海洋假交替单胞菌端生和侧生鞭毛对生物膜形成的影响

doi:10.11978/2022149

热带海洋学报 ›› 2023, Vol. 42 ›› Issue (3): 126-135.doi: 10.11978/2022149CSTR: 32234.14.2022149

海洋假交替单胞菌端生和侧生鞭毛对生物膜形成的影响

郭云学¹^,²(), 蔡兴盛¹, 古嘉瑜¹^,², 王晓雪¹^,²

1.中国科学院热带海洋生物资源与生态重点实验室, 中国科学院南海海洋研究所, 广东广州 510301
2.中国科学院大学, 北京 100049

收稿日期:2022-07-03 修回日期:2022-08-31 出版日期:2023-05-10 发布日期:2022-09-14
作者简介:
郭云学(1982—), 男, 河北省承德市人, 副研究员, 博士, 从事海洋微生物环境适应机制研究。email: yunxueguo@scsio.ac.cn
基金资助:
国家自然科学基金(31970037); 国家自然科学基金(91951203); 广东省基础与应用基础研究重大项目课题(2019B030302004)

Effects of polar and lateral flagella on biofilm formation in marine Pseudoalteromonas

GUO Yunxue¹^,²(), CAI Xingsheng¹, GU Jiayu¹^,², WANG Xiaoxue¹^,²

1. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
2. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2022-07-03 Revised:2022-08-31 Online:2023-05-10 Published:2022-09-14
Supported by:
National Natural Science Foundation of China(31970037); National Natural Science Foundation of China(91951203); Guangdong Major Project of Basic and Applied Basic Research(2019B030302004)

摘要/Abstract

摘要：

海洋假交替单胞菌广泛分布于各种海洋环境中, 能分泌多种生物活性物质和胞外酶类, 多具有较强的生物膜形成能力。这些生物膜具有诱导矿化和吸引腐蚀生物幼体附着的功能, 在海洋生态系统的能量和元素循环中扮演特殊角色。细菌的鞭毛系统介导细菌的运动行为, 在营养的获取以及生物膜和浮游状态转化过程中发挥作用。有研究表明, 鞭毛也是生物膜基质的有机组分, 但目前海洋假交替单胞菌鞭毛系统在生物膜形成中的作用尚不清楚。本文以南海表层来源的假交替单胞菌(Pseudoalteromonas sp. SCSIO 11900)和深海沉积物来源的近源假交替单胞菌(Pseudoalteromonas. sp. SM9913)为研究对象, 分别选取了编码鞭毛不同组分的基因进行敲除, 研究鞭毛缺失对细胞游动性和生物膜形成能力的影响。结果表明, 无论来自表层还是深海的假交替单胞菌都使用端生鞭毛维持细胞游动性, 而且其缺失促进生物膜的形成。此外, 深海来源的SM9913还具有侧生鞭毛。尽管这种鞭毛有助于提高细胞游动性, 却不影响细菌的生物膜形成能力。这些端生鞭毛系统在近缘海洋假交替单胞菌中广泛分布, 可能为该属菌株在不同海洋环境中获取营养、定殖及适应环境的过程中发挥重要作用。

关键词: 假交替单胞菌, 鞭毛, 游动性, 生物膜

Abstract:

Pseudoalteromonas are widely distributed in diverse marine environments. They can produce an array of bioactive compounds and enzymes, and have strong ability to form biofilms in general. The biofilms formed by Pseudoalteromonas are able to induce biomineralization and inhibit the settlement of fouling organisms. Flagella are essential for bacterial motility, mediating nutrient acquisition, and the transformation from planktonic living style to biofilm state. Flagella is also considered as a critical component of biofilm matrix. However, the function of flagella during biofilm formation of marine Pseudoalteromonas remains unclear. In this study, we focused on Pseudoalteromonas sp. SCSIO 11900 and SM9913, which were isolated from the surface water and the sediment of deep oceans, respectively. The influence of flagella on biofilm formation and motility was explored by analyzing mutants lacking the genes encoding components of different flagellar portions in both strains. The results showed that the polar flagella are essential for swimming motility, and inhibit biofilm formation in SM9913 and SCSIO 11900. In addition, the lateral flagella in SM9913 also plays a role in swimming motility, but has no effects on biofilm formation. We further showed that these polar flagella systems are present in different Pseudoalteromonas strains, implying that they may be important for these bacteria for nutrient acquisition, for colonization and environmental adaptation in diverse marine habitats.

