产碱杆菌HO-1菌株好氧转化氨为氮气的代谢途径与分子机制

Transformation of ammonia to nitrogen gas driven by microbes is a critical part of the nitrogen cycle. There were diverse pathways and mechanisms for this process, such as aerobic nitrification-anaerobic denitrification, anammox, etc. However, all these processes require the cooperation of several microorganisms and never a single microbe can fulfill the processes. With the advances on nitrogen cycle research, a few bacterial strains were reported to be able to transform ammonia to dinitrogen gas solely. Alcaligenes sp. HO-1 was a such bacterial strain. It could transform ammonia to dinitrogen gas under aerobic condition, suggesting that itself could finish the whole process of denitrification aerobically, with total nitrogen removal around 50% and more than 97% of which was transformed to dinitrogen gas. There was a transient accumulation of hydroxylamine in this process. It suggested that HO-1 could transform ammonia to hydroxylamine and subsequently oxide to dinitrogen. In it genome, there were genes annotated for the reduction of nitrite to N2, but that for nitrate reduction. It was noted that there were not known genes/gene cluster for ammonia oxidation in the genome, despite strain HO-1 could oxidize ammonia to hydroxylamine. This inferred that the genes/gene cluster involved in ammonia oxidation in HO-1 are completely different from known ones, as well as the metabolism pathway and molecular mechanism of ammonia oxidation behind. In order to reveal the novel pathway and molecular mechanism of transforming ammonia to N2 by HO-1, following studies are proposed: 1, metabolism pathway and nitrogen balance when HO-1 metabolizes ammonia; 2, clone and detail study of the genes/gene cluster involved in transforming ammonia to N2, especially those involved in ammonia oxidation, heterologous expression of these genes for further detail investigation on their products, mainly including their catalyzing properties and kinetics characteristics; 3, bio-information analyses and experimental determination of these genes, especially their distributions among microorganisms and environments, then combined with the nitrogen removal ability of HO-1, to explore the probable contribution of such microbes to nitrogen removal in aerobic environments. By finishing all above, the novel metabolism pathway and molecular mechanisms of ammonia oxidation and aerobic denitrification would be found and revealed, as well as the contribution of such microbes represented by HO-1 to nitrogen removal in aerobic aqua environments. It would be great significant for the elucidation of microbial driving nitrogen cycle in aqua ecosystems.

微生物驱动的从氨到氮气的转化是氮素循环的重要环节，具有复杂而多样的途径与机制，如：好氧硝化-厌氧反硝化、厌氧氨氧化等过程。然而这些过程不是单独一种微生物能够完成的，都需要多种微生物在不同条件下协同作用才能完成。随着研究的深入，也发现了少数微生物可以独自将氨转化为氮气。产碱杆菌HO-1菌株就是这样的一种微生物，它可以在好氧条件下把氨转化为氮气，即独自完成全程脱氮，能够将约50%的氨氮转化为氮气，再该过程中具有羟胺的短暂积累，说明该菌株可以将氨转化为羟胺并进一步转化为氮气。在该菌株的基因组中，具有还原亚硝酸到氮气过程的编码基因，但无硝酸盐还原酶基因；没有已知的与氨氧化相关的基因，说明其涉及氨氧化的基因，与已知的完全不同，意味着新的氨异化代谢途径。本项目提出，在已有工作的基础上，进行以下方面的研究：1、探讨其氨异化代谢途径与氮平衡；2、通过构建Fosmid 文库、功能筛选的方法，获得该菌株转化氨形成羟胺甚至氮气的基因簇；3、对基因簇进行亚克隆并在大肠杆菌中进行表达，研究该菌株中与脱氮相关的基因/基因簇，特别是与氨转化有关的基因/基因簇，进一步研究这些基因产物的催化特性与动力学特性；4、通过氮代谢相关基因/基因簇的生物信息学分析和对环境样品的测定，探讨这些基因在不同微生物和环境中的分布与表达情况，结合实验室模拟其好氧脱氮性能，探讨其对好氧环境脱氮的可能贡献。通过以上研究，希望获得一些重要结果：1）发现新的氨异化代谢途径；2）阐述全新的氨氧化和好氧脱氮的分子机制；3）探讨这一类微生物在好氧环境中脱氮的可能贡献，4）发表2-3篇高端论文。本研究对于阐明微生物驱动好氧水圈的氮循环具有重要的意义。

分离自反应器的HO-1菌株可以单独在好氧条件下把氨转化为氮气，即独自完成全程脱氮，将约50%的氨氮转化为氮气，说明该菌株可以将氨转化为羟胺并进一步转化为氮气。在该菌株的基因组中，具有还原亚硝酸到氮气过程的编码基因，但无硝酸盐还原酶基因；没有已知的与氨氧化相关的基因，说明其涉及氨氧化的基因，与已知的完全不同，意味着新的氨异化代谢途径。从HO-1的基因组中克隆获得了能使大肠杆菌具有产生氮气的阳性克隆子，来源于HO-1菌株的基因簇dnfT1RT2ABCD编码了其转化氨为氮气的代谢途径。DnfA以羟胺为底物，在DnfB的辅助下，可直接将羟胺氧化为氮气，氧同位素实验表明该反应需要氧的参与（2NH2OH + 18O2 + NADH + H+ N2 +NAD+ + 2H218O + 2H2O），其中DnfB也可以用其他氧还蛋白（如Fd/Fdr）替代。DnfA氧化羟胺为氮气的动力学常数Km为92.9 ± 3.0 µM、kcat为1.66 ± 0.03 min-1。与硝化-反硝化过程中羟胺需先氧化为亚硝酸/硝酸，然后再进行反硝化才能生成氮气的途径完全不同，本课题发现的羟胺直接氧化为氮气是一个新的反应，意味着HO-1菌株的氨到氮气的转化为新的途径与机制。近期结果表明，基因簇dnfT1RT2ABCD在Alcaligenes属高度保守，属内基因簇的相似度为81.7%-99.7%。dnf基因簇与Alcaligenes属成员关联进化，Dirammox活性普遍存在于Alcaligenes属菌株，并非菌株HO-1的特例，并且菌株Dirammox能力大小展现出分类单元相关性。体外多种DnfA同源蛋白的羟胺氧化酶活性测定，说明DnfA蛋白是菌株Dirammox活性的必要条件而非充分条件；Dirammox功能基因簇还广泛分布于Delftia、Pseudomonas和Microvirgula为代表的Betaproteobacteria和Gammaproteobacteria，而这些菌属广泛存在于各样环境中，以上结果强烈表明Dirammox对氮循环和环境的重要性。