海洋假交替单胞菌SM9913中基因岛GIPspSM9913的鉴定和功能研究

doi:10.11978/2022101

热带海洋学报 ›› 2023, Vol. 42 ›› Issue (2): 45-53.doi: 10.11978/2022101CSTR: 32234.14.2022101

海洋假交替单胞菌SM9913中基因岛GIPspSM9913的鉴定和功能研究

王鹏霞¹^,²^,³^,⁴(), 赵逸⁵, 杜晓飞¹^,³, 王伟权¹^,³, 王晓雪¹^,²^,³

1.中国科学院南海海洋研究所, 中国科学院热带海洋生物资源与生态重点实验室, 广东广州 510301
2.南方海洋科学与工程广东省实验室(广州), 广东广州 511458
3.中国科学院大学, 北京 100049
4.三亚海洋生态环境工程研究院, 海南三亚 572000
5.河北师范大学, 生命科学学院, 河北石家庄 050024

收稿日期:2022-05-06 修回日期:2022-06-29 出版日期:2023-03-10 发布日期:2022-07-01
通讯作者: 王鹏霞。email: wangpengxia@scsio.ac.cn
作者简介:
王鹏霞(1986—), 女, 安徽省太湖县人, 博士, 从事海洋细菌中可移动遗传元件的功能研究。email: wangpengxia@scsio.ac.cn
基金资助:
海南省科技计划三亚崖州湾科技城联合项目(320LH047); 广东省自然科学基金项目(2019A1515011912); 广州市科技计划项目(202002030493)

Identification and functional study of the genomic island GIPspSM9913 in Pseudoalteromonas sp. SM9913

WANG Pengxia¹^,²^,³^,⁴(), ZHAO Yi⁵, DU Xiaofei¹^,³, WANG Weiquan¹^,³, WANG Xiaoxue¹^,²^,³

1. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
2. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
3. University of Chinese Academy of Sciences, Beijing 100049, China
4. Sanya Institute of Ocean Eco-Environmental Engineering, Sanya 572000, China
5. College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China

Received:2022-05-06 Revised:2022-06-29 Online:2023-03-10 Published:2022-07-01
Contact: WANG Pengxia. email: wangpengxia@scsio.ac.cn
Supported by:
Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City(320LH047); Natural Science Foundation of Guangdong Province(2019A1515011912); Science and Technology Planning Project of Guangzhou(202002030493)

摘要/Abstract

摘要：

海洋细菌由于所处环境的复杂性和多变性, 进化出独特的环境适应机制。基因岛通常携带宿主细菌环境适应性有关的基因, 在推动细菌环境适应性和基因组多样化中起重要作用。假交替单胞菌属(Pseudoalteromonas)在各种海洋生境中广泛分布, 具有重要的工业应用价值和生态修复潜力。文章以深海沉积物中分离的假交替单胞菌SM9913为研究对象, 通过与表层海水中分离的假交替单胞菌TAC125进行比较基因组学分析, 发现SM9913基因组上yicC基因内部整合了一个18kb的基因岛, 我们命名为GIPspSM9913。该基因岛编码整合酶、切离酶以及多套限制修饰系统。数据库检索分析发现GIPspSM9913同源的基因岛在假交替单胞菌、希瓦氏菌(Shewanella)、弧菌(Vibrio)和气单胞菌(Aeromonas)等细菌中广泛存在。定量PCR结果显示, 当切离酶表达时, GIPspSM9913会从基因组上大量切离, 产生无基因岛的SM9913突变株。测序分析表明, GIPspSM9913的切除不会影响整合位点侧翼基因的表达。比较切离突变株与野生菌的运动能力、电转化频率和接合转移效率等, 发现GIPspSM9913可以提高宿主细菌的泳动能力和对外源DNA入侵的抵御能力。

关键词: 海洋细菌, 假交替单胞菌, 基因岛, 泳动能力, 环境适应性

Abstract:

Due to the complexity and variability of marine environments, marine bacteria may have evolved unique environmental adaptation mechanisms. Genomic islands usually carry genes related to the environmental adaptation of host bacteria and play an important role in driving bacterial adaptation and genome diversification. Pseudoalteromonas is an important genus that is widely distributed in various marine habitats, and has attracted attention due to the important industrial application and ecological restoration potential. In this study, we focused on Pseudoalteromonas sp. SM9913 isolated from marine sediments. By comparative genomics analysis of SM9913 with the closely related strain Pseudoalteromonas haloplanktis TAC125 isolated from surface seawater, an 18-kb genomic island GIPspSM9913 integrated in the yicC gene was identified. This genomic island encodes an integrase and an excisionase, as well as multiple restriction modification systems. Sequence analysis showed that GIPspSM9913 homologs are widely distributed in marine bacteria such as Pseudoalteromonas, Shewanella, Vibrio and Aeromonas. Quantitative PCR assays showed that GIPspSM9913 can excise when the excisionase is produced, resulting in the removal of GIPspSM9913. Sequencing analysis indicated that excision of GIPspSM9913 did not affect the expression of flanking genes. We also compared the swimming motility, electroporation efficiency and conjugation efficiency of the GIPspSM9913 deleted strain and the wild-type strain SM9913, and found that the presence of GIPspSM9913 can increase the swimming motility of the host bacteria and offer the host defense against the invasion of foreign DNA.

