海产品嗜冷菌波罗的海希瓦氏菌中3个冷激蛋白功能分析

Roles of three cold shock proteins in the psychrophilic seafood bacterium Shewanella baltica

摘要:

【目的】波罗的海希瓦氏菌是冷藏海产品中常见的腐败菌，而该菌中关于冷激蛋白的功能研究尚未见报道。本研究从分子生物学角度分析波罗的海希瓦氏菌中3个冷激蛋白各自的功能。【方法】采用BEAST软件分析γ-变形菌纲中部分食源性微生物的冷激蛋白进化时间，接着利用实时荧光定量PCR方法检测波罗的海希瓦氏菌3个冷激蛋白基因的表达规律，进而构建3个冷激蛋白的基因敲除株，分析敲除株在不同温度和不同环境胁迫条件下的生长状况、群体感应现象以及致腐能力，最后构建3个冷激蛋白的异源表达菌株并分析它们在不同温度和不同环境胁迫条件下的生长状况。【结果】波罗的海希瓦氏菌中鉴定到3个冷激蛋白，分别为cspC、cspD、cspG。所有γ-变形菌纲的cspD基因单独聚成一支，并于1 109.6百万年前与其他csp基因相分离，波罗的海希瓦氏菌的cspC和cspG在858.8百万年前互相分开。cspG基因是波罗的海希瓦氏菌低温生存的必需基因，且广泛响应环境胁迫条件；cspC基因对cspG基因功能的实施起辅助作用；cspD不响应冷激，但却会随生长阶段的变化而发生变化。此外，cspC基因和cspG基因在低温条件下与细菌的致腐能力相关。【结论】波罗的海希瓦氏菌3个冷激蛋白基因各有不同，且cspC基因和cspG基因与该菌致腐能力有关，这为今后研究腐败菌的冷适应和致腐机制提供了新思路。

Abstract:

[Objective] Shewanella baltica is a specific spoilage microorganism commonly detected in refrigerated seafood at the end of shelf life, while the knowledge about the roles of cold shock proteins (CSPs) in this bacterium is limited. This study aims to unravel the roles of three csp genes in S. baltica SB-19. [Methods] BEAST was employed to study the molecular evolutionary of csp genes in food-derived microorganisms of Gammaproteobacteria, and qPCR to determine the expression patterns of the three csp genes in S. baltica SB-19. The strains with csp gene knockout were constructed, and their growth rates, quorum sensing, spoilage ability were analyzed under different temperatures and environmental stresses. Finally, three strains with heterologous expression of csp were constructed, and their responses to different temperatures and environmental stresses were examined. [Results] Three csp genes, cspC, cspD, and cspG, were identified in S. baltica. All the cspD genes of Gammaproteobacteria formed a monophyletic clade and segregated from other csp genes approximately 1 109.6 million years ago (MYA). The cspC and cspG of S. baltica segregated approximately 858.8 MYA. Both cspC and cspG were involved in the response to cold shock, whereas cspD did not. The knockout and overexpression experiments revealed that cspG was essential for S. baltica survival at low temperature and played a universal role in the response to environmental stress, and cspC aided the bacterial response to stress. The cspD gene was associated with bacterial growth. Furthermore, the result of the experiments with inoculation of knockout strains in fish juice indicated that both cspC and cspG were associated with the spoilage ability of S. baltica below room temperature. [Conclusion] The three csp genes play different roles in S. baltica. The findings pave new ways for the research on the cold adaptation and spoilage mechanism of spoilage microorganisms.