海洋细菌生物被膜可拉酸含量影响厚壳贻贝稚贝附着

解静仪; 王小雨; 李局; 杨金龙; 梁箫

doi:10.12284/hyxb2023104

海洋细菌生物被膜可拉酸含量影响厚壳贻贝稚贝附着

doi: 10.12284/hyxb2023104

解静仪^{1, 2, 3,},
王小雨^{1, 2, 3},
李局^{1, 2, 3},
杨金龙^{1, 2, 3},
梁箫^{1, 2, 3, ,}

1.
上海海洋大学国家海洋生物科学国际联合研究中心，上海 201306
2.
上海海洋大学水产种质资源发掘与利用教育部重点实验室，上海 201306
3.
上海海洋大学水产动物良种创制与绿色养殖协同创新中心，上海 201306

基金项目: 上海市学术带头人项目（20XD1421800）；国家自然科学基金（41876159）；国家重点研发计划（2022YFE0204600）。

详细信息

作者简介:
解静仪（1997-），女，山东省淄博市人，研究方向为海洋贝类分子生物学。E-mail: m200100009@st.shou.edu.cn

通讯作者:
梁箫（1983-），女，博士，副教授，主要从事海洋微生物与海洋贝类互作关系的研究。E-mail: x-liang@shou.edu.cn

中图分类号: S968.3；P714⁺.5
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出版历程
- 收稿日期: 2023-01-29
- 修回日期: 2023-04-17
- 网络出版日期: 2023-08-22
- 刊出日期: 2023-08-31

Effect of the content of colanic acid in marine bacterial biofilms on the settlement of Mytilus coruscus plantigrades

Xie Jingyi^{1, 2, 3
,},
Wang Xiaoyu^{1, 2, 3},
Li Ju^{1, 2, 3},
Yang Jinlong^{1, 2, 3},
Liang Xiao^{1, 2, 3
, ,}

1.
International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2.
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
3.
Shanghai Collaborative Innovation Center for Cultivating Elite Breeds and Green-Culture of Aquaculture Animals, Shanghai Ocean University, Shanghai 201306, China

摘要

摘要: 可拉酸是生物被膜上重要的胞外多糖之一，但细菌可拉酸对海洋无脊椎动物附着过程的影响还鲜少研究。本研究从自然生物被膜中分离出8株海洋细菌，对其种属进行鉴定及聚类分析，并测定其生物被膜的可拉酸含量及对稚贝附着的诱导能力。筛选所得海洋细菌形成生物被膜并测定其成膜能力及胞外产物含量，发现β-多糖的生物量与厚壳贻贝稚贝附着率呈显著正相关趋势（p < 0.05）。8株海洋细菌生物被膜中可拉酸含量的定量结果显示，3株革兰氏阳性菌无法产生可拉酸，5株革兰氏阴性菌均可检测到不同含量的可拉酸，其中革兰氏阴性菌Shewanella marisflavi的可拉酸含量最高，为1 076.43 μg/mL。不同可拉酸含量的海洋细菌单一生物被膜与厚壳贻贝稚贝附着率之间关系的研究结果显示，海洋细菌生物被膜对厚壳贻贝稚贝附着率的诱导效果与其可拉酸含量呈显著正相关（p < 0.05）。以上结果表明，细菌生物被膜中的可拉酸能够参与诱导厚壳贻贝稚贝的附着。本研究为探究海洋细菌生物被膜的化学物质与海洋贝类附着之间的相互作用及其对贝类附着机制提出了新的见解。
- 生物被膜 /
- 厚壳贻贝 /
- 稚贝 /
- 可拉酸 /
- 附着
Abstract: Colanic acid is one of the vital exopolysaccharides in biofilms, yet the effect of marine bacterial colanic acid on the settlement of invertebrates is still rarely covered. In this study, eight strains of marine bacteria isolated from natural biofilms were identified and the phylogenetic analysis was carried out, the colanic acid content and inducing ability of biofilms were also determined. Before that, the biofilm formation capacity and the extracellular products of the screened bacteria were detected. It was found that β-polysaccharide had a significant positive correlation with the settlement rate of Mytilus coruscus plantigrades (p < 0.05). The quantitative results of colanic acid content showed that among the five Gram-negative bacteria which could produce colanic acid, Shewanella marisflavi had the highest colanic acid yield of 1076.43 μg/mL, and three Gram-positive bacteria could not generate it. The results of plantigrade settlement induced by different biofilms showed that the inducing activity of marine bacterial biofilms on the settlement rate of M. coruscus was positively correlated with the content of colanic acid (p < 0.05). These findings prove that the Gram-negative bacteria with colanic acid can positively regulate the settlement of M. coruscus plantigrades, while the Gram-positive bacteria without colanic acid have no inducing activity. This study provides further insights to figure out the interaction between the chemical cues of biofilms and marine molluscs.
- biofilm /
- Mytilus coruscus /
- plantigrade /
- colanic acid /
- settlement

HTML全文

图 1 8株细菌的菌落形态及邻接法构建的系统发育树

a. 8株细菌的表型特征; b. 邻接法构建16S rDNA基因序列系统发育树

Fig. 1 The colony morphological and phylogenetic tree constructed by the Neighbor-Joining method of the eight marine bacterial strains

a. The morphological characteristics of eight bacterial species; b. phylogenetic tree of 16S rDNA gene sequences constructed by the Neighbor-Joining method

