猪链球菌MnmE蛋白功能的初步研究

Preliminary Study on the Function of MnmE Protein in Streptococcus suis

为了解析猪链球菌中MnmE蛋白的生物学功能,通过同源重组技术获得了mnmE基因缺失菌株,采用活菌计数、高效液相色谱、质谱分析、荧光定量PCR等技术,鉴定MnmE蛋白在猪链球菌中的主要功能。结果显示,与野生菌株(SC19)相比,mnmE基因缺失菌株(△mnmE)在酸性条件pH 4.5下的存活能力显著下降;氨基酸靶向代谢组学共检测到23种氨基酸及其衍生物,其中△mnmE缺失菌株中有17种氨基酸具有显著差异(P<0.05,倍数差异>1.2),尤其是精氨酸上调4.8倍,丝氨酸上调2.9倍。荧光定量PCR发现△mnmE缺失菌株中与环境适应性相关的精氨酸分解代谢基因ArcA下调表达显著,丝氨酸合成酶SerB上调表达显著。初步鉴定了MnmE蛋白与猪链球菌对酸性环境适应性相关,MnmE蛋白缺失后,精氨酸分解代谢基因下调表达导致精氨酸在菌体里积累,不能被分解成CO_(2)、ATP、NH_(3),使得细菌代谢紊乱、适应性减弱。

In order to understand the biological function of MnmE protein in Streptococcus suis,the mnmE gene deleted strain was obtained by homologous recombination,the major functions of MnmE protein in Streptococcus suis were identified by counting viable bacteria,high-performance liquid chromatography analysis,mass spectrometry and fluorescence quantitative PCR.The results showed that compared with the wild strain(SC19),the survival ability of the mnmE(△mnmE)was significantly decreased at pH 4.5,and a total of 23 amino acids and their derivatives were detected by amino acid targeted metabolomics,among them,17 amino acids in△mnmE deletion strains showed significant difference(P<0.05,fold difference>1.2),particularly arginine was up-regulated 4.8-fold and serine was up-regulated 2.9-fold.RT-PCR showed that the expression of arginine catabolic gene ArcA,which is related to environmental adaptability,was significantly down-regulated and the expression of serine synthetase SerB was significantly up-regulated in mnmE deficient strains.In this study,we identified that MnmE protein was involved in acid environment adaptation of Streptococcus suis.After mnmE gene was deleted,arginine catabolism related gene arcA was down-regulated,serine synthetase serB was up-regulated,and arginine was accumulated in Streptococcus suis,that arginine could not be decomposed into CO_(2),ATP,NH_(3),leading to metabolic disorders and weakened adaptability in bacteria.