCR反应和琼脂电泳分析,得到了一个碱基数为3个单片段总和的连接片段,测序结果显示连接片段为预期的comD同源重组片段。结论成功构建了变形链球菌UA159comD基因同源重组DNA片段,可直接用于细菌转化构建comD基因缺陷菌株。     

Genetic variation in comC, the gene encoding competence-stimulating peptide (CSP) in Streptococcus mutans

The genetic variability in comC , the gene encoding the quorum-sensing molecule, competence-stimulating peptide (CSP) in Streptococcus mutans is reported. Seven comC alleles encoding three distinct mature CSPs were identified among 36 geographically diverse strains, although, compared with Streptococcus pneumoniae , the amount of predicted amino acid sequence variation is low. In agreement with other studies, significant variation was found in the natural competence for DNA uptake in these strains. However, there was no correlation between the CSP genotype and the ability to transform these strains. Representative strains encoding each of the CSP variants became competent in response to synthetic CSPs of each type. Therefore, in contrast to S. pneumoniae , comC alleles in S. mutans are functionally equivalent and there is no evidence of pherotype specificity.     

Cloning,Nucleotide Sequence,and Disruption of Streptococcus mutans Glutathione Reductase Gene (gor)

We cloned and sequenced the glutathione reductase gene (gor) of an oxygen-tolerant Streptococcus mutans, and constructed a gor-disruption mutant by homologous recombination. The gor gene consisted of 1,350 bp, coding for a protein of 450 amino acid residues. The deduced amino acid sequence of the S. mutans gor gene product showed extensive similarity with those of glutathione reductases from prokaryotes and eukaryotes. Although the mutant could grow aerobically, it showed no growth in the presence of 2 mM diamide, a thiol-specific oxidant. In contrast, growth of the wild-type strain was not significantly inhibited by 2 mM diamide, and glutathione reductase activity was increased 2.2-fold under these conditions. In addition, the level of glutathione reductase activity in the wild-type strain was increased 3.6-fold upon exposure to air, and the elevated level of the enzyme was retained throughout the aerobic growth. Thus, glutathione reductase may be important in protection of S. mutans against oxidative stress.     

ATP-dependent protein kinase activities in the oral pathogen Streptococcus mutans

ATP-dependent protein kinase activities were detected in both membrane and cytoplasmic fractions from the oral pathogen Streptococcus mutans. Different polypeptides were phosphorylated by endogenous kinase(s) in the two fractions. In membranes, five phosphoproteins were detected with apparent masses of 82, 37, 22, 12, and 10 kilodaltons (KD). In cytoplasm, two major acid-stable phosphoproteins were found. One was identified as HPr of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), while the other had an apparent mass of 61 KD. Both of these proteins were phosphorylated on a seryl residue. Fructose 1,6-bisphosphate stimulated phosphorylation of HPr by the kinase and inhibited phosphorylation of the 61-KD protein. In contrast, fructose 1-phosphate, 2-phosphoglycerate, 3-phosphoglycerate, and dihydroxyacetone phosphate inhibited phosphorylation of HPr and stimulated phosphorylation of the 61-KD protein. Several other glycolytic intermediates as well as inorganic phosphate inhibited phosphorylation of either or both proteins. Preincubation of cytoplasm with PEP prior to incubation with ATP reduced the amount of phospho-(seryl)-HPr formed, but not that of the 61-KD phosphoprotein. The latter protein has not yet been identified but has properties that suggest that it may be the protein kinase itself. These results provide evidence for one or more soluble ATP-dependent protein kinases in S mutans that are regulated by glycolytic intermediates and that may play a role in the modulation of carbohydrate uptake and metabolism in this organism. A model for feedback regulation of sugar transport in S mutans, mediated by an allosterically regulated kinase, is presented.     

Application of in vitro mutagenesis to identify the gene responsible for cold agglutination phenotype of Streptococcus mutans

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold-agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold-agglutination-negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR-based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N-terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.     

