巴氏醋杆菌核酸修复酶UvrA与菌体高醋酸耐受性的关系研究

Acetic acid which is the major component of vinegar shows a severe toxicity to microorganism by affecting the cell growth and metabolism. However, Acetobacter pasteurianus is usually used to acetic acid fermentations due to its high acetic acid tolerance. In our previous study, the transcription level of gene UvrA coding excinuclease (UvrA) was found to be up-regulated by high concentration acetic acid with the method of differential display PCR. For bacteria, UvrA is required for the repair of DNA damage. Moreover, uvrA is an acid-inducible and involved in the adaptive response to Low pH in Streptococcus mutans, indicating the high acetic acid tolerance of A. pasteurianus is related with the nucleotide repair system. Thus, the goal of this research is focused on the relationship of UvrA with the high acetic acid tolerance of A. pasteurianus. Firstly, the transcription and translation of UvrA in different concentrations of acetic acid was compared. Secondly, the strain with UvrA disruption was constructed. And then, the effects of uvrA disruption on the cell growth, mass of protein and nucleic acid in cell were studied. The result was verified by the experiment of complement expression. Eventually, considering the performance on acetic acid formation of A. pasteurianus under different initial acetic acid concentrations, the relationship of UvrA with the high acetic acid tolerance was clarified by thoroughly analyzing the effects of UvrA on the cell growth, DNA repair, protein expression, and so on. Moreover, this is the first time trying to study the mechanism of acetic acid tolerance based the DNA repair system.

食醋中主要成分醋酸对细胞有很强的毒性，影响菌体生长和代谢。巴氏醋杆菌对醋酸有很好的耐受性，常用于食醋生产。项目组前期利用差示反转录PCR方法发现巴氏醋杆菌核酸修复酶（UvrA）基因在高醋酸浓度下转录水平提高。细菌中UvrA参与DNA的损伤修复，链球菌中UvrA为酸可诱导的，且有利于菌体适应低pH环境，因此巴氏醋杆菌的高醋酸耐受性可能与其DNA修复系统有关。本项目以巴氏醋杆菌UvrA为对象，研究其在不同醋酸浓度下转录表达水平差异；然后利用基因敲除技术构建巴氏醋杆菌UvrA基因敲除菌株，分析UvrA敲除对菌体生长以及胞内蛋白、核酸浓度的影响，并利用回补表达实验验证；最后结合不同醋酸浓度下菌体生长和醋酸发酵规律，系统分析UvrA对菌体生长、DNA修复、蛋白表达等方面的影响，阐明巴氏醋杆菌UvrA与菌体醋酸耐受性的关系。该项目特色在于首次提出对巴氏醋杆菌基于DNA修复系统的醋酸耐受性机制进行研究。

本课题以巴氏醋杆菌AC2005为研究主体，以核酸修复酶UvrA为研究对象，研究醋酸对菌体生长和代谢的影响，利用基因工程的方法构建获得巴氏醋杆菌uvrA基因敲除与过表达菌株，研究UvrA对巴氏醋杆菌醋酸耐受性及醋酸发酵的影响。.研究发现，随着醋酸浓度的增加AC2005基因组完整性逐渐降低，说明醋酸会导致菌体基因组断裂损失。加入醋酸后核酸修复酶UvrA的蛋白表达水平增加了约130%。在1%、2%醋酸浓度下，与无醋酸条件相比uvrA转录水平分别增加了146%和275%，UvrA可能与其醋酸耐受性相关。来源于巴氏醋杆菌的核酸修复酶UvrA能够提高大肠杆菌对压力环境耐受性。分别构建了巴氏醋杆菌uvrA基因敲除菌株AC2005-ΔuvrA和重组表达菌株AC2005（pMV24-uvrA）以及对照菌株AC2005（pMV24）。在初始添加1%、2%醋酸条件下培养48h，与对照相比AC2005-ΔuvrA的生物量分别下降了84.85%和47.13%，而AC2005（pMV24-uvrA）则分别提高了17.1%和24.2%。6%醋酸处理40 min，AC2005-ΔuvrA的存活率仅为2.56×10-5，而AC2005（pMV24-uvrA）的存活率为1.1×10-3，大约是对照的2倍。与对照相比，醋酸条件下UvrA重组菌株基因组完整性提高，而敲除菌株基因组完整性降低。醋酸发酵实验表明，AC2005（pMV24-uvrA）产酸量比对照提高17%；而AC2005-ΔuvrA在醋酸浓度达到0.45 g/100mL时发酵停止。与对照相比，AC2005-ΔuvrA中ADH和ALDH酶活分别降低了66.8%和66.6%，而AC2005（pMV24-uvrA）则分别增加了17%和1.5%。.结果表明，醋酸会引起菌体基因组损伤，导致菌体生长受到抑制甚至死亡，而UvrA是醋酸诱导的，与基因组损伤修复有关。UvrA缺失导致基因组损伤加剧，菌体对醋酸敏感性增加，生长和醋酸发酵停滞；过表达UvrA可以减少醋酸导致的基因组损伤，保证ADH等醋酸耐受相关蛋白的表达，从而保证菌体的代谢活性，提高菌体醋酸耐受性。