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题名	氨氧化细菌的群体感应机制及在亚硝化过程中产生的效应
作者	高婕
学位类别	博士
答辩日期	2014-05
授予单位	中国科学院研究生院
授予地点	北京
导师	庄国强
关键词	群体感应 信号分子AHLs 群体淬灭 氨氧化细菌 活性污泥 quorum sensing autoinducer AHLs quorum quench ammonia-oxidizing bacteria activated sludge
其他题名	Quorum sensing (QS) mechanism in ammonia-oxidizing bacteria and the QS effect in nitrosification
中文摘要	微生物是驱动氮元素等物质循环的引擎，N循环中的生物过程及其中物质流与信息流的解析是当前研究微生物生态系统功能最为关注的领域。其中微生物间互作机制是理解物质循环与生物群落耦合及协调控制的最为重要的科学问题之一。微生物通过各种生物活性分子来实现微生物的互作和代谢变化来应对环境过程的变化，而在N循环的各个过程中，QS在其中的调控作用却没有一个完整的科学认识。本论文首先以氨氧化细菌个体作为研究对象，利用生物信息学及分子生物学方法对预估的酰基高丝氨酸内酯信号分子（AHLs）合成酶、AHLs识别受体进行异源表达，完成I/R调控系统的验证工作。同时采用多种分子生态学技术相结合的方法，系统地研究了QS对实际水处理的活性污泥样品氨氧化功能群群落结构及功能的影响。本研究获得了以下创新性结果： 1）首次在氨氧化的亚硝化螺菌（Nitrosospira multiformis）中发现功能性的AHLs合成酶基因。选取了四种硝化模式细菌（亚硝化螺菌N. multiformis, 亚硝化单胞菌Nitrosomonas europaea, 维氏硝化杆菌Nitrobacter winogradskyi, 汉堡硝化杆菌Nitrobacter hamburgensis）进行AHLs信号分子合成酶基因的验证。利用大肠杆菌异源表达系统，发现其中位于N. multiformis中的AHLs合成酶同源序列（nmuI）能够在大肠杆菌体系中成功表达，NmuI所催化合成的信号分子为带有C14-及3-oxo-C14-侧链的AHL。然而，采用相同的抽提方法，利用AHL报告菌生物检测以及液质化学检测，在N. multiformis原菌菌液中均未检测到相应的AHL信号分子。采用反转录扩增验证了nmuI在N. multiformis中能够功能性表达，而外源添加的C14-homoserine lactone (HSL) 以及3-oxo-C14-HSL在N. multiformis菌液中4 h及8 h时出现了不同程度的降解，说明在N. multiformis菌中可能存在AHLs的降解系统。 2）通过生物信息学分析，找到了可能的LuxR型的识别蛋白NmuR。经氨基酸序列比对，发现此LuxR型蛋白具有高度保守的氨基酸残基，而这些氨基酸残基在与AHLs、DNA结合上具有重要作用。采用蛋白溶解性试验，验证了NmuI所合成信号分子与NmuR蛋白同源，能够提高其稳定性和溶解性。 3）外源添加AHLs能够改善活性污泥的氨氧化速率，施用2 μM的信号分子能够使活性污泥样品中的氨氧化速率出现不同程度的提高。其中短链AHL处理组提高了约24 %，而长链AHL处理组（C14/3-oxo-C14-HSL）以及长短链AHL混合处理组（C6/3-oxo-C6+C14/3-oxo-C14-HSL）的增长更为明显，提高了60 %至70 %。AHL处理16天后，氨氧化细菌（ammonia-oxidizing bacteria, AOB）及氨氧化古菌（ammonia-oxidizing archea, AOA）中的amoA基因拷贝数与对照组相比，均出现了明显的增长。AOA中的amoA基因拷贝数增长速度要高于AOB。 4）不同AHLs处理的活性污泥样品间氨氧化功能微生物群落构成存在差异。在整个AHL处理阶段，外源添加的AHLs信号分子对活性污泥系统中主要细菌的群落结构并没有显著影响。而污泥样品中的AOA对不同的信号分子类型、不同的信号分子处理周期出现了明显的响应，其中的泉古菌优势菌群发生了明显的群落演替现象。AOB中亚硝化螺菌属与亚硝化球菌属的群落结构对不同类型的AHLs添加显示出较大差异，长短链AHLs混合处理组（C6/3-oxo-C6+C14/3-oxo-C14-HSL）样品中所占比例最大。说明这种AHLs的添加方式对污泥中这两种典型的氨氧化细菌影响更大。
英文摘要	Nowadays, more attention has been focused on the analysis of biologcal process, matter flow and information flow in the nitrogen biogeochemical cycling, due to the dominant driver in nitrogen transformation – microorganisms. Amazingly complex interactions exist between microbial communities in the substance cycle system, and it is always the core content in the research of ecosystem. A wide range of bioactive molecules, often through complex mechanisms, are the main effectors of these associations and modulations. However, the scientific understanding of quorum seensing (QS) regulatory effect is incomplete in microbial transformation processes of nitrogen. In this study, we report the existence and characterization of a functional QS signal synthase and receptor in Nitrosospira multiformis by heterologous expression in Escherichia coli. Moreover, the role of N-acyl homoserine lactones (AHLs) in ammonia oxidizing microbial community dynamics in activated sludge is addressed. The major contents and findings of the dissertation are as follows: 1) An AHLs synthase gene (nmuI) has been found in a chemolithotrophic ammonia-oxidizing microorganism (N. multiformis) as a long-chain AHLs producer. We chose four nitrified