Key words: Pseudoalteromonas, flagella, swimming motility, biofilm formation

郭云学, 蔡兴盛, 古嘉瑜, 王晓雪. 海洋假交替单胞菌端生和侧生鞭毛对生物膜形成的影响[J]. 热带海洋学报, 2023, 42(3): 126-135.

GUO Yunxue, CAI Xingsheng, GU Jiayu, WANG Xiaoxue. Effects of polar and lateral flagella on biofilm formation in marine Pseudoalteromonas[J]. Journal of Tropical Oceanography, 2023, 42(3): 126-135.

图/表 8

表1

实验中用到的菌株和质粒"

		描述	来源
菌株	SCSIO 11900	野生型, 分离自中国南海4m水深的珊瑚黏液层	Zeng et al, 2014
	11900ΔmotAB	敲除motAB的SCSIO 11900菌	本研究
	11900 ΔfliF	敲除fliF的SCSIO 11900菌	本研究
	11900 ΔflgK	敲除flgK的SCSIO 11900菌	本研究
	11900 ΔflaA	敲除flaA的SCSIO 11900菌	本研究
	SM9913	野生型, 分离自冲绳海槽附近1855m水深的沉积物	Qin et al, 2011
	9913ΔmotAB	敲除motAB的SM9913菌	本研究
	9913 ΔfliF	敲除fliF的SM9913菌	本研究
	9913 ΔflgK	敲除flgK的SM9913菌	本研究
	9913 ΔflaA	敲除flaA的SM9913菌	本研究
	ΔLF	敲除侧生鞭毛基因簇的SM9913菌	本研究
	ΔLFΔfliF	在ΔfliF中敲除侧生鞭毛基因簇基因的SM9913菌	本研究
	ΔLFΔflgk	在ΔflgK中敲除侧生鞭毛基因簇基因的SM9913菌	本研究
	ΔLFΔflaA	在ΔflaA中敲除侧生鞭毛基因簇基因的SM9913菌	本研究
	WM3064	接合转移用的DAP营养缺陷型大肠杆菌, 37°C	实验室保存
基因敲除质粒	pK18mobsacB-Ery	pK18mobsacB 质粒插入pHT304来源的红霉素抗性基因, Kan^r, Ery^r	Wang et al, 2015
	pK18Ery-11900 motAB	pK18mobsacB-Ery 携带SCSIO 11900 motAB基因上下游片段	本研究
	pK18Ery-11900 fliF	pK18mobsacB-Ery 携带SCSIO 11900 fliF基因上下游片段	本研究
	pK18Ery-11900 flgK	pK18mobsacB-Ery 携带SCSIO 11900 flgK基因上下游片段	本研究
	pK18Ery-11900 flaA	pK18mobsacB-Ery 携带SCSIO 11900 flaA基因上下游片段	本研究
	pK18Ery-9913 motAB	pK18mobsacB-Ery 携带SM9913端生鞭毛 motAB基因上下游片段	本研究
	pK18Ery-9913 fliF	pK18mobsacB-Ery 携带SM9913端生鞭毛fliF基因上下游片段	本研究
	pK18Ery-9913 flgK	pK18mobsacB-Ery 携带SM9913端生鞭毛flgK基因上下游片段	本研究
	pK18Ery-9913 flaA	pK18mobsacB-Ery 携带SM9913端生鞭毛flaA基因上下游片段	本研究
	pK18Ery-9913 LF	pK18mobsacB-Ery 携带SM9913侧生鞭毛基因簇上下游片段	本研究

表1

表2

实验中用到的引物"

