漂浮浒苔内共生假单胞菌的硝酸异化通路及其促生效应研究

The NO3- concentration of the Yellow Sea presents an increasing trend in recent years, however, the preferred nitrogen souce of Ulva prolifera is NH4+. We isolated a Pseudomonas stutzeri strain from the floating U. prolifera, and had finished its genome sequencing. Two nitrate dissimilatory pathways are presented in the genome, dissimilatory nitrate reduction to ammonium (DNRA) and denitrification. NO3- is turned into gaseous compounds through denitrification pathway, resulting in nitrogen loss. While, DNRA process reduces NO3- into NH4+, a more favorable and stable nitrogen form for plants and bacteria. Our focus will be posited on exploring whether bacterial DNRA, by supplying NH4+, helps the explosion of U. prolifera. In this project, we will first verify the genome sequencing results by constructing corresponding mutants and phenotypes analysis. Second, the expression profiles of genes involved in these two pathways will be observed when co-cultured with U. prolifera, to figure out what kind of nitrogen compound NO3- turns into. In the last, U. prolifera will be co-cultured with wild-type or corresponding mutants of P. stutzeri. The growth curves of U. prolifera will be obtained to display the effect of DNRA on promoting U. prolifera’s growth. We believe such a proposal, if granted, will elucidate the potential of bacterial DNRA on promoting the growth of U. prolifera in molecular level. This proposal will provide important clues for exploring the roles played by epiphytic and endophytic bacteria on the explosion of U. prolifera.

近年来黄海NO3-水平明显升高，而黄海绿潮优势种浒苔偏好NH4+。漂浮浒苔内生菌Pseudomonas stutzeri基因组测序发现其同时拥有硝酸异化成铵（DNRA）和反硝化两条硝酸异化通路。反硝化将NO3-转化为气态氮化合物，造成系统氮流失，而DNRA将NO3-转化为更易吸收、更稳定的NH4+，使氮保留。本课题旨在揭示微生物DNRA过程是否通过提供NH4+“帮助”浒苔迅速暴发。具体地，首先通过基因敲除和表型分析确定两种硝酸还原酶在DNRA和反硝化中的分工；其次，确定与浒苔共生时，DNRA和反硝化通路基因的表达情况，确定NO3-的去向；最后，在NO3-为唯一氮源条件下，观察不同突变株对浒苔生长的影响，确定DNRA在浒苔氮供给方面的作用。本课题的立项和实施将从分子水平上揭示浒苔共附生菌DNRA过程在促进浒苔生长方面的潜力，为探究浒苔共附生菌在浒苔暴发过程的作用提供重要线索和思路。

细菌与藻类共存现象在海洋中普遍存在，然而细菌在大型藻类暴发中的作用却鲜有研究。自2007年以来，连年周期性暴发的黄海绿潮因其规模之大、影响范围之广，引起世界范围内的关注。藻类与其共附生菌关系密切，藻菌互作一直是藻类学研究的热点。本文以一株分离自黄海绿潮浒苔的潜在内生假单胞菌为研究对象，完成了其基因组测序，并通过比较基因组分析，揭示了其适应在浒苔内部生存的基因组特点。在分析该假单胞菌的氮代谢通路时发现其同时拥有反硝化和硝酸异化成铵两条竞争性的硝酸异化通路。我们建立了高效的基因敲除方法，结合多种分子生物学手段，对其两条硝酸异化通路基因功能和调控机制进行了初步研究。本项目研究的主要结论是：1）通过乙醇加漂白剂有效去除藻体表面附生菌后，分离到一株漂浮浒苔内生假单胞菌，并获得了全基因组序列；2）通过比较基因组学分析表明该假单胞菌具有增强的转录调控和信号转导网络、丢失了大部分芳香族化合物降解基因等与在宿主内部生存相关的基因组特点，提示该菌株是研究浒苔藻菌互作的合适材料；3）通过分子生物学和生理学研究证明了两条硝酸异化通路的存在，并对关键酶基因功能进行了验证。证明了反硝化通路是无氧条件下唯一的硝酸异化通路。证明了反硝化通路亚硝酸还原酶基因nirS和硝酸异化成铵通路亚硝酸还原酶大亚基基因nirB表达受氧气影响，且二者之间存在相互调控。本项目为研究浒苔共附生菌在浒苔暴发过程中的作用提供了重要研究材料和线索。