珊瑚共附生嗜盐单胞菌原噬菌体上的HicAB毒素-抗毒素系统的鉴定及其结构分析

doi:10.11978/2025022

热带海洋学报 ›› 2025, Vol. 44 ›› Issue (6): 120-131.doi: 10.11978/2025022CSTR: 32234.14.2025022

珊瑚共附生嗜盐单胞菌原噬菌体上的HicAB毒素-抗毒素系统的鉴定及其结构分析

张雨¹^,²(), 刘自尧², 王晓雪², 陈冉²()

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 出版日期:2025-11-10 发布日期:2025-12-03
通讯作者: 陈冉。email: chenran@scsio.ac.cn
作者简介:
张雨(1999—), 男, 江西省上饶市人, 硕士, 从事海洋微生物生理与生化研究。email: yzhang0517@126.com
基金资助:
国家自然科学基金项目(42188102); 中国科学院南海海洋研究所专项基金(SCSIO2023QY03); 海洋负排放国际大科学计划(ONCE); 海南大学南海海洋资源利用国家重点实验室开放项目(MRUKF2023001)

Identification and 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 College of Life Science and Technology, Jinan University, Guangzhou 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 Online:2025-11-10 Published:2025-12-03
Contact: CHEN Ran. email: chenran@scsio.ac.cn
Supported by:
National Natural Science Foundation of China(42188102); Special Fund of the South China Sea Institute of Oceanology, Chinese Academy of Sciences(SCSIO2023QY03); Ocean Negative Carbon Emissions Program(ONCE); Open project of State Key Laboratory of South China Sea Marine Resources Utilization, Hainan University(MRUKF2023001)

摘要/Abstract

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, 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(electrophoretic mobility shift assay, EMSA)和DNaseⅠ足迹实验表明, HmHicB通过特异性结合hicAB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 specifically binds to palindromic sequences upstream of the -35 and -10 regions of the hicAB 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

中图分类号:

Q939.9

张雨, 刘自尧, 王晓雪, 陈冉. 珊瑚共附生嗜盐单胞菌原噬菌体上的HicAB毒素-抗毒素系统的鉴定及其结构分析[J]. 热带海洋学报, 2025, 44(6): 120-131.

ZHANG Yu, LIU Ziyao, WANG Xiaoxue, CHEN Ran. Identification and structural analysis of the HicAB toxin-antitoxin system encoded by a prophage in coral-associated Halomonas meridiana[J]. Journal of Tropical Oceanography, 2025, 44(6): 120-131.

图/表 10

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菌株	描述	来源
Hm43005	Isolated from Coral samples of G. fascicularis.	实验室保存
WM3064	thrB1004 pro thi rpsL hsdS lacZΔM15 RP4-360 Δ(araBAD)567 ΔdapA1341:: [erm pir]	实验室保存
BL21 (DE3)	F^-ompT hsdS_B(r_B^-m_B^-) gal dcm λ(DE3) Ω P_tacUV5::T7 polymerase	Novagen
BW25113 (K-12)	lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78 rph-1	Baba et al, 2006
Top10	F^-, mcrAΔ(mrr-hsd RMS-mcrBC), ϕ80, lacZΔM15, ΔlacX74, recA1, araΔ139Δ(ara-leu)7697, galU, galK, rps, (Strr) endA1, nupG	实验室保存

质粒	描述	来源
pHGECm	Cm^R, IPTG inducible expression vector in E.coli and H.meridiana SCSIO 43005 strains	实验室保存
pHGECm-hicA	Cm^R; over-expression vector for CTT34_05265	本研究
pHGECm-hicB	Cm^R; over-expression vector for CTT34_05260	本研究
pHGECm-hicAB	Cm^R; over-expression vector for CTT34_05260 and CTT34_05265 as one operon	本研究
pUT18C-hicA	Amp^R; interaction assay vector for CTT34_05265	本研究
pKT25-hicB	Km^R; interaction assay vector for CTT34_05260	本研究
pET28b	Km^R, IPTG inducible expression and purified vector	Novagen
pET28b-hicA-his	Km^R; pET28b P_T7-lac::hicA with C-terminal His-tagged	本研究
pET28b-hicB-his	Km^R; pET28b P_T7-lac::hicB with C-terminal His-tagged	本研究
pET28b-his-hicAB	Km^R; pET28b P_T7-lac:: hicAB with N-terminal His-tagged	本研究

引物名称	序列(5′-3′)	用途
pHGECm-hicA-F	TAACAATTTCACACAGGAGAGATGAACAGCAGAGCACTGATC	毒性验证
pHGECm-hicA-R	ATCCGCCAAAACAGCCAAGCTTCATTCGAGGCCAGCGCTTTTC	毒性验证
pHGECm-hicB-F	TAACAATTTCACACAGGAGAGATGTTATTTCCCATTGCCATTG	毒性验证
pHGECm-hicB-R	ATCCGCCAAAACAGCCAAGCTTTAGCTGGCTTGTTTGTTCCTG	毒性验证
pUT18C-hicA-F	ACTCTAGAGGATCCCCGGGTACCGATGAACAGCAGAGCACTGATC	细菌双杂交
pUT18C-hicA-R	ATTACTTAGTTATATCGATGAATTTCATTCGAGGCCAGCGCTTTTC	细菌双杂交
pKT25-hicB-F	CTAGAGGATCCCCGGGTACCTATGTTATTTCCCATTGCCATTG	细菌双杂交
pKT25-hicB-R	GAATTCTTAGTTACTTAGTTAGCTGGCTTGTTTGTTCCTG	细菌双杂交
pET28b-hicA-his-F	CTTTAAGAAGGAGATATACCATGAACAGCAGAGCACTGATC	蛋白表达纯化
pET28b-hicA-his-R	CTCGAGTGCGGCCGCAAGCTTTTCGAGGCCAGCGCTTTTCC	蛋白表达纯化
pET28b-hicB-his-F	CTTTAAGAAGGAGATATACCATGTTATTTCCCATTGCCATTG	蛋白表达纯化
pET28b-hicB-his-R	CTCGAGTGCGGCCGCAAGCTTGCTGGCTTGTTTGTTCCTGGC	蛋白表达纯化
pET28b-his-hicAB-F	GGTGGACAGCAAATGGGTCGGATGAACAGCAGAGCACTGATC	Pull-down分析
pET28b-his-hicAB-R	CTCGAGTGCGGCCGCAAGCTTTTAGCTGGCTTGTTTGTTCCTG	Pull-down分析

珊瑚共附生嗜盐单胞菌原噬菌体上的HicAB毒素-抗毒素系统的鉴定及其结构分析

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

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