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

一氧化二氮（N2 O;笑气）是一种有效的大气温室气体，并导致平流层臭氧消耗。人为N2 O排放主要是由农业土壤中的微生物引起的，并受到氮肥施用的影响。许多不同的微生物代谢途径已被证明有助于在不同的生境中产生N2 O，并且已报道多种酶产生N2 O。另一方面，只有一种类型的N2 O还原酶被描述为还原N2 O，产生N2.微生物N2 O的产生在缺氧条件下从硝酸盐的反硝化或异化硝酸盐还原为铵（DNRA）的结果。在反硝化剂中，硝酸盐通过中间体亚硝酸盐、一氧化氮（NO）和N2 O还原为N2，尽管N2 O还原的最后一步可能不存在或受损。在DNRA过程中，N2 O似乎是亚硝酸盐和NO解毒的副产物。一些执行DNRA的物种含有NosZ酶，但它们的环境作用在很大程度上是unsolved.This项目的目的是表征两个DNRA模型细菌Wolinella succinogenes和Bacillus vireti的N2 O生产和消费的分子基础。这两种生物体已被证明在硝酸盐充足的条件下产生NO，并将N2 O还原与细胞生长（笑气呼吸）结合起来。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。野生型细胞和W.琥珀酸类和B.将在各种呼吸方式下以及任选地在添加的NO或N2 O存在下生长的病毒网进行RNA-seq。基因表达动力学的分析将允许定义硝酸盐氨化，NO还原，亚硝化应激防御，N2 O产生和N2 O呼吸的代谢特征的基础上的调节子，包括前所未有的功能N2 O传感W。产琥珀酸细胞。除了mRNA分析，RNA-seq还将揭示W.琥珀酸类和B.第一次见到维雷蒂。该项目旨在与目前资助的项目（“硝酸盐氨化微生物中一氧化二氮的生产和消费”）协同作用，该项目结合了基因工程、微生物生理学和生物化学以及对生长中的W.琥珀酸类和B.这项工作将扩大我们对DNRA生物体在N2 O释放和消耗中未充分探索的作用的认识。可以想象，研究结果将显示在全球气候变化的背景下减少一氧化二氮排放的国际挑战的影响。

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.