typical strains, N. multiformis, Nitrosomonas europaea, Nitrobacter winogradskyi, and Nitrobacter hamburgensis to search the functional AHLs synthase gene. Our results suggest that the nmuI gene is functional in N. multiformis and two AHLs (C14-HSL and 3-oxo-C14-HSL) were detected using AHLs biosensor and liquid chromatography-mass spectrometry (LC-MS), when nmuI, producing a LuxI homologue, was introduced into E. coli. However, by extracting N. multiformis culture supernatants with acidified ethyl acetate, no AHLs product was obtained capable of activating the biosensor or detected by LC-MS. According to reverse transcription-PCR, the nmuI gene was transcribed in N. multiformis, and the degradation experiment demonstrated that the absence of AHL signals might be attributed to the possible AHL-inactivating activities of this strain. 2) We have found a LuxR homolog (NmuR) in N. multiformis and sequence alignments have revealed that the reported nine highly conserved amino acid residues, shared by most LuxR-type proteins, were included in NmuR. The R protein was overexpressed in E. coli strain and a proportion of the overexpressed NmuR was detected in the soluble fraction when in the presence of their cognate signals or a closely related AHLs. 3) Our results indicated that AHLs have a more important role in mediating ammonia oxidizing rate of activated sludge. Activated sludge samples from sewage treatment plant incubated with 2 μM varied AHLs were performed to evaluate the variations of ammonium oxidation rate. The addition of long-chain AHLs and total mixture of C6-HSL, 3-oxo-C6-HSL, C14-HSL, and 3-oxo-C14-HSL significantly increased the ammonium oxidation rate by 67% and 73%, respectively, in 16 days treatment. In contrast, the addition of short-chain AHLs (C6-HSL and 3-oxo-C6-HSL) led to a smaller proportion of increase (~ 24 %). Abundance of both archaeal and bacterial amoA genes increased significantly by 16 days AHLs treatment, and archaeal amoA gene copies has been growing much faster than bacteria. 4) Changes of ammonia oxidizer communities in the AHLs treated activated sludge were investigated by DNA fingerprints and pyrosequencing technology analysis. The results showed that the major composition of bacterial communities did not find much of a difference between AHLs treated and untreated samples. However, archaeal ammonia oxidizer varied with AHLs type and treatment time, indicating changes in community composition of ammonia-oxidizing archea (AOA). Phylogenetic analysis revealed that the predominant archaeal members, affected by AHLs additive, were associated with the Crenarchaeota phylum. The abundance of two typical ammonia-oxidizing microorganisms, Nitrosospira and Nitrosococcus, were relatively higher in samples with total mixture of C6-HSL, 3-oxo-C6-HSL, C14-HSL, and 3-oxo-C14-HSL, compared with other samples. That indicated AHLs influence became more remarkable on these ammonia-oxidizing bacteria (AOB) members.
公开日期	2015-07-08
内容类型	学位论文
源URL	[http://ir.rcees.ac.cn/handle/311016/15605]
专题	生态环境研究中心_中国科学院环境生物技术重点实验室
推荐引用方式 GB/T 7714	高婕. 氨氧化细菌的群体感应机制及在亚硝化过程中产生的效应[D]. 北京. 中国科学院研究生院. 2014.

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