大肠杆菌对声环境暴露的应激响应与机制研究

To explore the mechanism of natural population’s adaptation and resistance to environmental conditions, especially the reaction of microorganisms to various environmental factors has come into focus in the fields of modern microbial ecology and molecular ecology. Facing the increasingly serious problem of acoustic pollution, very little efforts have been put forth in studying the relation of single cell organisms and sound field. In the project, the primary objective is to develop a dose-response model to estimate the biological effects induced by sound wave and reveal their general principles. So, using a single cell organism as a model, we have exposed E. coli to sound, to test whether sonic waves of different dose affect basal cell metabolism and growth. Then, instantaneous kinetic equation should be established to analyze the dose-effect relationship between sound environmental exposure and biological effects.The developed models will be adapt to describe the change in effects on bacteria cell caused by differing levels of exposure to audible sound after a certain exposure time. .In addition, understanding the underlying mechanisms of sound bio-effects is another essential goal of our research. Therefore, observation of biological effects induced by sound will be carried out in the presence of calcium channel blocker or some agent to inhibit mechanosensitive ion channel (Msc), respectively. Meanwhile, influence of intracellular free calcium change on control gene expression in response to audible sound stimuli, especial response regulator sigma S, should be investigated. The role of Msc in signal transduction of mechanical stimuli will be estimated by single-gene deletions. The genes regulated by RpoS will be identified by transcriptome analysis. This study will propose the potential pathway and molecular mechanism according to above research findings. And it will be served for the rational development and efficient utilization of sound effects.

探索自然种群对环境的调整与适应机制，特别是微生物对各类环境因子的反应已成为近代微生物生态学和分子生态学共同关注的焦点。然而面对日益严峻的环境声污染问题，人们却很少关注它对微生物所带来的影响。基于此，本项目拟以单细胞原核生物大肠杆菌为对象，研究不同声环境暴露对其生长代谢的影响，建立菌体生长、蛋白合成等相关瞬时动力学方程，分析声环境暴露的剂量效应关系，揭示微生物声应激效应的一般特征和规律；借助钙通道阻断、机械敏感性通道(Msc)失活及转录组学分析等，研究钙离子在声应激中的作用，分析Msc在机械信号传导中的功能，考察σS参与的胞内应激调控行为，探讨微生物声应激的作用途径及调控机制。该研究对理解包括微生物在内的生物体对声环境的调整与适应机制具有重要意义，对声效应的合理开发和利用具有指导作用，它有利于探索和建立基于单细胞生物的声暴露风险评估及预警体系，开发基于声效应的促生长、促代谢、促合成的新型生物技术等。

本项目在自行研制的声频发生装置试验平台上，研究了大肠杆菌在声环境暴露下相关生物学作用的剂量效应关系及应激响应机制。研究发现，大肠杆菌可通过机械敏感性通道来实现对声环境暴露条件的响应，且其对声频、声功率和声强度响应效应存在明显差异，其中声功率和声强度对大肠杆菌的生长促进效应为非线性的单窗口效应，而声频率却呈现出非线性的双窗口效应。当声强在70-80dB间时，生物量随声强的增加明显升高；超过80dB时，尽管μmax随声强的增加持续增加，但对数生长期维持时间却开始快速下降。当声功率级处于55dB-61dB时，E.coli的生物量随功率的增加而快速上升，在61dB处出现峰值，当声功率级为63dB时，生物量急剧下降；μmax和对数生长期维持时间呈类似非线性、单窗口效应。当声频率在250Hz-16KHz时，声促生长作用具有明显的频率依懒性，当频率为2KHz和8KHz时，这种生长促进作用最为明显，与对照组相比，生物量分别提高了21%和27.1%。该发现对开发基于声效应的促生长、促代谢、促合成的新型生物技术具有重要意义。. 研究发现，大肠杆菌细胞膜上的大电导率机械敏感性离子通道（MscL）和小电导率机械敏感性离子通道（MscS）是其声应激响应的最初受体，当这两类通道同时缺失时，大肠杆菌对声刺激的响应几乎完全消失。进一步研究发现，当声刺激时机械敏感性通道的开启诱发钙离子内流，进而引起了随后的次级效应。转录组数据分析表明，声刺激信号的胞内传达，可能通过一系列磷酸化和去磷酸化来实现，尤其是一些与膜结构、功能和应急响应相关的基因在声刺激下响应尤为明显。分析发现声刺激明显促进了膜上ABC转运体的合成以及ATP酶的合成，提高了细胞吸收底物的能力；同时，声刺激还加强了大肠杆菌的有氧呼吸能力，使细胞能够产生更多的能量参与菌体的生长和繁殖。该研究对理解包括微生物在内的生物体对声环境的调整与适应机制具有重要意义。