Cloning of the phospho-β-galactosidase gene in Escherichia coli from lactose-negative mutants of Streptococcus mutans isolated following random mutagenesis with plasmid pVA891 clone banks

Abstract In order to mutagenize Streptococcus mutans a marker rescue plasmid, pVA891, was employed. The plasmid was ligated with Sau 3AI digested chromosomal DNA fragments from S. mutans GS-5IS3 and the resultant plasmids were amplified in Escherichia coli . These plasmids were then randomly integrated into the chromosome of strain GS-5IS3 following transformation. Lactose-negative transformants were isolated as white colonies on lactose-BTR-Xgal agar plates containing erythromycin. Six lactose-negative mutants representing three different chromosomal sites of integration were isolated from about eight thousand transformants. Mutant chromosomal DNA fragments flanking the plasmids were recovered by a marker-rescue method in E. coli and exhibited phospho-β-galactosidase activity.     

变形链球菌F-ATPase启动子荧光蛋白载体的构建及表达

目的构建由质子移位膜ATP酶(membrane-bound proton-translocating ATPase,F-ATPase)启动子启动的绿色荧光蛋白报告基因穿梭表达载体,观察其在大肠埃希菌中的表达同时鉴定表达产物。方法以变形链球菌(UA159)基因组为模板,扩增F-ATPase启动子片段,构建由F-ATPase启动子启动的绿色荧光表达载体pFgfp,酶切F-ATPase启动子及绿色荧光蛋白编码基因,连接到穿梭质粒pDL276,构建重组载体pLFgfp。结果重组质粒pLFgfp酶切及基因序列分析证实目的片段成功插入,重组载体转化后的大肠埃希菌有绿色荧光蛋白的表达,并能随着细菌传代继续表达。结论 F-ATPase启动子启动的绿色荧光蛋白穿梭表达载体pLFgfp构建成功,为研究生物膜环境中耐酸菌F-ATPase毒力因子的表达奠定基础。     

Comparative study of the effect of green and roasted water extracts of mate (Ilex paraguariensis) on glucosyltransferase activity of Streptococcus mutans

Glucosyltransferase (GTF) plays an important role in the development of dental caries. This study was carried out to compare the efficiency of green mate (GM) and roasted mate (RM) water extracts, drinks rich in polyphenolic compounds consumed in the subtropical region of South America, on the extracellular GTF activity from Streptococcus mutans. The RM extract exhibited a greater inhibitory effect (IC₅₀ of 10?mg/mL) despite presenting lower polyphenolic content. The kinetic analysis showed that there were significant differences (P?<?0.05) between the extracts with respect to the values for K_m and K_i, whereas the values for V_max were the same, implying the competitive nature of GTF inhibition. GTF activity was also measured using selected polyphenols as inhibitors, and the most effective inhibitors were rutin and caffeoylshikimic acid. The characterization of the extracts by ESI-MS and UPLC-MS showed that the compounds formed during roasting, possibly shikimic acid derivatives and other unindentified compounds formed by the Maillard reaction, appeared to contribute to the inhibition of GTF activity.     

Identification of A New Gene Responsible for the Oxygen Tolerance in Aerobic Life of Streptococcus mutans

Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.     

Protective immunity in rats by intranasal immunization with Streptococcus mutans glucan-binding protein D encapsulated into chitosan-coated poly(lactic-co-glycolic acid) microspheres

Recombinant Streptococcus mutans glucan-binding protein D (rGbpD) was incorporated into poly(lactic-co-glycolic acid) (PLGA) microspheres which then were surface-coated with chitosan. The microspheres, with a mean diameter of ca. 1.8 μm, were intranasally administered in rats. There were elevated salivary immunoglobulin A and serum immunoglobulin G antibody responses to rGbpD, as well as lower molar caries scores in immunized animals as compared to sham immunized ones. The chitosan-coated PLGA microspheres are thus potentially useful for antigen delivery in dental caries vaccination.