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

我已经通过随机诱变鉴定了编码葡聚糖结合蛋白C的gbpC基因，该蛋白C参与变形链球菌的葡聚糖（葡聚糖）依赖性聚集（ddag）。一般认为变形链球菌不表现出这种ddag表型，因为这种表型仅在某些胁迫条件下出现。我用整合载体pVA 891将随机突变导入变异链球菌，并筛选ddag阴性表型的突变体，以分离与该特性相关的基因。将含有Sau 3AI消化的宿主DNA片段的pVA 891插入染色体中，在同源重组后通过Campbell样机制发生。然而，我们获得的大多数突变体似乎是由于染色体重排以及pVA 891插入引起的。我发现，几十个表现出非聚集表型的突变体含有完整的gbpC基因，这些突变体具有一个大的和特征性的重复， 关于我们 染色体上负责表现型的区域。基于这些重复的特征，我开发了一种策略，将重复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性重复突变体的ddag（阳性或阴性）表型，在60 kb区域内鉴定了负责ddag-表型的690 bp基因。该基因是双组分调节系统中的一个反应调节基因，被命名为gcrR基因。我还发现了一个新的现象，即在木糖醇存在下反复培养的细胞进化为表现出葡聚糖依赖性聚集表型升高的细胞。通过构建携带gbpC：：lacZ融合基因的变形链球菌同基因突变体，证实该表型是由gbpC基因表达引起的。我发现这些细胞的gbpC表达量提高了20倍。令人感兴趣的是，当在蔗糖存在下摇动生长时，这些细胞也表现出对玻璃表面的粘附性降低。这可能是一些变形链球菌从牙菌斑中去除的方式之一。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