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

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RpoS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rpoS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℃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rpoS基因缺失突变株。rpoS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/mL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℃和20% NaCl的耐受性。荧光假单胞菌在rpoS基因缺失突变后长链信号分子C₁₀-HSL、C₁₂-HSL和C₁₄-HSL的含量增加。在灭菌三文鱼汁中的腐败活性检测表明rpoS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RpoS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。

Abstract:

[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 RpoS in spoilage caused by P. fluorescens.[Methods] An rpoS 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℃, and total viable count (TVC) and total volatile basic nitrogen (TVB-N) were assayed.[Results] The in-frame deletion rpoS mutant strain was successfully constructed in P. fluorescens. Deletion of rpoS gene significantly decreased the resistance to 10 mmol/L H₂O₂and 15% alcohol, increased the resistance to 150 μg/mL crystal violet and 175 mmol/L acetic acid, and had no influence on the resistance to 47℃ and 20% NaCl. The ropS mutant produced higher contents of long chain AHLs C₁₀-HSL, C₁₂-HSL and C₁₄-HSL than the wild type one. In addition, the mutant reduced TVB-N production in sterilized salmon juice stored at 4℃.[Conclusion] These results indicated that RpoS was not only involved in the resistance to multiple stresses, but also influenced quorum sensing and spoilage potential of P. fluorescens.