希瓦氏菌内源性质粒pMR-1中两对毒素-抗毒素系统的功能研究

Toxin-antitoxin systems (TAs) are prevalent in bacteria and archaea, and they are are small genetic elements encoded in prokaryotic chromosomal and plasmid DNA. The primary function of a plasmid TA module is to stabilize plasmid by eliminating plasmid-free daughter cells through a post segregation killing mechanism. In recent years, TAs were proved to act on crucial cellular processes including translation, replication, cytoskeleton formation, membrane integrity, and cell wall biosynthesis, their physiological roles remain enigmatic. Bacterial strain Shewanella oneidensis is a target of extensive research in the fields of bioelectrochemical systems and bioremediation. An endogenous plasmid pMR-1 was identified with several potential TAs presences, but studies on identification and functional characterization of TAs are largely lack in this strain. Based on previous progress, this project will focus on the functional studies of two pairs of TAs in the pMR-1 plasmid. The aim of this project is to identify and characterization of two pairs of TAs encoded by endogenous plasmid, and to study the toxic effect of toxin as well as the antitoxic activity of the antitoxin. In addition, specific efforts will be made to study the expression and regulation of the TA loci under various stresses (oxidative stress, pH stress antibiotics stress or heavy metal stress) and during biofilm formation. The proposed study will help us gain knowledge about the functions of plasmid-encoded TAs, and aid us to understand the physiological relevance of harboring endogenous plasmid especially under stress conditions. Potential industrial application of plasmid-encoded TAs to replace the use of antibiotics will also be explored in this study.

毒素-抗毒素系统首先发现于低拷贝质粒，是细菌和古细菌内丰富的小型遗传元件，由染色体或质粒编码。近年发现，毒素-抗毒素系统参与细胞内多个关键的生物学过程，包括复制、转录、翻译、细胞骨架形成、细胞膜的完整性及细胞壁的形成等，但其生理功能仍需完善。希瓦氏MR-1为环境微生物的模式菌，含有稳定的内源性质粒pMR-1。pMR-1编码多对潜在毒素-抗毒素系统，但缺乏功能研究。在前期工作基础上，本项目以鉴定的两对pMR-1编码的毒素-抗毒素系统为研究对象，运用传统微生物学和现代分子遗传学手段，研究毒素的毒性机理及抗毒素的拮抗机理；探讨环境胁迫条件下毒素-抗毒素系统的响应机制；并初步揭示pMR-1编码的毒素-抗毒素系统对生物被膜形成的调控作用。本项目不仅有助于进一步解析质粒编码的毒素-抗毒素系统功能，也有助于了解质粒及其编码的毒素-抗毒素在细菌环境适应过程中的作用，并有助于挖掘二者潜在的工业应用价值。

毒素-抗毒素系统广泛分布于细菌染色体和内源性质粒中，结构和功能上具有多样性，但被深入研究的毒素-抗毒素系统非常有限。基于本项研究：（1）鉴定了大质粒pMR-1编码的多对毒素-抗毒素系统，阐明其中2对的结构特征和调控方式；(2) 发现其中1对毒素-抗毒素系统SO_A0040-SO_A0041与我们发现的调控MR-1低温适应的原噬菌体CP4So编码的毒素-抗毒素系统ParESo-CopASo通过编码区相互调控；(3) 阐释环境菌株中的2对毒素-抗毒素系统MqsRA和 VapBC的功能和调控方式，前者参与生物膜和细菌多重耐药性形成，后者激活大肠杆菌多对毒素-抗毒素系统；（4）基于毒素-抗毒素系统的研究，2017年受邀撰写毒素-抗毒素领域研究新进展，总结了不同类型毒素-抗毒素系统的相互作用；（5）拓展了研究内容，揭示了丝状噬菌体编码切离酶，控制噬菌体的溶原和裂解循环；也发现了C-端锚定的内膜蛋白ElaB参与细菌的多重耐药性形成以及氧化胁迫和温度胁迫，并发现其受OxyR和RpoS的调控，且OxyR的调控具有RpoS依赖性。本项目执行期间，共发表6篇SCI论文，影响因子全部大于3，还发表2篇中文核心期刊。这些杂志主要包括国内外微生物领域的权威期刊Environmental Microbiology, Molecular Microbiology, Journal of Bacteriology，微生物学报等。除发表学术论文外，本项目负责人郭云学在项目执行期间评为副研究员，并协助培养硕士和博士研究生共3名。