Investigating widespread regulation of nitrogen assimilation at the level of RNA in bacteria

Nitrogen is one of the key elements in biological systems since it is required in large quantities for the building blocks of life. Humans are rather limited in the sources of nitrogen they can use to live and grow, and we obtain nitrogen we need from complex organic compounds in the food we eat. By contrast, microbial metabolism is more adaptable and bacteria can capture the nitrogen they need from a range of simpler forms present in nature such as nitrate. In recent times, the amount of man-made nitrogen in the environment has increased dramatically due to industrial fixation of the relatively inert nitrogen gas present in the atmosphere. As the world population has grown, more intensive agricultural practices have been used to increase crop yields to meet this demand, including the widespread use of synthetic nitrate-based fertilisers in modern agricultural practice.

Nitrate is highly soluble in water and readily lost from fields as run-off where it accumulates in rivers, lakes and oceans and provides an abundant nutrient for bacteria that are able to use it for growth. In agricultural fields the use of nitrate by soil microbes, instead of the crops for which it was intended, is not only economically wasteful but may also have a number of unintended consequences. One such consequence is predicted to be the increased consumption of dissolved organic carbon by particular bacteria in the environment. This poses a significant risk to the fertility and integrity of many soil types through catabolic breakdown of their organic constituents by nitrate-utilising bacterial populations. A significant decrease in soil organic matter would not only have a negative impact on the long-term sustainability of commercially valuable agricultural land, but increased microbial action on these carbon dioxide sinks may also contribute to global climate change.

In this research programme we will address the role of a key gene we have identified that is essential for the use of nitrogen by the model nitrate-utilising soil bacterium Paracoccus denitrificans, which is widely studied in laboratories around the world. This gene is predicted to produce a regulatory protein that may control how bacteria use nitrate and carbon to grow. Understanding this process will enable us to develop strategies to minimise the impact of nitrate-utilising bacteria on our agricultural soils and the wider environment.

The intensive use of nitrate-based fertilisers in modern agriculture has led to an abundance of bioavailable nitrogen (N) in soils and an imbalance in the global N-cycle. Nitrate is readily soluble and may leach from agricultural land and accumulate in ground water. It can support the growth of wide-ranging bacteria and is now receiving increasing attention as a key nutritional component of the N-cycle. The long-term consequences of high-level N input into the biosphere are not fully understood, but evidence has emerged that heterotrophic bacteria may be significant consumers of nitrate globally, in environments where there are high levels of dissolved organic carbon (C) relative to N. The model heterotrophic soil bacterium Paracoccus denitrificans can use nitrate or nitrite as the sole N-source for growth. Previous work has revealed that the assimilatory nitrate/nitrite reductase pathway requires NADH and is thus directly linked to C metabolism. Therefore, increasing availability of nitrate may be a significant driver for C catabolism.

We have found that expression of a putative member of the dihydrouridine synthase family (Dus), termed NifR3, is induced during nitrate assimilation in P. denitrificans. Although the function of NifR3 is unknown, Dus proteins may bind and modify tRNAs and mutation of nifR3 abolishes growth of the bacterium with nitrate or nitrite. RNA control of bacterial nitrate assimilation is widely accepted at the level of transcription, but very little is known about its role in translation. The goal of this research programme is to understand the role of NifR3. The project will combine microbial physiology with transcriptomic and proteomic analyses to reveal how nifR3 expression is controlled and the regulatory targets for NifR3. Protein biochemistry will test the hypothesis that NifR3 performs an unprecedented N-responsive and dihydrouridine mediated protein-RNA signalling role to promote the assimilation of inorganic-N in diverse bacteria.

The long-term sustainability of agriculture is under threat at a time where we are more dependent than ever on staple crops to feed an increasing global population. It is clear that current strategies to artificially increase productivity by using chemically-fixed nitrogen in agriculture have led to a range of unintended consequences for the environment. The impact of humans has created an imbalance in the global biogeochemical nitrogen cycle that can result in the accumulation of nitrate in ground water. Nitrate and nitrite are bioavailable nitrogen sources that are used for growth by a wide range of bacteria that possess an assimilatory nitrate/nitrite reductase system. In Paracoccus denitrificans, a model nitrate-utilising heterotrophic soil bacterium, assimilatory nitrate and nitrite reduction are NADH-dependent reactions and thus directly linked to respiratory carbon metabolism. This is in agreement with field-based studies where there is emerging evidence that heterotrophic bacteria may be significant consumers of nitrate globally, particularly in environments where there are high levels of dissolved organic carbon relative to nitrogen (e.g. soils, waste water treatment plants and estuaries).

The impact of this work will be the establishment of a link between nitrate assimilation and RNA metabolism that drives the process of nitrate-dependent growth in heterotrophic bacteria, most of which can use a variety of carbon sources. In addition, many of these bacteria also perform the nitrate-dependent anaerobic respiratory process, denitrification, associated with the emission of the greenhouse gases carbon dioxide and nitrous oxide. There is growing evidence that long-term application of nitrate-based fertilisers are enriching these populations of bacteria in the environment so this emission of greenhouse gases will be compounded in the future if left unchecked.

The research programme focuses on elucidating the role of a key protein required for assimilation of abundant forms of inorganic nitrogen by nitrate-utilising bacteria. The role of RNA metabolism in control of nitrate-utilising bacteria is an expanding field of scientific study and a number of research groups world-wide study the model denitrifying organism P. denitrificans. There will be diverse beneficiaries of knowledge arising from this research including organisations such as the Environment Agency (EA), DEFRA, Natural England, The National Institute of Agricultural Botany (NIAB) Group and Local Authorities (LA) and other bodies (including the farming community) with an interest in the management of the impact of nitrate-utilisation by microbes on sustainability of agricultural practices. In addition, there will be academic beneficiaries in subject areas ranging from Environmental Science to Biochemistry. For example, this research programme may inform other BBSRC programmes funded through the "Nitrogen: improving on nature" strategic initiative.

The nitrous oxide focus group and Nitrous Oxide Research Alliance (NORA) have received press coverage worldwide. These groups will be major conduits for dissemination of the outcomes of this research and the applicants will develop a linked website on bacterial nitrate assimilation. We will also ensure that major developments are publicised on the University web site, which is regularly updated. Given the relevance of this work to agricultural sustainability, food security and global climate change, the research outcomes will be published in high-impact journals and oral communications will be given at international conferences. Furthermore, we aim to develop links with local schools to raise awareness of the importance of nitrogen metabolism by microbes in the environment. All investigators will take every opportunity to talk to the general public about gene regulation in nitrate-utilising microbes and how nitrate assimilation may impact on bacterial communities and contribute to climate change.