rpoS基因缺失突变对荧光假单胞菌胁迫耐受性、群体感应及腐败活性的影响  

Effects of rpoS deletion on stress resistance,quorum sensing and spoilage potential of Pseudomonas fluorescens

作　　者：沈碧妙 吴淘栩 朱俊杰 徐琳娜 刘小香[2] 朱军莉 Bimiao Shen;Taoxu Wu;Junjie Zhu;Linna Xu;Xiaoxiang Liu;Junli Zhu(College of Laboratory Medicine,Hangzhou Medical College,Hangzhou 310053,Zhejiang Province,China;Faculty of Basic Medicine,Hangzhou Medical College,Hangzhou 310053,Zhejiang Province,China;Food Safety Key Laboratory of Zhejiang Province,College of Food Science and Biotechnology,Zhejiang Gongshang University,Hangzhou 310035,Zhejiang Province,China)

机构地区：[1]杭州医学院检验医学院,浙江杭州310053 [2]杭州医学院基础医学部,浙江杭州310053 [3]浙江工商大学食品与生物工程学院,浙江省食品安全重点实验室,浙江杭州310035

出　　处：《微生物学报》2018年第9期1563-1572,共10页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31501581); 浙江省卫生高层次创新人才培养工程(浙卫发[2016]63号); 2017年浙江省大学生科技创新活动计划暨新苗人才计划(2017R436005); 2015年度高校国内访问学者项目(2015FX124)

摘　　要：【目的】微生物活动是引起食品腐败的主要原因,研究食品腐败菌的腐败作用调控机制对于保证食品的质量和安全具有重要意义。荧光假单胞菌是一种代表性的食品腐败菌,本文旨在研究RNA聚合酶的选择性sigma因子Rpo S在荧光假单胞菌致腐败过程中的作用。【方法】运用同源重组的方法构建荧光假单胞菌冷藏鱼分离株的rpo S基因缺失突变株,比较野生型和突变株暴露于不同胁迫条件下的存活率;通过液相色谱-串联质谱(LC-MS/MS)分析野生型和突变株产生高丝氨酸内酯类(AHLs)群体感应信号分子的种类和含量;检测野生型和突变株接种于灭菌三文鱼汁后4°C贮存过程中的菌落总数和挥发性盐基氮的生成量。【结果】成功构建了荧光假单胞菌rpo S基因缺失突变株。rpo S基因的缺失导致荧光假单胞菌对10 mmol/L H2O2和15%乙醇的耐受性显著降低,对150μg/m L结晶紫和175 mmol/L醋酸的耐受性有一定程度增强,不影响其对47°C和20%Na Cl的耐受性。荧光假单胞菌在rpo S基因缺失突变后长链信号分子C_(10)-HSL、C_(12)-HSL和C_(14)-HSL的含量增加。在灭菌三文鱼汁中的腐败活性检测表明rpo S基因缺失可导致荧光假单胞菌挥发性盐基氮的生成量显著降低。【结论】荧光假单胞菌的Rpo S不仅调节细菌对多种胁迫条件的耐受性,还影响AHL群体感应和腐败活性。[Objective] Microbial activities are considered the main cause of most food spoilage.Study of the regulation mechanism of spoilage is valuable to maintain quality and safety of food.Pseudomonas fluorescens is a typical Gram-negative spoilage bacterium.The aim of this study was to elucidate the role of the alternative sigma factor Rpo S in spoilage caused by P.fluorescens.[Methods] An rpo S mutant of P.fluorescens isolated from refrigerated fish was constructed by homologous recombination.Survival rates of the wild type and the mutant under different stress conditions were compared.The types and contents of extracellular autoinducers acylated homoserine lactones（AHLs） were analyzed by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.In addition,the wild type and the mutant were inoculated into the sterile salmon juice stored at 4 °C,and total viable count（TVC） and total volatile basic nitrogen（TVB-N） were assayed.[Results] The in-frame deletion rpo S mutant strain was successfully constructed in P.fluorescens.Deletion of rpo S gene significantly decreased the resistance to 10 mmol/L H_2O_2 and 15% alcohol,increased the resistance to 150 μg/m L crystal violet and 175 mmol/L acetic acid,and had no influence on the resistance to 47 °C and 20% Na Cl.The rop S mutant produced higher contents of long chain AHLs C_（10）-HSL,C_（12）-HSL and C_（14）-HSL than the wild type one.In addition,the mutant reduced TVB-N production in sterilized salmon juice stored at 4 °C.[Conclusion] These results indicated that Rpo S was not only involved in the resistance to multiple stresses,but also influenced quorum sensing and spoilage potential of P.fluorescens.

关 键 词：RPOS 荧光假单胞菌 耐受性 食品腐败 群体感应 

分 类 号：TS201.3[轻工技术与工程—食品科学]