乳酸亚致死性损伤大肠杆菌修复相关基因及信号传导的研究

Sublethal injured bacteria are difficult to be sterilized thoroughly, causing great potential risk of food safety. It has caused great concern and become research focus in recent years. The establishment of plate counting and novel molecular detection methods to detect sublethal injured bacteria is based on the revelation of related genes and signal transduction involved in injury recovery. Related genes and signal transduction involved in recovery of lactic acid-sublethal injured Escherichia coli will be studied, which is common bacterium in food and gut. Screening of recovery sensing genes and study of metabolic pathway of oxidative stress release will be two main focuses in this project. The functions of related genes involved in recovery will be determined through gene knockout. In addition, the effect of added materials on injury recovery will be determined through comparation of recovery rates. On the other hand, differential expressed genes between injured and recovered cells will be studied on whole genome level, by RNA sequencing and reverse transcription-PCR. Novel recovery sensing genes and recovery targets in metabolic pathway will be explored in this process. The above research results will provide significant evidence for establishment of the regulation network of signal transduction. This study will provide important scientific evidence for detection method development of lactic acid-sublethal injured E. coli and other sublethal injured bacteria, and will also provide strategy for inhibition of injury recovery or improvement of bacteria activity in food field.

亚致死性损伤菌难以彻底消除，会造成极大的食品安全隐患，已引起高度重视，成为近年来的研究热点。亚致死性损伤菌修复相关基因及信号传导的揭示是建立平板计数法和新型分子检测方法的关键。乳酸亚致死性损伤大肠杆菌是食品及肠道中的常见菌，本项目将以该菌为对象进行修复相关基因及信号传导的研究。以修复感应基因的筛选研究、缓解氧化应激的代谢途径研究为重点。通过基因敲除，确定相关基因在修复中的功能；通过修复速率的比较，确定外源物质的添加对损伤修复的作用。另一方面，使用转录组测序结合反转录PCR技术，对亚致死性损伤与修复后的菌体进行全基因组水平的基因表达差异研究，发掘新的修复感应基因及代谢途径中的修复靶点，为建立修复信号传导的调控网络提供重要依据。本研究将为乳酸亚致死性损伤大肠杆菌及其他类型亚致死性损伤菌分子检测新策略的开发提供科学依据，也为食品领域中抑制损伤菌修复或提高菌体整体活性提供策略。

亚致死性损伤菌难以彻底消除，会造成极大的食品安全隐患，已引起高度重视，成为近年来的研究热点。亚致死性损伤菌修复机制的揭示是建立其检测方法的关键。本项目研究了修复的分子机制并得到以下结论：（1）大肠杆菌以亚致死性损伤状态广泛存活于不同pH值的乳酸溶液中。乳酸亚致死性损伤大肠杆菌O157:H7和K12处于37°C下的胰酪胨大豆肉汤培养基（TSB）培养基中时，均可在60 min之内完全修复。通过荧光素二乙酸酯/碘化丙啶双染色法，观察到损伤菌的代谢活性降低，但其修复后，恢复正常的代谢活性。损伤菌的修复率与修复温度、修复液的营养水平有关。另外，丙酮酸钠、吐温80、过氧化氢酶（CAT）、锰离子、铁离子、锌离子能够显著提高乳酸亚致死性损伤大肠杆菌的修复率，但是镁离子和钙离子对修复时间没有影响。相反地，金属离子螯合剂N,N,N',N'-四（2-吡啶甲基）乙二胺（TPEN）可以螯合细胞内外的离子，导致修复率下降。（2）通过突变体筛选，证实双组分系统BarA/UvrY以及编码RpoS σ因子的RpoS是修复过程中的重要感应基因。而在BarA/UvrY及RpoS调控下游的酶缓解氧化应激系统也参与到修复过程中，编码合成CAT的katE、katG和编码合成Cu/Zn超氧化物岐化酶（Cu/ZnSOD）的sodC在修复中起着重要作用。（3）证实了CAT和SOD在修复过程中的作用。损伤菌产生大量活性氧（ROS），在修复过程中，ROS逐渐被清除，完全修复菌的ROS水平恢复到与未处理菌一致。一方面，外源添加CAT可通过直接清除ROS的方式提高修复率；另一方面，外源添加锌离子（Cu/ZnSOD的金属辅因子）可以提高SOD活性，从而降低ROS含量并提高修复率。本项目揭示了乳酸亚致死性损伤大肠杆菌复活的分子机制，也为亚致死性损伤菌分子检测新策略的开发提供了科学依据。