Table_4_Involvement of RpoN in Regulating Motility, Biofilm, Resistance, and Spoilage Potential of Pseudomonas fluorescens.XLSX

Pseudomonas fluorescens is a typical spoiler of proteinaceous foods, and it is characterized by high spoilage activity. The sigma factor RpoN is a well-known regulator controlling nitrogen assimilation and virulence in many pathogens. However, its exact role in regulating the spoilage caused by P. fluorescens is unknown. Here, an in-frame deletion mutation of rpoN was constructed to investigate its global regulatory function through phenotypic and RNA-seq analysis. The results of phenotypic assays showed that the rpoN mutant was deficient in swimming motility, biofilm formation, and resistance to heat and nine antibiotics, while the mutant increased the resistance to H2O2. Moreover, the rpoN mutant markedly reduced extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized fish juice at 4°C; meanwhile, the juice with the rpoN mutant showed significantly higher sensory scores than that with the wild-type strain. To identify RpoN-controlled genes, RNA-seq-dependent transcriptomics analysis of the wild-type strain and the rpoN mutant was performed. A total of 1224 genes were significantly downregulated, and 474 genes were significantly upregulated by at least two folds at the RNA level in the rpoN mutant compared with the wild-type strain, revealing the involvement of RpoN in several cellular processes, mainly flagellar mobility, adhesion, polysaccharide metabolism, resistance, and amino acid transport and metabolism; this may contribute to the swimming motility, biofilm formation, stress and antibiotic resistance, and spoilage activities of P. fluorescens. Our results provide insights into the regulatory role of RpoN of P. fluorescens in food spoilage, which can be valuable to ensure food quality and safety.

荧光假单胞菌（Pseudomonas fluorescens）是一类典型的蛋白质类食品腐败菌，具有极强的腐败活性。σ因子（sigma factor）RpoN是众多病原菌中调控氮同化与致病力的经典调控因子，但其在荧光假单胞菌介导的食品腐败过程中的具体调控作用仍未明确。本研究构建了rpoN的框内缺失突变株，通过表型分析与RNA测序（RNA-seq）技术探究其全局调控功能。表型检测结果显示，rpoN突变株的游泳运动能力、生物膜（biofilm）形成能力、耐热性以及对九种抗生素的抗性均出现缺陷，而其对过氧化氢（H₂O₂）的抗性则显著提升。此外，在4℃条件下的灭菌鱼汁体系中，rpoN突变株的胞外蛋白酶与总挥发性盐基氮（TVB-N）产量显著降低；与此同时，接种该突变株的鱼汁感官评分显著高于接种野生型菌株的样本。为鉴定RpoN调控的靶基因，本研究对野生型菌株与rpoN突变株开展了基于RNA-seq的转录组学分析。与野生型菌株相比，rpoN突变株中共有1224个基因在RNA水平上显著下调，474个基因显著上调（差异倍数≥2）。转录组分析结果表明，RpoN参与调控多种细胞生理过程，主要包括鞭毛运动、黏附、多糖代谢、抗性响应以及氨基酸转运与代谢；上述调控功能或与荧光假单胞菌的游泳运动、生物膜形成、胁迫与抗生素抗性以及腐败活性密切相关。本研究阐明了荧光假单胞菌中RpoN在食品腐败过程中的调控作用，可为保障食品品质与安全提供理论参考。