两株溶藻弧菌噬菌体的生理特性和基因组研究

doi:10.11978/2020067

热带海洋学报 ›› 2021, Vol. 40 ›› Issue (4): 22-34.doi: 10.11978/2020067CSTR: 32234.14.2020067

两株溶藻弧菌噬菌体的生理特性和基因组研究

钟宛宣^1,²(), 杨芸兰³, 李祥付^1,⁴, 徐杰^1,^2,⁴()

1.中国科学院南海海洋研究所, 热带海洋环境国家重点实验室, 广东广州 510301
2.中国科学院大学海洋学院, 北京 100049
3.厦门大学环境与生态学院, 近海海洋环境科学国家重点实验室, 福建厦门 361102
4.中国科学院南海生态环境工程创新研究院, 广东广州 510301

收稿日期:2020-06-24 修回日期:2020-07-26 出版日期:2021-07-10 发布日期:2020-07-29
通讯作者: 徐杰
作者简介:钟宛宣(1995—), 女, 广东省广州市人, 硕士研究生。email: zhongwanxuan17@mails.ucas.ac.cn
基金资助:
广州市科技计划项目(20190420029);国家自然科学基金(41506095);国家自然科学基金(41676075);中国科学院南海生态环境工程创新研究院自主部署项目(ISEE2019ZR02);广东省省级科技计划项目(2018B030320005);中国博士后科学基金(2019M662237)

Physiological and genomic characteristics of two lytic phages infecting Vibrio alginolyticus

ZHONG Wanxuan^1,²(), YANG Yunlan³, LI Xiangfu^1,⁴, XU Jie^1,^2,⁴()

1. State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
2. College of Marine Science, University of Chinese Academy of Sciences, Beijing 100049, China
3. College of the Environment and Ecology, State Key Laboratory for Marine Environmental Science, Xiamen University, Xiamen, 361102 China
4. Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou, 510301 China

Received:2020-06-24 Revised:2020-07-26 Online:2021-07-10 Published:2020-07-29
Contact: XU Jie
Supported by:
Project of Program of Science and Technology of Guangzhou(20190420029);National Natural Science Foundation of China(41506095);National Natural Science Foundation of China(41676075);Project of Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences(ISEE2019ZR02);Project of Department of Science and Technology of Guangdong Province(2018B030320005);China Postdoctoral Science Foundation(2019M662237)

摘要/Abstract

摘要：

利用噬菌体治疗水产养殖细菌病害具有专一性强、自我增殖能力强等优点, 可以有效减少对环境的污染。为了获得可防治水产病原体溶藻弧菌(Vibrio alginolyticus)的生物杀菌剂, 本研究以Vibrio alginolyticus ATCC 17749^T为宿主, 从福建省东山岛的对虾养殖场与水产市场污水环境中分离得到两株巨型噬菌体vB_ValM_R10Z和vB_ValM_R11Z, 并分别从噬菌体形态、宿主范围、生命周期和基因组信息等方面进行了鉴定和分析。实验结果显示, 噬菌体R10Z和R11Z具有相似的噬菌斑形态和电镜形态, 属于肌尾噬菌体; 除宿主之外, 噬菌体R10Z和R11Z均能侵染另一株欧文氏弧菌Vibrio owensii JL3186; 两株噬菌体均对氯仿不敏感, 表明其衣壳蛋白不含脂类物质; 噬菌体R10Z和R11Z的潜伏期均为20min, 但裂解量却相差一倍, 分别为45PFU·cell^-1和114PFU·cell^-1; 两株噬菌体的基因组大小分别为247167bp和246831bp, G+C含量分别为41.30%和41.33%, 两者基因组相似性达99.45%, 均未检测出毒力基因与抗药性基因; 系统发育分析显示两株噬菌体均属于Myoviridae(科), Tevenvirinae(亚科), Schizotequatrovirus(属), 同源基因在环境中分布广泛。两株噬菌体性状优良, 在水产病害的噬菌体治疗领域具有重要的研究价值和潜在的应用价值。

关键词: 弧菌, 噬菌体, 噬菌体治疗, 生理特性, 基因组

Abstract:

Phage therapy of bacterial diseases was considered to be effective and environmentally friendly in aquaculture. To obtain the biological agents that prevent Vibro alginolyticus, two jumbo phages vB_ValM_R10Z and vB_ValM_R11Z were isolated from prawn farming waters and the sewage in fishery markets in Dongshan Island, Fujian Province, using Vibrio alginolyticus ATCC 17749 ^T as the host. The morphology, host range, life cycle, and genome were identified and analyzed. Phages R10Z and R11Z had similar morphology of plaques and virions, and shared the same host range of infecting other V. owensii strain JL3186. Both phages were not sensitive to chloroform, indicating that the capsid did not contain lipids. They had the same latent period of 20 min but different blast sizes of 45 PFU·cell ^-1 for R10Z and 114 PFU·cell^-1 for R11Z. The genomic sizes of phages R10Z and R11Z were 247167 bp and 246831 bp with G+C content of 41.30% and 41.33%, respectively. Furthermore, their genomes had a similarity of 99.45%, without virulence genes and antibiotic resistance genes. Our phylogenetic analysis showed that they belonged to genus Schizotequatrovirus, subfamily Tevenvirinae, family Myovirdae. Their homologous genes were distributed in the environment widely. Phages R10Z and R11Z had good capability and great potentials for their usage as phage therapy agents of vibriosis in aquaculture.

Key words: Vibrio, phage, phage therapy, physiological properties, genome

中图分类号:

P735.531

钟宛宣, 杨芸兰, 李祥付, 徐杰. 两株溶藻弧菌噬菌体的生理特性和基因组研究[J]. 热带海洋学报, 2021, 40(4): 22-34.

ZHONG Wanxuan, YANG Yunlan, LI Xiangfu, XU Jie. Physiological and genomic characteristics of two lytic phages infecting Vibrio alginolyticus[J]. Journal of Tropical Oceanography, 2021, 40(4): 22-34.

图/表 8

图1

表1

图2

图3

表2

噬菌体vB_ValM_R10Z和vB_ValM_R11Z的tRNA基因"

vB_ValM_R10Z				vB_ValM_R11Z
tRNA基因序号	位置	氨基酸	反密码子	tRNA基因序号	位置	氨基酸	反密码子
1	164151~164264	Phe	AAA	1	163682~163794	Phe	AAA
2	164275~164399	Asn	GTT	2	163805~163929	Asn	GTT
3	164784~164861	Met	CAT	3	164314~164391	Met	CAT
4	166706~166779	Pro	TGG	4	166236~166309	Pro	TGG
5	166789~166865	Pro	TGG	5	166319~166395	Pro	TGG
6	166873~166948	Arg	ACG	6	166404~166479	Arg	ACG
7	167302~167378	Leu	TAG	7	166833~166909	Leu	TAG
8	167380~167454	Phe	GAA	8	166911~166985	Phe	GAA
9	167544~167618	Lys	TTT	9	167075~167149	Lys	TTT
10	167630~167706	Lys	TTT	10	167161~167237	Lys	TTT
11	167770~167844	Glu	TTC	11	167301~167375	Glu	TTC
12	167851~167936	Tyr	GTA	12	167382~167467	Tyr	GTA
13	168039~168115	Met	CAT	13	167570~167646	Met	CAT
14	168411~168509	Ser	TGA	14	167942~168040	Ser	TGA
15	168574~168647	Ile	GAT	15	168105~168178	Ile	GAT
16	168654~168727	Trp	CCA	16	168185~168258	Trp	CCA
17	168803~168877	Asn	GTT	17	168334~168408	Asn	GTT
18	168879~168954	Asn	GTT	18	168410~168485	Asn	GTT
19	169024~169099	Asp	GTC	19	168554~168629	Asp	GTC
20	169119~169192	Gln	TTG	20	168649~168722	Gln	TTG
21	169322~169396	Thr	TGT	21	168850~168925	Thr	TGT
22	169968~170042	Ala	TGC	22	169167~169241	Ala	TGC
23	170117~170192	Arg	TCT	23	169316~169391	Arg	TCT
vB_ValM_R10Z				vB_ValM_R11Z
tRNA基因序号	位置	氨基酸	反密码子	tRNA基因序号	位置	氨基酸	反密码子
24	170717~170792	Met	CAT	24	169915~169990	Met	CAT
25	170982~171055	Gly	TCC	25	170180~170253	Gly	TCC
26	171062~171135	Val	TAC	26	170260~170333	Val	TAC
27	171551~171627	His	GTG	27	170749~170825	His	GTG
28	171640~171713	Cys	GCA	28	170838~-170911	Cys	GCA
29	171716~171792	Leu	TAA	29	170914~170990	Leu	TAA
30	171909~171985	Leu	CAA	30	171107~171183	Leu	CAA
31	172154~172242	Ser	GCT	31	171352~171440	Ser	GCT