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图 2 不同海洋细菌生物被膜对厚壳贻贝稚贝附着率

不同字母代表显著性差异

Fig. 2 Percentages of settlement of Mytilus coruscus plantigrade on the different marine bacterial biofilms

Difference letters represent significant difference

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图 3 不同海洋细菌生物被膜形成能力

A. 不同海洋细菌形成生物被膜CLSM 拍摄图像；B. 不同海洋细菌形成生物被膜的终密度；C. 不同海洋细菌生物被膜膜厚；不同字母代表显著性差异

Fig. 3 Biofilm formation ability of different marine bacteria

A. Confocal laser scanning micrographs of biofilms; B. the density of eight monospecific bacterial biofilms; C. the thickness of biofilms; difference letters represent significant difference

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图 4 不同海洋细菌生物被膜胞外产物生物量体积

不同字母代表显著性差异

Fig. 4 Biomass volume of extracellular products of different marine bacterial biofilm

Difference letters represent significant difference

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图 5 8株海洋细菌的可拉酸染色（A）及可拉酸含量测定（B）

A. 光镜下观察可拉酸在媒染生物膜上的分布，细菌被染成红色，可拉酸被染成蓝色；B. 生物被膜可拉酸浓度，红色箭头表示未检测到可拉酸，不同字母代表显著性差异

Fig. 5 The dyeing (A) and determination of the content (B) of colanic acid of eight marine pacteria

A. The distribution of colanic acid on mordant-stained biofilms under light microscope, the strains were stained red and the colanic acid was stained blue; B. the colanic acid concentration of biofilms, the red arrow indicates no colanic acid was detected, difference letters represent significant difference

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图 6 厚壳贻贝稚贝附着率与细菌生物被膜可拉酸含量的关系

Fig. 6 Interaction between settlement rate of Mytilus coruscus plantigrades and colonic acid content of baicterial biofilm

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A1 激光共聚焦扫描显微镜拍摄生物被膜胞外产物（黄色：α-多糖；蓝色：β-多糖；绿色：蛋白；红色：脂质）

A1 CLSM reveals extracellular substances of biofilm ( yellow: α-polysaccharide; blue: β-polysaccharide; green: protein; red: lipid)

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表 1 激光扫描共聚焦显微镜荧光染料信息

Tab. 1 Confocal laser scanning microscope fluorescence dye information

染料	结合物质	波长/nm
碘化丙啶（PI）	死细菌	561
四甲基罗丹明共轭物刀豆蛋白（ ConA-TMR）	α-多糖	552/578
卡尔科弗卢尔荧光增白剂（CFW）	β-多糖	254/432
1,1'−双十八烷基−3,3,3',3'− 四甲基吲哚二碳花菁高氯酸盐（DiD’oil）	脂质	648/670
异硫氰酸荧光素（FITC）	蛋白质	495/519

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表 2 不同海洋细菌的16S rDNA序列分析

Tab. 2 16S rDNA sequence analysis of different marine bacteria

序列号	登录号	对照菌名	比对序列号	相似度/%	来源
ECSMB14101	CP041153	Shewanella marisflavi	CP041153	99.58	自然生物被膜
ECSMB14103	CP023558	Pseudoalteromonas marina	CP023558	99.79	自然生物被膜
ECSMB14105	MT820512	Vibrio splendidus	MT820512	99.93	自然生物被膜
ECSMB14107	CP034970	Vibrio chagasii	MN938232	99.24	自然生物被膜
ECSMB20101	OP209745	Planococcus sp. 1	MH938046	99.93	自然生物被膜
ECSMB20104	OP209750	Pseudoalteromonas sp. 27	KX889969	99.93	自然生物被膜
ECSMB20107	OP209748	Bacillus sp. 9	MG309326	99.86	自然生物被膜
ECSMB21103	OP209747	Salinicoccus sp. 1	DQ001316	98.22	自然生物被膜

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表 3 8株海洋细菌遗传距离

Tab. 3 Genetic distance of the eight marine bacterial strains

测试菌株	Pseudoalteromonas marina	Planococcus sp. 1	Vibrio splendidus	Vibrio chagasii	Salinicoccus sp. 1	Bacillus sp. 9	Shewanella marisflavi	Pseudoalteromonas sp. 27
Pseudoalteromonas marina	0
Planococcus sp. 1	0.262	0
Vibrio splendidus	0.126	0.259	0
Vibrio chagasii	0.139	0.267	0.036	0
Salinicoccus sp. 1	0.262	0.109	0.250	0.257	0
Bacillus sp. 9	0.256	0.088	0.263	0.261	0.113	0
Shewanella marisflavi	0.099	0.267	0.140	0.144	0.254	0.259	0
Pseudoalteromonas sp. 27	0.021	0.262	0.119	0.132	0.259	0.256	0.093	0

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表 4 胞外产物与厚壳贻贝稚贝附着率相关性分析

Tab. 4 Correlation analyses between biofilm extracellular products and settlement rate of Mytilus coruscus plantigrades

胞外产物	附着率
胞外产物	r	p
α-多糖	0.584	0.129
β-多糖	0.742	0.035^*
蛋白质	−0.656	0.077
脂质	0.579	0.132
注：p为检验值；r为相关系数；*表示显著性差异（p < 0.05）。

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