Epigenetic control of nitrous oxide emission by denitrifying bacteria

The structure of DNA, first described by Watson and Crick, is an iconic aspect of science and many people are familiar with the DNA 'double helix' housing the genetic information for life. However, it is less commonly known that DNA is polymorphic and can adopt many different structures necessary for biological function.

DNA is composed of four different 'building blocks' called bases that include adenine, guanine, thymine and cytosine. These are joined together to form elongated chains, called polymers, that intertwine to give a 'twisted ladder' helical structure. In particular, DNA sequences rich in the bases guanine and cytosine can form alternative secondary structures that may resemble 'knots', called G-quadruplexes and i-motifs. In our genomes, these types of sequence are widespread and may play various roles from regulating gene expression to defining the stability of our chromosomes and the life span of our cells. The majority of past work by scientists on G-quadruplexes and i-motifs has been performed in animals. Since DNA encodes the genetic instructions for all forms of life, alternative DNA structures may also play a key role in how genes are read in microorganisms such as bacteria. This is an area of research which is important, but has so far received less attention than studies in animals.

We have chemicals that bind these special DNA structures and wish to understand exactly how they determine the structure of DNA and how this can affect whether genes are switched on or off. In particular, we focus on how we can use these chemical tools to stop bacteria releasing the potent greenhouse gas nitrous oxide that contributes to global warming and climate change. Nitrous oxide is better known as laughing gas. It is approx. 300-times more powerful molecule for molecule compared to carbon dioxide and is stable in the atmosphere for many years. Nitrous oxide is released when the bacteria grow in an excess of nutrients in the environment, typically caused by the use of nitrogen-based fertilisers in farmed soils. Use of fertilizers in the environment is required to maintain food supply and sustain our expanding populations world-wide, so we need to understand how we can counteract the side-effects of fertilizers and help prevent release of the greenhouse gas. Our land and water are inextricably linked, and nitrous oxide is also produced by microorganisms in sewage treatment works, where they use nitrogen compounds derived from human waste and agricultural run-off to grow.

The focus of this project is to understand how G-quadruplex and i-motif DNA control how genes for nitrous oxide destruction in bacteria are read, and how we can use chemical tools that target these alternative DNA structures to stop bacteria releasing the potent climate-damaging gas nitrous oxide when they grow in environments rich in nitrogen nutrients. Our work will advance our understanding about how bacteria use DNA structures to determine how their genes are read. It will also allow chemists and microbiologists to control this process inside bacteria, using compounds that can enter cells, without the need to genetically modify organisms. The knowledge we generate could change how people manage soil fertility and treat sewage, to help reduce greenhouse gas emissions, global warming and climate change, which is an important priority for countries around the world.

DNA/RNA secondary structures are diverse and have numerous biological roles. G-quadruplex (GQ) structures are widely-accepted to modulate gene expression in eukaryotic systems and GQ-ligands can attenuate gene expression at the level of transcription and translation. A number of biologically relevant molecules, including metal cations can bind and influence the stability of GQ DNA in in vitro experiments. Despite many GQ sequences being predicted in the genomes of microorganisms, so far, limited progress has been made in understanding the breadth of possible biological function in bacteria.

Paracoccus denitrificans is a model denitrifying bacterium that can assimilate nitrate as sole nitrogen source for biosynthetic purposes during aerobic growth. Importantly, it can also respire nitrate as an alternative electron acceptor to oxygen during anaerobic growth in the process of denitrification, which results in emission of the potent greenhouse gas nitrous oxide (N2O) if biosynthesis of the denitrification enzyme NosZ that performs the reduction of N2O to dinitrogen is insufficient.

Nitrate assimilation and N2O respiration are dependent on the nas and nos genes, respectively. Our published work identified GQ sequences that are predicted to form stable structures. A GQ-forming sequence upstream of nasT can bind GQ-ligands, and biophysical properties and biological functions determined. However, the role of GQs upstream of nosC and nosX remain to be addressed. Also, an i-motif-forming sequence is present upstream of nosF, within genes required for biosynthesis of NosZ that allows denitrifying cells to consume/destroy the potent and stable greenhouse gas N2O that contributes to global warming.

The central aim of this proposal is to establish the mechanism by which nos GQ/iM DNA structures influence NosZ biosynthesis and N2O (and wider) metabolism, and how chemical tools can be applied as biotechnology to enhance bacterial N2O-capture in denitrifying bioreactors.

To realise the "1.5-degree limit" enshrined in the UN Paris climate agreement 2016, emission of greenhouse gases must decrease significantly and rapidly. Human activities drive the production of greenhouse gases, carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O) and halocarbons, which promote global warming and climate change. This research programme focuses on understanding the role of alternative DNA secondary structures as targets for non-GM 'chemical microbiology' strategies and biotechnology to enhance the consumption and destruction N2O, by bacteria. The concentration of N2O in the atmosphere has increased by 20% since it was discovered in 1772. Although the atmospheric concentration of N2O is much smaller (328 ppb in 2015) compared with CO2 (400 ppm), its global warming potential (cumulative radiative forcing) is ~300-times greater than that of CO2 in a 100-year time horizon. Currently, N2O contributes about 10% of the overall global warming effect. Of that, almost 70% of N2O is emitted from agricultural lands. N2O is primarily produced in soil by the activities of microorganisms during the denitrification process that converts nitrate to N2O and dinitrogen (N2) gas. The ratio of N2O to N2 production depends on oxygen supply or water-filled pore space, decomposable organic carbon, nitrogen substrate supply, temperature, pH and trace metal availability.

Denitrifying microbes are important sources and sinks for N2O and express N2O reductase (NosZ), the only dedicated enzyme for the destruction of this important climate-active gas. Most mitigation strategies for N2O focus on promoting N2 product formation in the natural process. However, we currently lack non-GM tools that can target and enhance gene expression and biosynthesis of the NosZ enzyme directly to expand the reservoir capacity of denitrifying cells to mop-up excess N2O from environments. The impact of this work will be the establishment of the link between alternative DNA secondary structures and the expression of proteins needed for biosynthesis of functional NosZ. There will be diverse beneficiaries of knowledge arising from this research, including the Environment Agency, DEFRA, Natural England, National Institute of Agricultural Botany/The Arable Group, Local Authorities and other bodies (including the farming community). We have also received interest from stakeholders in the water sector, such as Anglian Water and the Advanced Water Management Centre in Queensland. There will be further academic beneficiaries in subject areas ranging from environmental science to biochemistry, which we will engage with directly through the Nitrous Oxide Research Alliance, Earth Life Systems Alliance and Tyndall Centre for Climate Change Research. Also, through using bespoke ligands that target DNA secondary structures to enhance NosZ expression in denitrifying bacteria, our knowledge will be applied to generate N2O-capture biotechnology, which may generate small innovative spin-out companies within the bioscience sector.

Investigators also coordinate the Nitrous Oxide Focus Group that has received press coverage world-wide and this will be a major conduit for dissemination of research outcomes from this project to a wide range of beneficiaries: academic scientists, industrial enterprise and government. Research will be published in high-impact open-access journals and oral communications will be given at international conferences. We will engage with young scientists through school visits and University open days. Engagement with the general public will be through presentations and workshops for lay audiences (e.g. the Norwich Science Festival, Royal Norfolk Show and science cafés), YouTube videos explaining research findings and post-publication press releases to promote media engagement. All investigators will take every opportunity to talk to the general public about 'chemical microbiology' and how N2O research can help prevent climate change.