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Investigation for regulatory mechanisms of GcrR protein for S.mutans gbp Cexpression

GcrR蛋白对变异链球菌gbp C表达的调控机制研究

基本信息

批准号：
13671952
负责人：
SATO Yutaka
金额：
$ 1.92万
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：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

项目摘要

The auther has investigated the regulatory mechanisms of the gcrR gene as a response regulator of two-component regulatory systems for gbpC gene expression. Signal transduction of response regulators is performed by their phosphoryl transfer from their counterparts, sensor-transmitter proteins. The 53rd amino acid aspartate of GcrR protein that is expected as the signal receptor site was changed to aranine by site-directed mutagenesis, and this mutation was introduced into S.mutans chromosome. Albeit this altered GcrR protein in this mutant should not be phosphorylated, this mutant exhibited the same dextran-dependent aggregation phenotype as the wild type strain. Therefore, the authers concluded that GcrR protein does not act as a response regulator but act as a represser. GbpC protein is a member of wall-anchored proteins tethered to peptidoglycan layer. The authers confirmed GbpC expression at cell wall by the Western blot analysis, and the S.mutans cells were indicated to be able t … More o bind to immobilized glucan mediated by the GbpC protein using the BiaCore system. Cell wall anchoring of the protein is mediated by the sortase enzyme. Sortase negative mutant of S.mutans did not exhibit dextran-dependent aggregation phenotype as expected. A nonsense mutation of the gbpC gene was initially found in strain GS-5 and this lead to detection of single nucleotide polymorphisms in the gbpC gene among S.mutans strains. Both conserved and polymorphic regions of the gbpC gene will be useful for an estimation of functional domains of GbpC protein and identification of S.mutans strains. A gbpC gene homologue from Streptococcus macacae was successfully identified by PCR method with primers designed from this conserved GbpC protein information. The authers found another aggregation phenotype designated as cold agglutination. The collagen-binding adhesin gene was identified from some strains of S.mutans by random mutagenesis method using in vitro transposition of Himarl transposon. This is the first report that demonstrates a collagen-binding adhesin from viridans streptococci in human oral indigenous flora. Less

作者研究了 gcrR 基因作为 gbpC 基因表达双组分调控系统的响应调控因子的调控机制。反应调节剂的信号转导是通过其对应物（传感器-递质蛋白）的磷酰基转移来进行的。通过定点诱变将GcrR蛋白中预期作为信号受体位点的第53个氨基酸天冬氨酸改变为丙氨酸，并将该突变引入到变形链球菌染色体中。尽管该突变体中的这种改变的 GcrR 蛋白不应被磷酸化，但该突变体表现出与野生型菌株相同的葡聚糖依赖性聚集表型。因此，作者得出结论，GcrR 蛋白不充当反应调节剂，而是充当阻抑剂。 GbpC 蛋白是束缚在肽聚糖层上的壁锚蛋白的成员。作者通过蛋白质印迹分析证实了细胞壁上的 GbpC 表达，并且使用 BiaCore 系统表明变形链球菌细胞能够与 GbpC 蛋白介导的固定化葡聚糖结合。蛋白质的细胞壁锚定是由分选酶介导的。变形链球菌的分选酶阴性突变体没有表现出预期的葡聚糖依赖性聚集表型。 gbpC 基因的无义突变最初在菌株 GS-5 中发现，这导致在变形链球菌菌株中检测到 gbpC 基因的单核苷酸多态性。 gbpC 基因的保守区和多态性区域都将有助于估计 GbpC 蛋白的功能域和鉴定变形链球菌菌株。根据该保守的GbpC蛋白信息设计引物，通过PCR方法成功鉴定了来自猕猴链球菌的gbpC基因同源物。作者发现了另一种聚集表型，称为冷凝集。利用Himarl转座子的体外转座随机诱变方法从一些变形链球菌菌株中鉴定出胶原结合粘附素基因。这是第一份证明人类口腔本土菌群中草绿色链球菌的胶原蛋白结合粘附素的报告。较少的