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

我已经通过随机突变鉴定了编码葡聚糖结合蛋白C的gbpc基因，该基因参与了变形链球菌的葡聚糖依赖聚集(DDAG)。人们普遍认为变形链球菌不会表现出这种ddag表型，因为这种表型只有在特定的胁迫条件下才会出现。我已经用整合载体pVA891将随机突变引入变形链球菌，并筛选了ddag阴性表型的突变体，以分离与这一特性相关的基因。含有Sau3AI消化的宿主DNA片段的pVA891在同源重组后通过类似Campbell的机制插入到染色体中。然而，我们获得的大多数突变体似乎是由染色体重排和pVA891插入引起的。我发现数十个表现为非聚集表型的突变体含有完整的gbpc基因，并且这些突变体具有大量的、具有特征的…复制。更多的是染色体上负责表型的区域。根据这些复制的特征，我制定了一种策略，将复制引入这些生物染色体的任何特定区域。通过观察连续构建的特异重复突变体的ddag表型(阳性或阴性)，在60kb的范围内鉴定了与ddag表型有关的690bp基因。该基因是双组分调控系统的反应调节基因之一，被命名为基因gcrR。我还发现了一个新的现象，即在木糖醇存在的情况下重复培养的细胞进化成那些表现出升高的葡聚糖依赖聚集表型的细胞。通过构建携带gbpc：：lacZ融合基因的变形链球菌等基因突变体，证实该表型是gbpc基因表达的结果。我发现这类细胞的gbpc表达增加了20倍。有趣的是，当在蔗糖存在的情况下摇动培养时，这些细胞对玻璃表面的粘附性也降低了。这可能是从牙菌斑中去除一些变形链球菌种群的方法之一。较少

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