P4类隐蔽性原噬菌体对宿主菌环境适应性的生理功能研究

Deep-sea bacteria can adapt to extreme habitats, but at present the study of deep-sea bacteria adaptation mechanism is limited. Bacteria in the same genus has the shared core genome, however, large differences are observed for the prophages with differences in their number, size, and gene content, suggesting that cryptic prophages may participate in the host adaptation to different environments. P4-like prophages have the following features: widespread in the marine bacteria, removable by natural excision, conservative insertional locus and similar genes. Therefore, this project will focus on deep-sea bacteria isolated from different habitats, with an aim to explore the physiological relevance of P4-like cryptic prophages in bacterial host. Bacteria from brackish estuary and land-source also harbor P4-like prophages will be compared to the deep-sea bacteria. The physiological function of P4-like cryptic prophages in five bacteria from different habitats will be assessed to reveal the potential relationship between the cryptic prophages and the adaptability of their host to specific habitats. Expression of these P4-like cryptic prophage genes in adverse environmental conditions will be assessed via quantitative RT-PCR to reveal the relationship between the gene expression regulation of cryptic prophages by their bacteria host and the environmental adaptability of their host. The natural excision frequency of these P4-like prophages will be quantified via qPCR to reveal the mechanism of controlling the stability of cryptic prophages on the host genome.

深海细菌能够适应极端的海洋生境，但目前对其环境适应机制的研究还较少。不同生境来源的同属细菌中核心基因组相同，但隐蔽性原噬菌体的个数、大小和基因组成差别很大，暗示隐蔽性原噬菌体可能参与了细菌对不同环境的适应过程。P4类原噬菌体具有在海洋细菌中广泛存在，可自然切离，插入位点保守和组成基因相似等特征。本项目拟以不同生境来源的深海细菌为研究对象，以河口和陆地来源的细菌为对照，开展P4类隐蔽性原噬菌体帮助细菌适应不同生境机制的研究。运用分子生物学手段检测不同生境来源细菌中P4类隐蔽性原噬菌体的生理功能，揭示原噬菌体与宿主菌环境适应性之间的关系；运用定量RT-PCR分析不同环境压力下原噬菌体基因的表达差异，揭示宿主菌对原噬菌体基因的调控与宿主菌对环境的适应能力之间的关系；运用定量PCR检测不同环境压力下原噬菌体自发切离的频率，揭示宿主菌通过调节原噬菌体的稳定性来适应环境变化的遗传学机制。

海洋细菌能够适应多变的海洋生境，但目前对其环境适应机制的研究还较少。隐蔽性原噬菌体在海洋细菌中广泛存在，可能在海洋细菌环境适应性中起重要作用。本研究开展了六株不同生境来源海洋细菌中P4类隐蔽性原噬菌体的功能研究，发现P4类隐蔽性原噬菌体通常整合在海洋细菌高度保守的yicC基因内，编码毒素抗毒素系统、限制修饰系统、调控因子等；一般情况下，P4类隐蔽性原噬菌体在野生菌中稳定存在，切离酶超表达后，可从基因组上自然切离；利用此原理，我们构建了P4类隐蔽性原噬菌体自然切离突变株；表型检测发现隐蔽性原噬菌体影响宿主菌运动性、生物膜形成或对外源DNA抵御等重要生命过程；进化分析发现，yicC位点的P4类隐蔽性原噬菌体至少分为两个亚类，其中，以11900_P4-like为代表的亚类可与SXT/R391家族整合型转移元件ICE相互作用。ICE编码的调控因子SetCD不仅可以激活11900_P4-like的切离和转移，还可以激活11900_P4-like的复制从而上调其携带的所有基因的表达。而以SM9913_P4-like为代表的亚类不与SXT/R391 ICE相互作用，可能通过其他机制被激活切离和转移。实验过程中，我们发现SCSIO 11900含有的另一个水平转移元件（基因岛GIprfC）为该菌株高产生物膜必须的基因片段，分析发现GIprfC不含任何转移有关基因，然而GIprfC可与SXT/R391 ICE串联而被带动转移，阐明了一种基因岛水平转移的新机制；研究还发现，大肠杆菌BW25113中P4原噬菌体上毒素抗毒素系统参与了宿主菌对抗氧化胁迫压力和生物膜形成。综上，海洋细菌可通过调控P4类隐蔽性原噬菌体的切离，转移及所含基因的表达来调控宿主菌的运动性、生物膜形成等来适应海洋环境。

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