Key words: marine bacteria, Pseudoalteromonas, genomic island, motility, environmental adaptation

王鹏霞, 赵逸, 杜晓飞, 王伟权, 王晓雪. 海洋假交替单胞菌SM9913中基因岛GIPspSM9913的鉴定和功能研究[J]. 热带海洋学报, 2023, 42(2): 45-53.

WANG Pengxia, ZHAO Yi, DU Xiaofei, WANG Weiquan, WANG Xiaoxue. Identification and functional study of the genomic island GIPspSM9913 in Pseudoalteromonas sp. SM9913[J]. Journal of Tropical Oceanography, 2023, 42(2): 45-53.

图/表 7

表1

图1

表2

基因岛GIPspSM9913上所含基因的信息"

基因名称	基因编号	产物长度/氨基酸数	最相似蛋白[菌株](蛋白编号)	一致性/%	功能/结构域
int	PSM_A2839	409	整合酶[Pseudoalteromonas atlantica] (WP_024601833.1)	99	P4家族整合酶
cds1	PSM_A2840	264	假定蛋白[Pseudoalteromonas spiralis] (KYL33016.1)	99	假定蛋白
Xis	PSM_A2841	77	转录调控因子[Pseudoalteromonas spiralis] (KYL33017.1)	99	切离酶, 原噬菌体CP4-57的调控蛋白(AlpA)
cds2	PSM_A2842	38	-	-	假定蛋白
cds3	PSM_A2843	293	假定蛋白[Pseudoalteromonas spiralis] (KYL33019.1)	88	未知功能结构域(DUF3296)
cds4	PSM_A2844	188	假定蛋白[Pseudoalteromonas spiralis] (KYL33020.1)	98	假定蛋白
cds5	PSM_A2845	276	假定蛋白[Pseudoalteromonas] (WP_024601828.1)	99	LabA_like蛋白
cds6	PSM_A2846	56	假定蛋白[Paraglaciecola chathamensis S18K6] (GAC12573.1)	55	双功能抗毒素、转录抑制因子RelB
cds7	PSM_A2847	126	假定蛋白[Pseudoalteromonas] (WP_024601827.1)	100	假定蛋白
cds8	PSM_A2848	44	HsdR家族Ⅰ型限制修饰系统中的脱氧核糖核酸酶, 部分序列[Pseudoalteromonas arctica A 37-1-2] (ERG10608.1)	95	HsdR家族Ⅰ型限制修饰系统中的脱氧核糖核酸酶
hsdR	PSM_A2849	1057	DEAD/DEAH家族解旋酶[Marinospirillum minutulum] (WP_027849446.1)	78	HsdR家族Ⅰ型限制修饰系统中的脱氧核糖核酸酶
cds9	PSM_A2850	412	SIR2家族蛋白[Pseudogulbenkiania sp. MAI-1] (WP_024304176.1)	43	SIR2-like结构域蛋白
hsdS	PSM_A2851	437	限制性内切酶S亚基[Shewanella sp. Sh95] (WP_055647049.1)	59	Ⅰ型限制修饰系统中HsdS蛋白，EcoKI限制性修饰系统蛋白
cds10	PSM_A2852	355	50S核糖体蛋白L31 [Pseudoalteromonas haloplanktis] (WP_050582305.1)	95	限制性内切酶类(RecB)超家族的核酸酶
hsdM	PSM_A2853	559	DNA甲基化酶[Thalassolituus oleivorans R6-15] (AHK15533.1)	77	Ⅰ型限制性修饰系统中N-6 DNA甲基化酶
Mrr	PSM_A2854	92	限制性内切酶[Pseudoalteromonas sp. S2292] (KJZ24913.1)	96	含有(D/E)-(D/E)XK 活性位点的Ⅱ型限制酶，限制性内切酶 Mrr
cds11	PSM_A2855	195	假定蛋白[Pseudoalteromonas] (WP_052201246.1)	95	假定蛋白
cds12	PSM_A2856	234	大蛋白[Pseudoalteromonas spiralis] (KYL33168.1)	97	具有保守 AHH的HNH/ENDO Ⅶ位点的核酸酶
cds13	PSM_A2857	38	-	-	假定蛋白

表2

图2

图3

图4

图5

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引物	序列 (5′—3′)	用途
xis-F	CGGAATTCATGAACAAACTAATTAAACCAAC	pXis的构建和ΔGIPspSM9913的验证
xis-R	CCCAAGCTTTCATAAAGTGGAGCTCATG	pXis的构建和ΔGIPspSM9913的验证
GI-wF	GCCTGGTGTTATGGAAGCAC	ΔGIPspSM9913的验证
GI-wR	GATATTGATTGGCAAGGCGC	ΔGIPspSM9913的验证
GI-qF	GCTGTAGAGCTAAAAGTCCTGATTGA	定量(附着位点B, attachment site B) attB, 计算GIPspSM9913的切离率
GI-qR	CCAGAAGGCGCAGATAAAATAAATA	定量(附着位点B, attachment site B) attB, 计算GIPspSM9913的切离率
gyrB-qF	AGTGGCAACGCTAATTACTGCAT	定量SM9913基因组上内参基因gyrB, 计算 GIPspSM9913的切离率
gyrB-qR	CGGTAGAAGAACGTTAGTAGCAAGGT	定量SM9913基因组上内参基因gyrB, 计算 GIPspSM9913的切离率

海洋假交替单胞菌SM9913中基因岛GIPspSM9913的鉴定和功能研究

Identification and functional study of the genomic island GIPspSM9913 in Pseudoalteromonas sp. SM9913

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