Charactorization of expression mechanisms for a novel glucan-binding protein C in S.mutans

变异链球菌中新型葡聚糖结合蛋白 C 表达机制的表征

基本信息

批准号：
09671869
负责人：
SATO Yutaka
金额：
$ 1.98万
依托单位：
TOKYO DENTAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

I have identified the gbpC gene encoding the glucan-binding protein C which is involved in dextran (glucan)-dependent aggregation (ddag) of Streptococcus mutans by random mutagenesis. It was generally thought that S.mutans does not exhibit this ddag phenotype, since this phenotype was appeared only under certain stress conditions. I have introduced random mutation into S.mutans with one of the integration vectors pVA891 and have screened the mutants for the ddag- negative phenotype to isolate genes involved in this property. Insertion of pVA891 containing a Sau3AI-digested host DNA fragment into the chromosome occurs following homologous recombination via a Campbell-like mechanism. However, most of mutants that we obtained appeared to have resulted from chromosomal rearrangements as well as pVA891 insertion. I found that several dozen mutants exhibiting the non-aggregation phenotype harbored the intact gbpC gene and that these mutants possessed a large and characteristic duplication of … More a region of the chromosome which was responsible for the phenotype. Based upon characterization of these duplications, I developed a strategy to introduce a duplication into any specific region of the chromosome of these organisms. The 690 bp gene responsible for the ddag- phenotype was identified within a 60 kb region by observing ddag (positive or negative) phenotypes of successively constructed specific duplication mutants. The gene was one of the response-regulator gene of the Two-component regulatory systems, and was designated as the gene gcrR.I also found a new phenomenon that cells repeatedly cultured in the presence of xylitol evolved into those exhibiting the elevated dextran-dependent aggregation phenotype. This phenotype was confirmed to result from expression of the gbpC gene by constructing of a S.mutans isogenic mutant carrying the gbpC : : lacZ fusion gene. I found that gbpC expression of the such cells was elevated 20-fold. It was of interest that these cells also exhibited decreased adhesion to glass surfaces when grown with shaking in the presence of sucrose. This may be one of the ways by which some populations of S.mutans are removed from dental plaques. Less

我已经鉴定出编码与链球菌突变体的葡聚糖（葡萄糖）依赖性聚集（葡萄糖）依赖性聚集（DDAG）的GBPC基因。人们普遍认为，S.Mutan不存在此DDAG表型，因为该表型仅在某些应力条件下出现。我已经将随机突变引入了S.Mutans，其中一个积分向量PVA891，并筛选了DDAG-阴性表型的突变体，以分离涉及该特性的基因。通过坎贝尔样机制在同源重组后，将插入含有SAU3AI消化的宿主DNA片段的PVA891插入染色体中。但是，我们获得的大多数突变体似乎是由于染色体重排以及PVA891插入而产生的。我发现几个表现出非聚集表型的十二个突变体具有完整的GBPC基因，并且这些突变体假定了……染色体的一个较大且具有特征性的重复，是染色体的一个区域，负责表型。根据这些重复的特征，我制定了一种策略，将重复的重复介绍给这些生物体的染色体的任何特定区域。通过观察成功构建特定重复突变体的DDAG（正或负）表型，通过观察DDAG（正或负）表型来确定负责DDAG-表型的690 bp基因。该基因是两个组分调节系统的响应调节基因之一，被设计为基因GCRR。我还发现了一种新现象，即在木糖醇存在的存在中反复培养的细胞演变为表现出升高的右右右旋丙烷依赖性聚集表型。通过构建携带GBPC ：： LACZ融合基因的S.Mutans同源性突变体的构造，该表型是由GBPC基因表达的。发现此类细胞的GBPC表达升高了20倍。令人感兴趣的是，这些细胞在蔗糖存在下摇动时也表现出对玻璃表面的粘合剂。这可能是从牙菌斑中删除一些S.Mutan人群的方式之一。较少的