Managing the Nitrogen economy of bacteria

We plan to address how the supply of one key nutrient for plant growth - nitrogen in a form that can be taken up by plants
(i.e. ammonia) - can be supplied by soil dwelling bacteria. A lack of nitrogen supply to plants frequently limits their growth,
and the use of chemically produced nitrogen fertilizers threatens the environment and is energetically expensive to
produce. Hence alternative methods to supply fixed nitrogen that are not dependent on fossil fuels or the application of
chemicals to the soil are desirable. We plan to investigate how the nitrogen economy of simple soil dwelling bacteria is
established through the network of control systems operating to achieve optimal levels of ammonia within cells, and to
modify these control systems to then allow the export of ammonia or amino acids to the soil and hence to plants. Ammonia
will be produced by the action of the bacterial nitrogen fixing enzyme nitrogenase, and we will work out how the bacterial
cell regulates this metabolic process in order to maximize its own resource use efficiency. This knowledge will allow us to
rewire the regulatory control for the purpose of enhancing agricultural productivity.To date some simple first pass attempts have been made to exploit bacterial ammonia export for plant growth, and somewhat surprisingly these one offs show promise in that plant growth is enhanced in a manner suggesting reduced nitrogen from the bacteria is becoming available to support plant growth.Hence successfully refining ammonia export by bacteria holds great promise.

Our work requires that we accurately quantify various key small molecules and proteins used to determine the cells
nitrogen economy, and produce a scheme whereby we can intervene and create a situation where some of the nitrogenase
derived ammonia is excreted from the bacterial cells to the outside without greatly sacrificing the growth and fitness of the
nitrogen fixing bacteria. To do so we will use methodologies which capture the various players of the control systems in
action, allowing us to work out where the major points of regulation occur and how they might be modified to gain an
ammonia export function without losing the fitness of the organism. This approach requires that we combine experiments
with modelling of the nitrogen control schemes, and also deploy synthetic biology tools to produce new master gene
regulator proteins which will allow us to capture the control of the entire complex networks of genes needed for the cells
management of its nitrogen economy. By combining these approaches with knowledge and parallel study of how soil dwelling bacteria
establish close associations with plant roots and are competitive , and in particular how they gain carbon as an energy source from plants, we
expect to be able to in a sustainable way improve nitrogen supply to plants in order to improve crop yields. In addition, we
plan to utilize the knowledge and understanding that is gained in this project to also develop renewable biotechnological
processes for industrial production of nitrogen containing chemicals that is driven entirely by solar energy.

Plant growth is often limited by the availability of a source of combined nitrogen. Low energy input agriculture and biotechnology would benefit from exploiting bacterial nitrogen fixation to supply N for plant growth and fine chemicals production as ammonia.Our systems and synthetic biology expertise in studying the regulation of N economy of the Ecoli and mycobacterial bacteria ( a prior BBSRC LoLa award) now places us in a strong position to engineer nitrogen fixing bacteria such that they give up some of the ammonia produced by the action of bacterial nitrogenase , firstly to the rhizosphere for plant growth and secondly to other N pathways to allow the biochemical synthesis of fine chemicals.

To achieve the above we will implement wet lab and modelling approaches we have developed to establish the systems behavour of the Ecoli and mycobacteria to nitrogen run out stress. By making use of our bespoke synthetic transcription control systems which uncouple stress signaling from transcription output, detailed metabolic and regulatory protein quantitation and metabolic flux determinations around the conserved N hub comprising the key proteins and associated enzymatic activities (to include:Glutamine Sythetase, PII, GlnK, AmtB, NtrC, GlnD, GlnE, NifA, NifL, N2ase) we will establish the base line systems behavours of four dioazotrophs: Klebsiella pneumoniae, Azotobacter, Anobena and Azorhrizobium. The choice of these organisms allows a systematic progression from the simple Ecoli cousin Kpneumoniae to the more complex life styles and metabolic capabilities of the three other chosen diazotrophs. We will introduce directed changes to the native transcriptional and PTM regulation schemes to achieve ammonia export. We will determine consequences of this engineered export capacity of fitness and competitiveness and the ability to support plant growth via a modified rhizosphere interaction and a photosynthetic coupled N compound production.

We believe there are a range of groupings who will benefit from this research. They include those groupings who champion the use of low input agricultural systems where reducing and then eliminating the polluting run offs from chemical fertilizers is an objective, alongside the closely associated aim of reducing energy inputs into agriculture with a view to establishing sustainable farming practices. Governments interested in improving energy security will also benefit from advances in such ares, where fossil fuel driven chemical synthesis of fertilizers represents an large commitment to C02 production, often mitigating efforts to reduce green house emissions.Less developed societies unable to produce, afford and distribute chemical fertilizers would also greatly benefit.

Groupings interested in using transformative technologies to tackle societal challenges would benefit from our intended use of data driven synthetic and systems biology treatments of the organisms we wish to, from a knowledge based perspective, re-purpose to yield low input solutions in agriculture. Our plans will offer an exemplar approach in addressing a real world problem using academically centric disciplines in maths, informatics, molecular biology, metabolomics and cell physiology integrated to achieve prescribed outcomes. We believe these integrated approaches will be of interest to Industry both because of theiir value in showcasing potential areas for collaboration but also importantly in being illustrative of the ways in which teams can be assembled to tackle particular problems in research and development.The staff involved in the project will benefit from the integrated working, so moving on from the columnar type of activity which can often help define a discipline but where the potential of the discipline can be constrained through not developing interfaces with other disciplines.We believe such exposure will benefit the organisations they ultimately work with and may be employed by, both nationally and internationally.

We intend to communicate our research activities to a range of audiences, and include amongst these audiences those in schools and cafe scientific environments to help raise the underlying issues and be illustrative of scientific biotechnological solutions to worldwide challenges in achieving a sustainable planet.For example we will make use of the outreach Laboratory at ICL to engage groupings and implement demonstrations and discussion sessions the annual ICL science fair.

The project centers around the N economy of particular types of bacterial cells. Although focused on relevance to plant growth, the N utilization pathways of microbes are of huge interest through the the role of N in pathogenesis (eg in mycobacterial caused lung infections), its role in the establishment and maintenance of the microbiota of animal guts, the N cycle in nature largely driven by microbial transformations, and not least its role in the successful fermentations used in the food industry and biotechnology industries deploying bacterial based production systems. Hence a very wide range of interests interface with the knowledge base around microbial N metabolism ranging from clinicians and vetinarian scientists , through environmental science to pure and applied microbiology.