表2

图4

表3

图5

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细菌种属	株系	致病性	vB_ValM_R10Z	vB_ValM_R11Z
V. alginolyticus	ATCC 17749^T	可致病	+	+
V. alginolyticus	JL2674	未知	-	-
V. campbellii	JL2671	未知	-	-
V. campbellii	JL3506	未知	-	-
V. parahemolyticus	ATCC 17802^T	可致病	-	-
V. parahemolyticus	BVP2	未知	-	-
V. parahemolyticus	20160623-13	可致病	-	-
V. parahemolyticus	20160719-2	可致病	-	-
V. parahemolyticus	20160615-5	可致病	-	-
V. harveyi	BVH1	可致病	-	-
V. harveyi	20150916-2	可致病	-	-
V. owensii	JL2581	未知	-	-
V. owensii	JL3186	未知	+	+
V. owensii	JL3187	未知	-	-
V. caribbeanicus	JL3219	未知	-	-
V. fortis	JL3515	未知	-	-
V. chagasii	JL3518	未知	-	-
V. inhibens	JL3707	未知	-	-
V. cholerae	20161020-5	可致病	-	-
V. variabilis	JL3833	未知	-	-

噬菌体	vB_ValM_R10Z	vB_ValM_R11Z	VH7D	φSt2	φGrn1	ValKK3	KVP40	φpp2	nt-1
宿主菌	V. alginolyticus	V. alginolyticus	V. sp. 7D	V. alginolyticus	V. alginolyticus	V. alginolyticus	V. parahaemolyticus	V. parahaemolyticus	V. natrigens
地点	中国东山岛	中国东山岛	中国厦门	希腊克里特岛	希腊克里特岛	马来西亚	日本Urado湾	中国台湾南部	大西洋
分离环境	养殖水体	污水	养殖水体	海水	海水	沉积物	污水	养殖水体	沉积物
头长/nm	147	148	155	151	138	140	140	150	120
头宽/nm	78	81	75	81	74	79	70	90	70
尾长/nm	120	114		132	134	120	130	110	110
潜伏期/min	20	20		30	30
裂解量/PFU·cell^-1	43	114		97	44
噬菌体	vB_ValM_R10Z	vB_ValM_R11Z	VH7D	φSt2	φGrn1	ValKK3	KVP40	φpp2	nt-1
GenBank 收录号	MT612988	MT612989	KC131129.1	KT919973.1	KT919972	KP671755.1	AY283928	JN849462	HQ317393
基因组大小/bp	247167	246831	246964	250485	248605	248088	244835	246421	247489
G+C值	41.30%	41.33%	41.31%	42.60%	38.80%	41.20%	42.60%	42.55%	41.33%
ORF数量/个	386	385	387	412	410	390	386	383	379
tRNA数量/个	31	31	27	29	28		30	30	28
毒力基因	无	无	无	无	无	无	无	无	无
抗药性基因	无	无	无	无	无	无	无	无	无
参考文献	本文	本文	Luo et al, 2015	Kalatzis et al, 2016; Skliros et al, 2016	Kalatzis et al, 2016; Skliros et al, 2016	Lal et al, 2016	Matsuzaki et al, 1992; Miller et al, 2003	Lin et al, 2012	Zachary, 1976

两株溶藻弧菌噬菌体的生理特性和基因组研究

Physiological and genomic characteristics of two lytic phages infecting Vibrio alginolyticus

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