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

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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.