海洋污损微生物膜中细菌群落结构分析及其代谢物DKPs作用初探

	海洋污损微生物膜中细菌群落结构分析及其代谢物DKPs作用初探
	于洋
学位类型	硕士
导师	夏传海
	2014-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	海洋化学
关键词	污损微生物膜污损细菌群落结构代谢物 Dkps 胞外聚合物
摘要	海洋生物污损是海事活动中的常见现象，给人类带来了巨大的经济损失和安全隐患。污损生物膜对后续大型污损生物的附着起着重要的调节作用，直接影响着大型生物污损层的形成，因而成为了探究生物污损发生机制的重要切入点。其中，污损细菌是污损生物膜的重要组成部分，与其分泌的胞外聚合物(Extracellular polymeric substances, EPS)统称为污损微生物膜，是本论文的研究对象。研究表明，污损微生物膜对大型污损生物的孢子和幼虫的附着起着重要作用，其作用方式与污损微生物膜中的群落组成及其产生的代谢物有关。本文以烟台海域污损微生物膜为研究对象，对其进行了群落结构分析和代谢物化学组成分析，进而探讨了这些代谢物对微生物膜形成的影响，具体研究结果如下：通过海上挂板采集了烟台海域污损微生物膜样品，采用分子生物学手段对污损微生物膜细菌群落结构进行了鉴定，发现其主要由芽孢杆菌属Bacillus sp.、假交替单胞菌属Pseudoalteromonas sp.、嗜冷杆菌属Psychrobacter sp.、盐单胞菌属Halomonas sp.、肠杆菌属Enterobacter sp. 五个菌属的细菌组成，其中Bacillus sp.和Pseudoalteromonas sp.为优势菌，在各组被测样品中均占到60%和20%以上。从污损微生物膜混合菌中分离得到了多株污损细菌，对典型污损细菌进行了菌种鉴定，得到了两株优势菌P. issachenkonii，B. anthracis和两株劣势菌P. nivimaris，H. meridiana。从天然污损微生物膜及典型污损细菌P. issachenkonii的发酵液中分离得到了多种化合物，包括典型的细菌群体感应信号分子二酮哌嗪(Diketopiperazines, DKPs)。扫描电镜观察结果及结晶紫定量结果显示，DKPs可以促进天然污损微生物膜的形成，而对4种典型污损细菌微生物膜的形成则具有不同程度的促进作用。其中，多种DKPs均可以促进P. issachenkonii微生物膜的形成；cyclo(Pro-Gla)、cyclo(4-OH-Pro-Leu)、cyclo(Pro-Phe)可以促进B. anthracis微生物膜的形成；P. nivimaris微生物膜的形成仅受到cyclo(Pro-Gla)、cyclo(Pro-Phe)的促进作用；cyclo(Pro-Gla)、cyclo(Pro-Phe)、cyclo(Pro-Tyr)可以促进H. meridiana微生物膜的形成，表明微生物膜的群落组成可能是多种DKPs共同作用的结果。对典型污损细菌微生物膜中EPS主要组分的定量分析表明，DKPs可以提高微生物膜中胞外多糖及胞外蛋白质的含量，对胞外多糖的促进作用更为明显，表明DKPs可能是通过促进EPS的产生从而促进污损细菌的成膜。综上所述，烟台海域污损微生物膜由多种多样的污损细菌及其代谢物共同组成，其中Bacillus sp.和Pseudoalteromonas sp.为优势菌种；DKPs为天然污损混合菌及P. issachenkonii代谢物的重要组分，可能通过促进EPS的分泌而调控污损混合菌、优势菌、劣势菌的附着，影响着天然污损微生物膜的细菌群落组成，是造成不同种类污损细菌优劣地位的重要诱因。
其他摘要	Marine biofouling is a common phenomenon in maritime activities, which has caused severe economic losses and security threats to human society. During the formation of biofouling, biofouling biofilms first develop and modify subsequent macrofouling processes. Therefore, biofouling biofilms are recognized as a key point in the investigation on the mechanisms of biofouling. In biofouling biofilms, bacteria are significant components. Biofouling bacteria and their extracellular polymeric substances (EPS) form bacterial biofilms which can influence the adhesion of macrofouling organisms greatly. We focused on the bacterial biofilms in this investigation. It has been verified that the effect of biofilms on macrofouling is closely related to bacterial species and their metabolites. The present study focused on bacterial biofilms in Yantai coastal waters. We conducted bacterial community analysis and the chemical constituents of their metabolites were purified and identified. Furthermore, the effects of the isolated metabolites on biofilms formation were also covered. Through an exposure to marine environment, bacterial biofilms were collected in Yantai coastal waters. The bacterial community was revealed by molecular biological method. Five bacterial species were identified, including Bacillus sp., Pseudoalteromonas sp., Psychrobacter sp., Halomonas sp. and Enterobacter sp., among which Bacillus sp. and Pseudoalteromonas sp. were dominant species occupying over 60% and 20% of all the biofilms samples, respectively. Numerous bacteria were isolated from the bacterial biofilms. Four typical biofouling bacteria were identified, including two dominant bacteria P. issachenkonii, B. anthracis, and two inferior bacteria P. nivimaris, H. meridiana. Several compounds were isolated from the fermentation of the natural mixed biofilms bacteria and dominant bacterium P. issachenkonii, including several quorum sensing autoinducers DKPs. SEM and crystal violet staining results showed that DKPs promoted the formation of natural mixed bacterial biofilms, while the promoting effect on 4 typical biofouling bacteria was different to some extent. All the DKPs used enhanced biofilms formation of P. issachenkonii. Cyclo(Pro-Gla), cyclo(4-OH-Pro-Leu) and cyclo(Pro-Phe) enhanced the biofilms formation of B. anthracis. Cyclo(Pro-Gla) and cyclo(Pro-Phe) enhanced the biofilms formation of P. nivimaris. Cyclo(Pro-Gla), cyclo(Pro-Phe) and cyclo(Pro-Tyr) enhanced the biofilms formation of H. meridiana. Quantification of significant bacterial EPS componants showed that DKPs improved exopolysaccharides and extracellular proteins amount, especially exopolysaccharides. Therefore, DKPs might promote bacterial biofilms formation through enhancing EPS amount. In a word, the biofouling bacterial biofilms in biofouling biofilms in Yantai coastal waters were comprised with a variety of biofouling bacteria, among which Bacillus sp. and Pseudoalteromonas sp. were recognized as the dominant species. DKPs were major compounds in the metabolites of natural mixed bacteria and P. issachenkonii. DKPs might promote the attachment of bacteria through enhancing EPS amount and further influence the bacterial community, which might explain the differences of biomass among dominant and inferior bacteria.
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/6852
专题	中国科学院烟台海岸带研究所知识产出_学位论文
推荐引用方式 GB/T 7714	于洋. 海洋污损微生物膜中细菌群落结构分析及其代谢物DKPs作用初探[D]. 北京. 中国科学院研究生院,2014.

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