Abstract：
Pseudomonas fragi is by far one of the most threatening species in the spoilage of chilled meat that is stored under aerobic conditions. The membrane protein AprD is a well-established regulator controlling protease secretion in Pseudomonas spp. However, its exact roles in modulating metabolic pathways and spoilage potential of P. fragi at the molecular level remain undefined. Here, an in-frame deletion mutation of aprD was used to explore the impacts on their biofilm structure, matrix secretion, and cell metabolism. The results showed that ΔaprD formed relatively disorganized loose aggregation in biofilm, resulting in a thinner structure and more dead cells. Meanwhile, marked changes in the content of extracellular carbohydrates and proteins were observed. Furthermore, intracellular metabolomic profiling revealed the involvement of aprD in several cellular metabolic pathways, mostly including the carbohydrate pathway, amino acid pathway, and nucleotide pathway, while the characterization of extracellular metabolism clarified the variations in the spoilage-related metabolites (e.g., creatine, IMP, spermine, fatty acids, amino acids, and oligopeptides) could be highly correlated with aprD deletion. In this finding, we indicated that aprD could be responsible for cell reproduction and in situ spoilage potential of P. fragi NMC25 during chilled storage by controlling related metabolism and nutrients utilization. Thus, our results will contribute to an improved understanding of the regulatory mechanism of aprD gene in meat spoilage contaminated with P. fragi, which can be valuable to ensure the quality and safety of meat.
摘要：
到目前为止，脆弱假单胞菌是在有氧条件下储存的冷冻肉腐败中最具威胁性的物种之一。膜蛋白AprD是控制假单胞菌属蛋白酶分泌的公认调节剂。然而,它在分子水平上调节重组假单胞菌的代谢途径和腐败潜能中的确切作用仍不明确。这里,用aprD的框内缺失突变来探索其对生物膜结构的影响，基质分泌，和细胞代谢。结果表明，ΔaprD在生物膜中形成了相对无序的松散聚集，导致更薄的结构和更多的死细胞。同时,观察到细胞外碳水化合物和蛋白质含量的显着变化。此外，细胞内代谢组学分析揭示了aprD参与几种细胞代谢途径，主要包括碳水化合物途径，氨基酸途径，和核苷酸途径，而细胞外代谢的表征阐明了腐败相关代谢物的变化(例如，肌酸,IMP,精胺,脂肪酸，氨基酸,和寡肽）可能与aprD缺失高度相关。在这个发现中,我们表明，aprD可能通过控制相关的代谢和养分利用，在冷藏过程中负责P.fragiNMC25的细胞繁殖和原位腐败潜力。因此，我们的研究结果将有助于更好地理解受P.fragi污染的肉类腐败中aprD基因的调控机制，这对确保肉类的质量和安全是有价值的。
