肺炎链球菌MarR家族蛋白SPD_0379通过调节基因rafX的表达调控磷壁酸的合成

Pneumococcal MarR family protein SPD_0379 regulating the expression of gene rafX and the biosynthesis of teichoic acids

摘要:

目的：明确肺炎链球菌MarR家族蛋白SPD_0379对细菌磷壁酸（teichoic acids，TA）的调控作用。方法：通过凝胶阻滞实验验证SPD_0379蛋白与rafX基因启动子序列的结合，通过LacZ报告基因观察细菌在spd_0379基因缺陷后的rafX基因表达变化，通过斑点杂交、ELISA和流式细胞术检测细菌磷壁酸的含量变化，通过生长曲线分析揭示SPD_0379蛋白在细菌生长中的作用。结果：SPD_0379蛋白与rafX基因启动子序列发生特异性结合，spd_0379基因缺陷后，细菌的rafX基因表达上调（t=10.010，P=0.001），免疫斑点与ELISA均显示磷壁酸含量明显升高（t=5.848，P=0.004；t=4.653，P=0.001），且缺陷菌的自溶较野生菌明显提前。结论：SPD_0379蛋白负调控rafX基因的表达，抑制细菌磷壁酸的合成，参与细菌自溶的调控。

Abstract:

Objective：To determine the regulative effect of Streptococcus pneumoniae MarR family protein SPD_0379 on the biosyn－thesis ofteichoic acid. Methods：EMSA was employed to analyze whether protein SPD_0379 bound to rafX gene promoter sequence. The change of bacterial rafX gene expression was observed by LacZ reporter gene. Dot blot，ELISA techniques and flow cytometry analysis were implemented to reveal the change of the concentration of teichoic acid. Results：A specific binding between proteins SPD_0379 and rafX gene promoter sequence was observed. After spd_0379 gene deficiency，the expressions of bacterial rafX gene were up-regulated（t=10.010，P=0.001），with the concentration of teichoic acid significantly up-regulated in D39Δspd_0379 detected by both dot blotting and ELISA（t=5.848，P=0.004；t=4.653，P=0.001），and the autolysis of spd_0379 deficient strains were signifi－cantly earlier than wide type D39. Conclusion：Protein SPD_0379 regulates the expression of rafX gene，inhibits the synthesis of bacterial teichoic acid，and is involved in the regulation of bacterial autolysis.