Production and consumption of nitrous oxide in nitrate-ammonifying microorganisms – a genome-wide transcriptome analysis

一氧化二氮(N2O；笑气)是一种有效的大气温室气体，导致平流层臭氧消耗。人为排放的N2O主要是由农田土壤微生物造成的，氮肥的施用有利于N2O的排放。许多不同的微生物代谢途径被证明有助于在不同的生境中产生N2O，并且已有多个酶被报道产生N2O。另一方面，只有一种类型的N2O还原酶被描述为还原N2O，产生N2。在缺氧条件下，硝酸盐产生微生物N2O是反硝化或异化硝酸盐还原为铵(DNRA)的结果。在反硝化菌中，硝酸盐通过中间体亚硝酸盐、一氧化氮(NO)和N2O还原为氮气，尽管N2O还原的最后一步可能不存在或受到损害。在DNRA过程中，N2O似乎是亚硝酸盐的副产物，没有解毒作用。一些DNRA执行物种含有NOSZ酶，但它们在环境中的作用在很大程度上尚未解决。本项目旨在表征两种DNRA模式细菌--琥珀酸狼疮杆菌和绿色芽孢杆菌产生和消耗N2O的分子基础。这两种生物都已被证明在硝酸盐充足的条件下产生NO，并将N2O还原与细胞生长(笑气呼吸)联系在一起。它的重点是通过进行全基因组转录组分析来调节相应的酶库存。在不同呼吸模式下生长的野生型细胞和适当的基因缺失突变体，以及可选的，在添加NO或N2O存在的情况下，将受到RNA-SEQ的影响。基因表达动力学的分析将有助于确定硝酸盐氨化、NO还原、亚硝化胁迫防御、N2O产生和N2O呼吸等代谢特征背后的规律，包括由W.琥珀酸菌细胞感受N2O的前所未有的特征。除了mRNA图谱外，RNA-seq还将首次揭示琥珀酸假单胞菌和绿色芽孢杆菌中sRNA和反义转录本等非编码调控RNA的丰富。该项目旨在与目前获得资助的项目(“硝酸氨化微生物中一氧化二氮的生产和消耗”)协同工作，该项目结合了基因工程、微生物生理学和生物化学，以及对产琥珀酸杆菌和绿色芽孢杆菌生长培养中硝酸盐、亚硝酸盐或N2O呼吸的气体产物的最新监测。这一努力将拓宽我们对DNRA生物在N2O释放和消耗中所扮演的未被探索的角色的认识。可以想象，研究结果将对在全球气候变化背景下减轻N2O排放的国际挑战产生影响。

Nitrous oxide (N2O; laughing gas) is an effective atmospheric greenhouse gas and contributes to ozone depletion in the stratosphere. Anthropogenic N2O emissions are mainly caused by microorganisms from agricultural soils and are favoured by the application of nitrogenous fertilizers. Many different pathways of microbial metabolism have been shown to contribute to N2O generation in diverse habitats and multiple enzymes have been reported to produce N2O. On the other hand, only one type of N2O reductase has been described that reduces N2O, yielding N2.Microbial N2O generation from nitrate under anoxic conditions results from denitrification or dissimilatory nitrate reduction to ammonium (DNRA). In denitrifiers, nitrate is reduced to N2 via the intermediates nitrite, nitric oxide (NO) and N2O, although the final step of N2O reduction might be absent or impaired. In the DNRA process, N2O appears to be a side product of nitrite and NO detoxification. Some DNRA-performing species contain a NosZ enzyme but their environmental role is largely unresolved.This project aims to characterize the molecular basis of N2O production and consumption in the two DNRA model bacteria Wolinella succinogenes and Bacillus vireti. Both organisms have been shown to produce NO under nitrate-sufficient conditions and to couple N2O reduction to cell growth (laughing gas respiration). It is focused on the regulation of the corresponding enzyme inventories by conducting a genome-wide transcriptome analysis. Wild-type cells and appropriate gene deletion mutants of W. succinogenes and B. vireti grown under various respiratory regimes and, optionally, in the presence of added NO or N2O will be subjected to RNA-seq. The analysis of gene expression dynamics will allow to define the regulons underlying the metabolic features of nitrate ammonification, NO reduction, nitrosative stress defence, N2O generation and N2O respiration including the unprecedented feature of N2O sensing by W. succinogenes cells. In addition to mRNA profiling, RNA-seq will also reveal the abundance of non-coding regulatory RNAs such as sRNAs and antisense transcripts in W. succinogenes and B. vireti for the first time. This project is designed to act synergistically with the currently funded project (“Production and consumption of nitrous oxide in nitrate-ammonifying microorganisms”), which combines genetic engineering, microbial physiology and biochemistry as well as state-of-the-art monitoring of gaseous products of nitrate, nitrite or N2O respiration in growing cultures of W. succinogenes and B. vireti.This effort will broaden our knowledge on the underexplored role of DNRA organisms in release and consumption of N2O. Conceivably, research outcomes will show an impact on the international challenge of mitigating N2O emissions in the context of global climate change.