名称	序列	用途
11900ΔmotAB-up-F	ACATGCATGCGCTAATGCTGCGAT	用于敲除SCSIO 11900中的motAB
11900ΔmotAB-up-R	CGGAATTCATTCCCTCCAAAGGTTTTAT
11900ΔmotAB-down-F	CGGAATTCAATGACTTGGCTATGTACCT
11900ΔmotAB-down-R	CTAG TCTAGATAAGTCACTGATCAATAAAC
11900ΔfliF-up-F	ACATGCATGCGCTAATAAAGTTGCTCGATC	用于敲除SCSIO 11900中的fliF
11900ΔfliF-up-R	CGGAATTCAATAATTCTCCACTACCTAGTT
11900ΔfliF-down-F	CGGAATTCTCAAGAACAAAAACAACTACC
11900ΔfliF-down-R	ACGCGTCGACGTACCAGCTGTCTTTCTGCT
11900ΔflgK-up-F	ACATGCATGCTGGTGGTTTAACAGTCACGA	用于敲除SCSIO 11900中的flgK
11900ΔflgK-up-R	CGGAATTCGTTACTCTACTCCTTGGCGG
11900ΔflgK-down-F	CGGAATTCTTATGCGTTTATCAAATAACT
11900ΔflgK-down-R	ACGCGTCGACCATTGAGGTTCAAACAATAA
11900ΔflaA-up-F	GACTAACATTGCTTACCGCATGAA	用于敲除SCSIO 11900中的flaA
11900ΔflaA-up-R	TTTACCATCTCCTAGATTTAAA
11900ΔflaA-down-F	ACATAGATGTTAGATTTAAATGAT
11900ΔflaA-down-R	TCCATTAGCAATACTAAATTCATT
9913ΔmotAB-up-F	ACATGCATGC GCTAGTGCTGCGATCATTT	用于敲除SM9913中端生鞭毛的motAB
9913ΔmotAB-up-R	CGGAATTCATTCCCTCCAAAGGTTTTA
9913ΔmotAB-down-F	CGGAATTCAATGACTTGGCTATGTACCT
9913ΔmotAB-down-R	CTAGTCTAGATTGCTCGATTACTTCATCA
9913ΔfliF-up-F	AGAAACCTTGGCACGCTGCCA	用于敲除SM9913中端生鞭毛的fliF
9913ΔfliF-up-R	AATAATTCTCCACTACCT
9913ΔfliF-down-F	TCAAGAACAAAAACAACTACCTC
9913ΔfliF-down-R	TCTTCTAAATTAGCAATACGC
9913ΔflgK-up-F	ACATGCATGC GAATAGTCAATCTGAAGGG	用于敲除SM9913中端生鞭毛的flgK
9913ΔflgK-up-R	CGGAATTC TTTACCATCTCCTAGTTTCA
9913ΔflgK-down-F	CGGAATTC TTTAACAATAAGCTCTATACTTT
9913ΔflgK-down-R	CTAG TCTAGA GATAATACTAACCGCTGCC
9913ΔflaA-up-F	GTTAGGTGGTTAATAATAATTAAAA	用于敲除SM9913中端生鞭毛的flaA
9913ΔflaA-up-R	ACGTTGTAGAATGTTTGATGACTATG
9913ΔflaA-down-F	TTTAACAATAAGCTCTATACTTTA
9913ΔflaA-down-R	TACCCTCTTAAATTACTGAAAAATT
9913LF-up-F	ACATGCATGCGCAGTTCGTGCTCTAGGTAA	用于敲除SM9913中侧生鞭毛基因簇
9913LF-up-R	CGGAATTC AAAATGTCGAAATAAAAGTT
9913LF-down-F	CGGAATTC TAAATTTTTAGGATTACAACAA
9913LF-down-R	ACGCGTCGACCAAGCATCAGTTGGCTAATA

表2

图1

图2

图3

图4

图5

表3

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菌株	基因组NCBI序列号	端生鞭毛	侧生鞭毛	分离环境	参考文献
P. sp. SCSIO 11900	JEMJ00000000	+	-	中国南海4m水深珊瑚黏液层	Zeng et al, 2014
P. lipolytica SCSIO 04301	JDVB00000000	+	-	中国南海63m水深沉积物	Zeng et al, 2014
P. rubra SCSIO 6842	CP013611/CP013612	+	-	孟加拉湾150m深海水	Li et al, 2016
P. sp. SM9913	CP001796	+	+	冲绳海槽附近1855m沉积物	Qin et al, 2011
P. tetraodonis UCD-SED8	LITK01000005	+	-	海洋沉积物	Lee et al, 2015
P. haloplanktis TAC125	CR954246	+	-	大西洋沿岸海水	Medigue et al, 2005
P. sp. PAMC 22718	AJTK00000000	+	+	喀拉海低温海水	Park et al, 2012
P. sp. OCN003	CP011011	+	-	夏威夷珊瑚礁区发病珊瑚	Beurmann et al, 2015
P. distincta ATCC 700518	NZ_JWIG01000001	+	-	白令海的海绵	Givan et al, 2015
P. elyakovii ATCC 700519	JWIH00000000	+	-	贻贝的体液	Givan et al, 2015
P. lipolytica UCD-48B	LJTC01000005	+	-	海草的叶子	Alexiev et al, 2016

海洋假交替单胞菌端生和侧生鞭毛对生物膜形成的影响

Effects of polar and lateral flagella on biofilm formation in marine Pseudoalteromonas

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