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热带海洋学报

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珊瑚共附生嗜盐单胞菌原噬菌体上的HicAB毒素-抗毒素系统的结构分析

张雨1, 2, 刘自尧2, 王晓雪2, 陈冉2   

  1. 1 暨南大学生命科学技术学院, 广东 广州 510632;

    2 中国科学院南海海洋研究所热带海洋生物资源与生态重点实验室, 广东 广州 511458

  • 收稿日期:2025-02-17 修回日期:2025-03-14 接受日期:2025-03-18
  • 通讯作者: 陈冉
  • 基金资助:

    国家自然科学基金（42188102）; 中国科学院南海海洋研究所专项基金(SCSIO2023QY03); 海洋负碳排放(ONCE)计划; 海南大学南海海洋资源利用国家重点实验室开放项目(MRUKF2023001)。

Structural analysis of the HicAB toxin-antitoxin system encoded by a prophage in coral-associated Halomonas meridiana

ZHANG Yu1, 2, LIU Ziyao2, WANG Xiaoxue2, CHEN Ran2   

  1. 1 College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, 510632, China

    2 Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 511458, China

  • Received:2025-02-17 Revised:2025-03-14 Accepted:2025-03-18
  • Contact: CHEN, Ran
  • Supported by:
    National Science Foundation of China(42188102); Special Fund of the South China Sea Institute of Oceanology, Chinese Academy of Sciences(SCSIO2023QY03); the Ocean Negative Carbon Emissions (ONCE) Program; Open project of State Key Laboratory of South China Sea Marine Resources Utilization, Hainan University(MRUKF2023001)

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摘要： 噬菌体与细菌宿主的相互作用对珊瑚健康和珊瑚礁稳定性具有重要影响，同时也是病毒学领域的研究热点之一。为应对噬菌体的感染压力，细菌进化出多种固有和适应性免疫系统，其中毒素-抗毒素（toxin-antitoxin, TA）系统是重要的防御机制之一。本研究通过生物信息学分析预测，珊瑚共附生嗜盐单胞菌Halomonas meridiana SCSIO 43005（Hm43005）所携带的原噬菌体Phm2中的CTT34_05265和CTT34_05260基因分别编码HmHicA和HmHicB蛋白，并通过大肠杆菌生长实验证实其构成TA系统。Pull-down和细菌双杂交实验进一步验证了HmHicA与HmHicB之间的相互作用。凝胶迁移阻滞实验（EMSA）和DNaseⅠ足迹实验表明，HmHicB通过特异性结合HmhicA启动子-35区上游和-10区的回文序列，发挥转录自调控功能。此外，HmHicB单独表达对宿主表现出轻微毒性，提示抗毒素蛋白在细胞内可能具有其他调控靶点。结构分析显示，HmHicA具有核糖体非依赖性RNA切割酶活性，而HmHicB包含N端毒素结合结构域和C端DNA结合结构域。HmHicB通过C端形成同源二聚体，并与HmHicA以2:2的化学计量比形成复合物。HmHicA带正电荷的活性口袋与HmHicB带负电荷的毒素结合结构域之间的相互作用，以及HmHicA His24活性位点的包埋，可能是抗毒素抑制毒素活性的分子机制。这些研究结果为深入解析该TA系统的性质及其生理生态功能奠定了重要基础。

关键词: 珊瑚共附生细菌, 嗜盐单胞菌, 毒素-抗毒素系统, HicAB系统

Abstract: The interaction between phages and bacterial hosts significantly impacts coral health and reef stability, representing a key focus in virology. To combat phage infection, bacteria have evolved diverse innate and adaptive immune systems, including toxin-antitoxin (TA) systems, a crucial defense mechanism. In this study, bioinformatics analysis predicted that the CTT34_05265 and CTT34_05260 genes within the prophage Phm2 of the coral-associated bacterium Halomonas meridiana SCSIO 43005 (Hm43005) encode HmHicA and HmHicB proteins, respectively. Their TA system functionality was confirmed through E. coli growth assays. Pull-down and bacterial two-hybrid experiments validated the interaction between HmHicA and HmHicB. Electrophoretic mobility shift assays (EMSA) and DNase I footprinting revealed that HmHicB binds specifically to palindromic sequences upstream of the -35 and -10 regions of the HmhicA promoter, mediating transcriptional autoregulation. Additionally, HmHicB alone exhibited mild toxicity, suggesting potential alternative regulatory targets for the antitoxin. Structural analysis indicated that HmHicA functions as a ribosome-independent RNase, while HmHicB contains an N-terminal toxin-binding domain and a C-terminal DNA-binding domain. HmHicB forms a homodimer via its C-terminus and assembles with HmHicA in a 2:2 stoichiometric complex. The molecular mechanism of antitoxin-mediated toxin inhibition likely involves electrostatic interactions between the positively charged active pocket of HmHicA and the negatively charged toxin-binding domain of HmHicB, as well as the burial of the HmHicA His24 active site. These findings provide a foundation for further exploration of the properties and physiological roles of this TA system.

Key words: coral-associated bacteria, Halomonas meridiana, toxin-antitoxin system, HicAB system