冷藏条件下肉源莓实假单胞菌nuoB对其生物被膜形成和细胞代谢的影响

doi:10.3864/j.issn.0578-1752.2023.11.011

摘要/Abstract

摘要：

【目的】研究nuoB对莓实假单胞菌生物被膜形成和细胞代谢的影响，进一步揭示nuoB对莓实假单胞菌介导的冷鲜肉腐败的调控机制，为优化冷鲜肉保鲜体系提供理论基础。【方法】以莓实假单胞菌NMC25野生株和nuoB突变株作为研究对象，利用共聚焦激光扫描显微镜（CLSM）观察生物被膜空间结构的差异；通过菌落细胞计数、胞外聚合物的表征解析生物被膜组分变化；基于超高效液相色谱联合质谱的非靶向代谢组学方法探寻与nuoB相关的代谢产物水平变化。【结果】CLSM显示，原位条件下野生株生物被膜细胞呈现密集排列、垂直化定殖的状态，而ΔnuoB生物被膜显示为相对无序、松散的结构。菌落中细胞计数结果显示，不同生长环境下的野生型菌株和突变型菌株的生物被膜细胞数量均无显著性差异，表明突变株在TSA或肉表面上以生物被膜形式生长的能力没有发生大的变化。与野生株相比，原位培养时突变株的蛋白质（P<0.01）和总糖（P<0.05）含量都显著升高，表明nuoB影响了莓实假单胞菌胞外聚合物的分泌。代谢组结果显示，野生组与突变组在正交偏最小二乘法判别分析模型中有明显的分离（R²X=0.481，R²Y=0.977，Q²=0.909），说明原位培养时突变株的代谢产物发生了明显变化。模型中组间差异显著的代谢物包括2-羟基肉桂酸、L-酪氨酸、L-苯丙氨酸、DL-色氨酸、17-羟基-二十碳四烯酸、5-OxoETE等。这些代谢物经过通路分析后发现，它们主要与脂肪酸的生物合成、不饱和脂肪酸的生物合成、核黄素代谢、2-氧代羧酸代谢、嘌呤代谢、氰基氨基酸代谢、苯丙氨酸代谢等有关。【结论】nuoB的破坏会导致莓实假单胞菌原位培养时生物被膜空间结构发生变化，促进胞外聚合物的生物合成，影响细胞内碳代谢、核苷酸代谢、脂质代谢和氨基酸代谢。

关键词: 莓实假单胞菌, nuoB, 生物被膜, 胞外聚合物, 细胞代谢

Abstract:

【Objective】This paper focused on the impacts of nuoB on the biofilm formation and cell metabolism in Pseudomonas fragi (P. fragi), so as to further reveal the regulatory mechanism of nuoB in the spoilage of chilled meat contaminated with P. fragi, and to provide a theoretical basis for developing effective preservation system of chilled meat. 【Method】P. fragi NMC25 and its nuoB-mutant strain were used in the present study, and the differences in the spatial structure of biofilms were observed by confocal laser scanning microscopy (CLSM). The changes in biofilm composition were tested by the cell enumeration and the analysis of the extracellular polymeric substances. In addition, the ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) was employed to investigate the alterations of nuoB-related metabolite abundance. 【Result】CLSM images showed that cells in the biofilms of wild-type strains cultivated in situ was highly dense, nematic ordering, whereas ΔnuoB displayed relatively disorganized and sparse arrangement. Additionally, cell enumeration revealed insignificant differences between the wild-type and mutant biofilms regardless of the difference of culture medium. The result indicated that the mutants did not change significantly in their ability to grow as biofilm on the surface of TSA or meat sample. For extracellular polymeric substances from biofilms in situ, the protein and carbohydrate contents of ΔnuoB were significantly higher (P<0.01, P<0.05, respectively) than those of wild-type strains, indicating that nuoB affected the secretion of extracellular polymers by P. fragi. The metabolomics results revealed a clear separation between the wild and mutant groups in an orthogonal partial least squares discriminant analysis model (R²X=0.481, R²Y=0.977, Q²=0.909), which suggested that the metabolites of the mutants had changed markedly. In the model, differentially expressed metabolites were screened, including 2-hydroxycinnamic acid, L-tyrosine, L-phenylalanine, DL-tryptophan, 17(S)-HETE, and 5-OxoETE. Pathway mapping analysis was conducted based on the chosen candidates. In total, the major metabolic pathways included fatty acid biosynthesis, unsaturated fatty acid biosynthesis, riboflavin metabolism, 2-oxocarboxylic acid metabolism, purine metabolism, cyanogenic amino acid metabolism, and phenylalanine metabolism. 【Conclusion】The disruption of nuoB stimulated significant variations in the spatial structure of the P. fragi biofilm grown in situ, promoting the biosynthesis of extracellular polymeric substances and affecting intracellular metabolic pathways, such as carbon, nucleotide, lipid, and amino acid metabolism.

Key words: Pseudomonas fragi, nuoB, biofilm, extracellular polymeric substances, cell metabolism

吴亚婕, 谭松, 陈愉平, 牛阿娟, 柳雨欣, 王光宇, 徐幸莲, 邱伟芬. 冷藏条件下肉源莓实假单胞菌nuoB对其生物被膜形成和细胞代谢的影响[J]. 中国农业科学, 2023, 56(11): 2172-2185.

WU YaJie, TAN Song, CHEN YuPing, NIU AJuan, LIU YuXin, WANG GuangYu, XU XingLian, QIU WeiFen. Effects of nuoB on the Biofilm Formation and Cellular Metabolism of Meat-Borne Pseudomonas fragi During Chilled Storage[J]. Scientia Agricultura Sinica, 2023, 56(11): 2172-2185.

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	细菌数量 Bacterial counts (log CFU/colony)	蛋白质 Protein (µg/mg-wet-cell-biomass)	总糖 Carbohydrate (µg/mg-wet-cell-biomass)	蛋白/总糖 Protein/Carbohydrate ratio
原位培养 In situ cultivation
NMC25	9.92±0.07	38.61±1.92	14.16±0.69	2.73±0.23
ΔnuoB	9.86±0.06	46.65±1.62**	16.55±0.93*	2.82±0.08
TSA培养 TSA cultivation
NMC25	10.11±0.09	0.61±0.36	17.23±0.80	0.04±0.02
ΔnuoB	10.05±0.03	2.10±0.63**	17.40±0.77	0.12±0.04**