【作者】 林琳；

【导师】 田兵；

【作者基本信息】 浙江大学 ， 生物物理学， 2016， 博士

【摘要】 耐辐射奇球菌(Deinococcus radiodurans,简称DR)属于奇球菌属,该属细菌通常对极端环境,如电离辐射、紫外照射、干燥和氧化剂等,具有无可比拟的超强抗性。多年来,耐辐射奇球菌已成为人们研究微生物在极端环境压力下抗性的模式生物之一。然而在菌群水平上,耐辐射奇球菌是如何应对极端环境压力的机制尚不清楚。本研究利用生化和分子生物学等手段研究了耐辐射奇球菌群体感应(Quorum Sensing,QS)系统,研究了其作为菌体细胞间信号通讯途径参与菌群水平氧化应激反应的功能与作用机制。首先,发现DR菌能够合成群体感应信号分子酰基高丝氨酸内酯(N-acyl-homoserine lactones, AHLs),研究鉴定了耐辐射奇球菌特殊的DqsIR群体感应系统并发现其对菌体的氧化胁迫抗性具有重要作用。DR菌具有不同于其他微生物中LuxⅠ, LuxM等的AHL合成酶DqsⅠ-1和DqsI-2;在没有氧化压力条件下,AHL水平由于群体感应信号降解酶(quorum quenching enzyme)的作用而被抑制,而在氧化胁迫下,DqsⅠ-1和DqsI-2表达上调导致AHL的积累,表现为一种特殊的氧化条件依赖的群体感应作用；DqsIR群体感应系统的AHL合成酶和AHL降解酶的表达呈现出一种动态模式,从而控制菌体在有无氧化压力条件下AHL信号的水平；AHLs通过与调控因子DqsR相互作用调节了抗逆基因的转录水平,参与了耐辐射奇球菌的氧化压力抗性作用。与野生菌株相比,dqsⅠ缺失突变株(DMDqsI)和dqsR缺失突变株(MDqsR)都对氧化压力更加敏感。外源添加AHL信号分子可以完全补偿DMDqsⅠ的氧化抗性表型。RNA-seq转录组数据表明,MDqsR中许多参与损伤响应、DNA修复过程等的基因转录水平都发生了改变。因此,DqsIR介导的群体感应系统参与了耐辐射奇球菌应对氧化压力的抗性机制,并且在奇球菌属细菌中可能是保守存在的。其次,自诱导物-2(AI-2)作为可以同时介导细菌种内和种间群体感应的信号分子,对参与调节各种细菌的行为起着重要作用。在本研究中,我们通过构建AI-2合成酶LuxS同源物基因的缺失突变株(DRALuxS),结合生存率表型实验和基因转录分析实验探究了AI-2信号在DR菌抗性中的作用。突变株DRΔLuxS不能合成AI-2。与野生型菌株相比,DRΔLuxS对伽马辐射和H202更加敏感；但向DRΔLuxS菌株中添加野生菌株的培养上清液可以完全补偿其抗性表型；在正常或氧化应激条件下,突变菌株中活性氧自由基的水平都高于野生株。实时定量PCR结果显示一些抗性相关基因的转录水平在DRΔluxS中下降,其中包括过氧化氢酶、胞外核酸酶、DPS-1和ABC转运蛋白等,表明AI-2参与调控了耐辐射奇球菌部分压力胁迫响应相关基因。因此,AI-2信号对耐辐射奇球菌极端环境抗性具有一定的贡献。本论文研究了耐辐射奇球菌群体感应系统及其对菌体氧化胁迫抗性基因的调控作用,研究结果为从群体感应水平上揭示耐辐射奇球菌应对氧化胁迫的抗性作用机制和深入了解菌体极端环境抗性机理提供了基础。更多还原

【Abstract】 Deinococcus radiodurans belongs to the Deinococcus, a genus of bacteria characterized by an unparalleled ability to survive lethal effects of extreme stresses, including ionizing radiation, ultraviolet (UV) radiation, desiccation, and oxidants. For years, D. radiodurans has been used as an ideal model organism for studying microbial resistance to extreme stresses. However, it remains unclear how does D. radiodurans response to the stresses on the level of bacterial population. In this work, we characterized quorum sensing circuits and investigated the function and regulation mechanism of the quorum sensing circuits involved in response of D. radiodurans to oxidative stress, using biochemical and molecular biological techiques.First, we show that AHLs can be employed by D. radiodurans. An AHL-mediated quorum-sensing system (DqsI/DqsR), which plays important roles in cell resistance to oxidative stress, was identified in D. radiodurans. D. radiodurans has special AHL synthesases (DqsⅠ-1 and DqsI-2), different from Luxl-type and LuxM-type AHL synthesases. We found that under non-stress conditions, the AHL level was "shielded" by quorum quenching enzymes, whereas AHLs accumulated by induction of DqsⅠ-1 and DqsI-2 when D. radiodurans was exposed to oxidative stress, indicating an oxidative stress-dependent quorum sensing. The dynamic expression of AHL synthesases and quorum quenching enzymes might be used by the bacterium to control AHL level under different conditions. The DqsR, acting as a regulator of quorum sensing, controls gene expression along with AHLs. Both dqsl mutant (DMDqsI) and dqsR mutant (MDqsR) were more sensitive to oxidative stress compared with the wild-type strain. Exogenous AHLs could completely restore the survival fraction of DMDqsI under oxidative stress. RNA-seq analysis showed that a number of genes involved in stress-response, cellular cleansing and DNA repair had altered transcriptional levels in the mutant of DqsR. Hence, the DqsIR-mediated quorum sensing is an adaptive strategy for D. radiodurans in response to oxidative stress and might be conserved in the extremophilic Deinococcus bacteria.Second, Autoinducer-2 (AI-2) serves as a quorum-sensing signaling molecule that mediates both intraspecies and interspecies communication among bacteria, and plays roles in regulating various bacterial behaviors. In the present study, we investigated the functions of AI-2 signaling in resistance of D. radiodurans to oxidative stress by construction of the AI-2 synthase (LuxS homolog) gene disruption mutant, survival phenotype assay, and gene transcription assay. The gene mutant (DRΔLuxS), which was unable to produce AI-2, was significantly more sensitive to both gamma radiation and H2O2 compared with the wild-type strain. Complementation of the wild-type-derived spent medium into the cell culture of DRΔLuxS completely restored the resistance phenotype of DRΔLuxS. A higher level of reactive oxygen species was accumulated in the mutant compared with the wild type under normal or oxidative stress. Quantitative real-time PCR assays showed that transcriptional levels of some resistant proteins, including catalase, extracellular nuclease, Dps-1 and ABC transporters were decreased in DRΔLuxS, indicating that AI-2 is involved in regulation of the resistant genes of D. radiodurans. Hence, AI-2 signaling may contribute to the extreme resistance of D. radiodurans to oxidative stresses.In sum, the present study investigated the quorum-sensing circuits and their regulation on resistant genes in D. radiodurans under oxidative stress.The results provide fundament for elucidation regulation mechanism of D. radiodurans in response to oxidative stresse from the point of view of quorum sensing and for understanding the resistance of D. radiodurans to extreme stresses.更多还原