操纵子comCDE调控肺炎链球菌转化的分子机制研究

Investigation on the molecular mechanism of operon comCDE regulating the transformation of Streptococcus pneumoniae

目的:探讨操纵子comCDE对调控肺炎链球菌转化的分子机制。方法:将D39野生菌株与comE基因缺陷菌株(△comE)分别用感受态刺激因子(Competence-stimulating peptide,CSP)诱导转化,记录不同时间点的转化率,并采用FQ-PCR技术检测其comE的mRNA表达水平,并通过双向电泳及图像分析比较CSP诱导后不同时间点的蛋白质表达谱差异及野生菌株与△comE菌株的蛋白质表达谱差异。结果:D39菌株在CSP诱导后不同时间点都能发生转化,其中0 min时转化率最高,其comE的mRNA表达量在CSP诱导10 min后表达显著增加,20 min时迅速回落(P<0.05)。△comE菌株不能被CSP诱导发生转化。蛋白质组学研究显示,△comE菌株有6个蛋白较D39菌株的表达量显著增加;CSP诱导10 min时有6个蛋白表达显著增加,而在20 min时又迅速回落;二者有2个蛋白相同;有1个蛋白在加入CSP后表达显著降低。结论:基因comE的存在是CSP诱导转化必需的,CSP诱导转化可能有非comE依赖途径,而comE可能有除转化以外调控功能,且不依赖于CSP。

Objective： To investigate the molecular mechanism of operon comCDE regulating S.pn transformation. Methods：The wild S. pn strains and their comE-defective derivations （△comE） were subjected to CSP-induced transformation. Transformation rates were calculated at different time points, and the mRNA expression levels of comE were detected by FQ PCR.The proteins of different expression among different strains were detected by two-dimensional electrophoresis and image analysis. Results ： D39 could undergo transformation at 0,10 min and 20 min after CSP was added, and the transformation rate at 0min was the highest. After 10 minutes induction with CSP the mRNA level of comE was significantly higher than that of basal level（P〈0.05 ）, and at 20minutes the mRNA level of comE was significantly decreased. For comE -defective strain,the transformation rate was 0. proteomics analysis displayed that six proteins were expressed significantly higher in △comE strains than that in D39 wild stains. The expression of six proteins increased significantly at 10 minutes after CSP was added, but reduced quickly at 20 minutes after CSP was added. The expression of two proteins were changed in both cases. The expression of another one protein was significantly decreased after CSP was added. Conclusion： ComE is an essential gene for CSP-induced transformation, CSP probably induces some transformation related genes by comE-independent pathway. And comE probably has other fuctions than regulatedtransformation, and it might be CSP-independent.