细菌反硝化脱氮与异化型硝酸盐还原氨化的分子决定机制研究

Aquatic microbes play a central role in the global biogeochemical cycles of carbon, nitrogen, phosphorus, sulfur and other elements on Earth because of their high diversity in species and metabolic pathways. Anammox (anaerobic ammonia oxidation) and comammox (complete ammonia oxidation) had been predicted to be possible thermodynamically before the reactions were really observed in specific microbes. Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are widespread in many bacteria and some bacteria such as Shewanella loihica PV-4 strain harbor both dissimilatory nitrate reduction pathways. Theoretically, dissimilatory nitrate ammonification and denitrification process are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been gradually depleted and the corresponding free energy changes are 35% and 7% lower than that of aerobic respiration. On the other hand, aerobic denitrification could occur in the presence of the oxygen-insensitive periplasmic nitrate reduction NapA in some heterotrophic facultative bacteria. The shorter pathway could increase the rate of ATP generation and thus the rate of growth while the longer pathway could maximize the energy yield and growth yield via multiple energy generation steps. However, the growth yield of planktonic dentrifying bacteria were actually lower than expected and even lower than that of DNRA strains. High energy and growth yields would be advantageous when bacteria grow in clonal clusters, as is typical for microbial biofilms and zoogloeal flocs formed by activated sludge bacteria as previously predicted. We intend to generate a series of homogenetic mutants in which either denitrifiation or DNRA genes are deleted to test whether microbial biofilms and bacterial flocs support complete denitrification that reduces nitrate to dinitrogen gas without high emission of harmful nitric oxide and nitrous oxide. The molecular mechanism underlying the choice of denitrification or DNRA in a single strain such as Shewanella loihica PV-4 and activated sludge bacterium Zoogloea resiniphila MMB based on the environmental factors such as carbon/nitrogen (C/N) ratios, pH value and carbon sources will also be investigated by combing molecular genetics and ecophysiological approaches. The expected results could provide insights into the global biogeochemistry cycles of nitrogen driven by aquatic microbes.

微生物具有极高的生物多样性，代谢途径多种多样，在元素生物地球化学循环中发挥主要作用。微生物氮循环途径复杂多样，基于理论计算和模拟对Anammox和Comammox现象及相关微生物的预言后来分别被实验所证实。反硝化脱氮和异化型硝酸盐还原成铵（DNRA）两种呼吸途径均广泛分布在细菌中并在某些细菌如罗希希瓦氏菌和动胶菌中共存。理论上反硝化产能比DNRA更多，因应环境条件变化，细菌可选择反硝化或者DNRA途径进行无氧呼吸和生长，但对其调节机制所知甚少。我们拟构建一系列遗传背景基本相同的相关基因突变株，进行分子遗传学和生理生态学研究，对希瓦氏菌和动胶菌中反硝化和DNRA两种异化硝酸盐还原途径的分子调控机制进行研究。与往常利用浮游生长菌群所进行的研究不同，我们将研究希瓦氏菌生物被膜和动胶菌菌胶团状态对其反硝化和DNRA途径的影响。预期结果可为理解水圈微生物驱动的氮循环机制和脱氮技术研发提供参考。

在无氧和缺氧条件下，许多兼性厌氧细菌可以利用硝酸根和亚硝酸根作为电子受体进行无氧呼吸，包括异化型硝酸盐还原成铵（DNRA）和反硝化脱氮两种相互竞争的途径，在氮元素生物地球化学循环中扮演重要角色，也可以应用于氮污染治理。大规模工业固氮和氮肥施用引起对大气存留时间长、增温效应强的温室气体氧化亚氮（N2O）释放提高的担忧，其百年期间全球变暖潜势约为二氧化碳的300倍。罗伊希瓦氏菌PV-4株同时拥有DNRA和反硝化脱氮两个途径。早期研究揭示，在高碳氮（C/N）比下，DNRA占优势，几乎无反硝化；在C/N比下，反硝化脱氮占优势。我们利用这个菌株作为模式，采用分子遗传学和生理生化分析等多种手段分析揭示其调控和途径抉择的分子机制。在我们前期的研究基础上，我们对细菌全局调节因子、细胞第二信使环状腺苷酸受体蛋白（cyclic AMP receptor protein）家族的两个旁系同源蛋白质CRP1（大肠杆菌CRP的直系同源蛋白）和CRP2在DNR途径抉择中的作用进行深入研究，我们构建一系列框内缺失突变株，然后对比分析这些突变株与野生型的异化型硝酸盐还原过程。发现CRP1（大肠杆菌CRP的直系同源蛋白）和CRP2分别对DNRA和反硝化脱氮具有正向调控，CRP1通过抑制CRP2而间接抑制反硝化。在PV-4株中敲除crp1基因后，发现DNRA同样受到抑制，而反硝化脱氮过程得到促进，脱氮相关基因（nirK、norBC和nosZ）的表达得到上调。分析发现含铜的NirK亚硝酸还原酶基因不受CRP1的直接调控，可能是间接的负调控。硝酸盐还原过程中出现的亚硝酸根暂时积累现象在crp敲除后不再出现。无论C/N比高与低，均伴随着一氧化氮（NO）和氧化亚氮气体的释放。说明CRP促进DNRA过程，却抑制反硝化脱氮过程。比较基因组学分析显示PV-4菌株中含有4个CRP同系物，而其他脱氮菌通常含有CRP2。进一步分析发现低C/N比会诱导crp2的表达；nirK上游含有保守的CRP2识别的结合位点，凝胶迁移实验证明CRP2与nirK的启动子结合，而且人为过量表达crp2可以诱导nirK的转录，因此CRP2可能参与对反硝化脱氮的正调控。这些结果说明CRP家族在该菌对环境C/N比的感知及DNRA和反硝化的途径抉择中发挥关键作用。生物力能学计算表明在氮源（电子受体）相对不足时，选择DNRA途径不但产能较多，所释放的铵