敲除aceE基因对猪链球菌丙酮酸代谢的影响

Effect of ace E gene knockout on pyruvate metabolism of Streptococcus suis

【目的】探究缺失编码丙酮酸脱氢酶蛋白的aceE基因对猪链球菌生长特性、三羧酸循环和丙酮酸代谢的影响。【方法】通过测量菌液的OD600值,绘制野生型菌株与aceE基因缺失突变株的生长曲线;利用试剂盒测定三羧酸循环和丙酮酸代谢旁路中乙酰CoA、琥珀酸CoA、延胡索酸、草酰乙酸、丙酮酸、乳酸和ATP的含量,通过荧光定量qRT-PCR确定柠檬酸合酶基因、苹果酸脱氢酶基因、琥珀酸脱氢酶基因、异柠檬酸脱氢酶基因、丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因的表达水平。【结果】与野生株相比,菌株ΔaceE在平台期OD600值下降;添加1 g/L乙酸盐能够显著提升菌株ΔaceE平台期OD600值。菌株ΔaceE的丙酮酸含量上升,ATP含量下降;三羧酸循环代谢中乙酰CoA、琥珀酸CoA、延胡索酸含量降低;柠檬酸合酶基因和苹果酸脱氢酶基因表达水平上升,琥珀酸脱氢酶基因和异柠檬酸脱氢酶基因表达水平下调;在丙酮酸代谢旁路中丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因表达水平上升。【结论】结果显示,菌株ΔaceE三羧酸循环活性降低,虽然能够通过PDH旁路将部分丙酮酸分解为乙酸盐,进一步转化为乙酰CoA进入三羧酸循环,但ATP含量仍未恢复至野生株水平,这为进一步研究代谢改变与细菌表型的关系提供了理论基础。

[Objective]To explore the effect of the knockout of aceE gene which encodes pyruvate dehydrogenase on the growth characteristics,tricarboxylic acid cycle,and pyruvate metabolism of Streptococcus suis.[Methods]The growth curves of the wild-type strain and strainΔaceE were established with the OD600 values of the bacterial liquids.The content of acetyl CoA,succinate CoA,fumarate,oxaloacetate,pyruvate,lactate,and ATP in the tricarboxylic acid cycle and the pyruvate metabolism bypass was measured.Quantitative RT-PCR was employed to determine the gene expression levels of citrate synthase,malate dehydrogenase,succinate dehydrogenase,isocitrate dehydrogenase,pyruvate decarboxylase,lactate dehydrogenase,alcohol dehydrogenase,and acetaldehyde dehydrogenase.[Results]Compared with the wild-type strain,strainΔaceE showed decreasing OD600 value during the platform phase.The OD600 value of strainΔaceE in the plateau phase significantly increased after the addition of 1 g/L acetate.Compared with the wild-type strain,strainΔaceE showed increased content of pyruvate while decreased content of ATP,acetyl CoA,succinate CoA,and fumarate.Moreover,it showed up-regulated expression levels of citrate synthase and malate dehydrogenase,while down-regulated expression levels of succinate dehydrogenase and isocitrate dehydrogenase.For the proteins involved in pyruvate metabolism bypass,pyruvate decarboxylase,lactate dehydrogenase,alcohol dehydrogenase,and acetaldehyde dehydrogenase presented up-regulated gene expression levels in strainΔaceE.[Conclusion]StrainΔaceE has reduced activity of tricarboxylic acid cycle.Although part of pyruvate can be decomposed into acetate through the pyruvate dehydrogenease bypass and further converted into acetyl CoA to enter the tricarboxylic acid cycle,the ATP content has not been restored to the wild-type strain level.This study provides a theoretical basis for further research on the relationship between metabolic changes and bacterial phenotypes.