植物乳杆菌环二腺苷酸合成酶的克隆表达与活性分析

Cloning, expression and characterization of c-di-AMP-synthesizing enzyme from Lactobacillus plantarum

摘要:

【背景】环二腺苷酸(Cyclic Diadenosine Monophosphate，c-di-AMP)是一种主要存在于革兰氏阳性菌中的重要的第二信使分子，其参与细菌的生长、生存、抗逆性等多种生理活动，但目前关于乳酸菌中c-di-AMP的研究甚少。【目的】从植物乳杆菌(Lactobacillus plantarum)中克隆得到c-di-AMP合成酶基因，在大肠杆菌中进行可溶性表达并研究其体外活性。【方法】使用高效液相色谱以及质谱分析对植物乳杆菌-YRA7细胞内容物中的c-di-AMP进行检测；以植物乳杆菌-YRA7基因组DNA为模板，克隆c-di-AMP合成酶基因(lpDacA)，构建重组表达载体pET-28a-lpDacA并在大肠杆菌BL21(DE3)中诱导表达，通过Ni-NTA亲和层析纯化后进行体外活性研究。【结果】在植物乳杆菌中检测到c-di-AMP分子；成功构建了c-di-AMP合成酶基因的重组表达质粒，该重组蛋白在大肠杆菌中得到可溶性表达；体外活性分析显示，该重组蛋白可以催化ATP生成c-di-AMP，其活性依赖于二价阳离子的存在，在Mg2+存在以及碱性环境下活性较强；RHR是合成酶活性的关键基序，是环二腺苷酸合成酶与ATP的结合位点。【结论】植物乳杆菌c-di-AMP合成酶的克隆表达及活性分析为进一步研究c-di-AMP在植物乳杆菌中的作用奠定了基础。

Abstract:

[Background] Cyclic diadenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced primarily within Gram-positive bacteria and is involved in cell growth, survival, stress resistance and many other aspects of bacterial physiology; however, there are few studies on c-di-AMP in lactic acid bacteria currently. [Objective] The gene of c-di-AMP-synthesizing enzyme from Lactobacillus plantarum was cloned and efficiently heterologous expressed in Escherichia coli, and its biochemical activity in vitro was studied. [Methods] c-di-AMP in L. plantarum-YRA7 was detected by HLPC and ESI-MS using the cell extract. Then the c-di-AMP-synthesizing enzyme gene (lpDacA) was cloned from the genomic DNA of L. plantarum-YRA7 and the recombinant plasmid pET-28a-lpDacA was constructed. The recombination protein was successfully expressed in E. coli BL21(DE3), and purified by Ni-NTA affinity chromatography. Then enzymatic characteristics in vitro was studied. [Results] c-di-AMP was detected in L. plantarum-YRA7. The recombinant expression plasmid pET-28a-lpDacA was constructed and the purified recombinant protein was obtained. The biochemical activity study showed that the purified recombinant protein converted ATP into c-di-AMP in vitro. Its c-di-AMP-synthesizing activity was dependent on divalent metal ions and exhibited higher activity in the presence of Mg2+ at a basic pH. We also found that RHR motif is essential for the c-di-AMP-synthesizing activity and is the ATP binding site of lpDacA. [Conclusion] Cloning, expression and characterization of c-di-AMP-synthesizing enzyme from L. plantarum will laid a solid foundation for future exploration on the physiological role of c-di-AMP in L. plantarum.