Managing the Nitrogen economy of bacteria

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Ways to improve exports of ammonia through the careful examination of the ways in which cells commit to producing ammonia and establish diazotrophy. We have discovered that N fixation can occur in what may be a novel biofilm, and under conditions where oxygen is present which would normally inhibit expression and activity of nitrogenase. This represents an important step in being able to establish N fixing bacterial communities around plant roots, even when conventional micro or anaerobic conditions cannot be met.

It is being explored through additional funding, a BBSRC-funded Impact Acceleration Account award at Imperial College London:

BB/S506667/1
Exploitation Route In supporting sustainable plant growths via low input N supply, including looking into beneficial associative interactions in the laboratory to better understand the limits on such beneficial interactions at the level of ammonia release from bacterial cells. The group will host a Rutherford Fellowship holder to extend the latter studies.Against progress in the basic understanding of establishing diazotrophy we are now well placed to take on plant root-bacteria interaction studies having worked out the necessary single cell gene expression approaches we need to probe populations of bacteria in mixed physiological states (using RNA FISH as a major tool).
Sectors Agriculture, Food and Drink,Environment

 
Description We have linked up with groups in India interested in inocula approaches to sustainable agriculture. In December 2018 Dr Joerg Schumacher visted the Indian partners and this year Dr Shraddah Gang has joined the group supported by a UKREI and BBSRC IAA funds to work on establishing a practical way of estimating the impact of bacterial N fixation on plant growth in no symbiotic settings, via inocula of the bacteria we are studying as core models for our sLoLa award.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Environment
Impact Types Economic

 
Description BBSRC GCRF IAA projects
Amount £70,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2017 
End 01/2018
 
Description Capacity building in rhizobacteria mediated plant stress resistance for Indian agriculture
Amount £70,000 (GBP)
Funding ID Unique reference no: 2017-DST-10362; DST-UKIERI Grant 
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 03/2020
 
Description Marie Curie fellowship
Amount € 130,000 (EUR)
Organisation European Union 
Sector Public
Country European Union (EU)
Start  
 
Description Rutherford Fellowship to Tufail Basir
Amount £60,000 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 03/2020
 
Description Bacterial World, Natural History Museum, Oxford, Dec 2018 (Phil Poole) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Phil gave a talk to school children on an open day at the museum as part of the Bacterial World exhibition.
Year(s) Of Engagement Activity 2018
URL http://www.oum.ox.ac.uk/bacterialworld/
 
Description Clever clover: Nitrogen for Life 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This was an exhibition at the Imperial College annual festival. It involved 15 scientists associated with the slola grant and was organised by Jorg Schumacher.

Summary of activities

• Working title: Clever Clover


• What type of activity are you proposing to use to engage this public around this research:

?Interactive Exhibit / Hands on demonstration
?Installation
?Talk
?Workshop activity
?Facility / Lab tour
? Music and Dance performance
? Roaming performance
?Other - Specify:


• If your primary activity isn't a talk would you or any member of your team still be interested in giving a 20-30 minute talk during your time at the Festival alongside your other contribution?
Primary activity is Exhibition, Talk/Discussion is planned alongside


• Please identify which audience group your activity is most suitable for. We will not exclude other visitors from taking part. It just helps us to promote parts of the festival to specific visitor groups.
? Families with young children (0-5)
? Families with young children (6 - 12)
? Families with older children (over 12)
? Adults


• Are there any potential non-academic collaborators you might be interested in working with on your Festival idea - science communication specialists, artists, family workshop designers.etc? The Festival Team may be able to put you in contact with them and help source funding. Not currently planned but will be explored


• How are you planning to fund your stand?

? Existing research funding budget
? Departmental budget (will be explored)
? Personal budget
? Public engagement specific funding (Many research councils, charities and learned societies offer pots of money for PE activities. Check out some of these listed here - https://www.imperial.ac.uk/about/leadership-and-strategy/provost/academic-partnerships/societal-engagement-funding-opportunities-/ will be explored
? Funding from central Festival budget (There are small amounts to support Festival content. Please indicate later on when describing your ideas how much money you might require) will be explored


• What are you hoping to get out of taking part in the Festival?
Research on Biological Nitrogen Fixation is a currently high priority for research councils and e.g. the Gates foundation. There are significant risks to the global environment and global food security associated with manmade imbalances in the global nitrogen cycle, of which the wider public is little aware when compared to e.g. global warming. This is despite imbalances in the global nitrogen cycle was deemed a the second most ecological barrier to human development, ahead of global warning (e.g. Rockstroem (2009) Nature 461: 472-475). Our main objective is to help rising awareness about the topic and to highlight mitigating solutions.
Year(s) Of Engagement Activity 2018
URL http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/eventssummary/event_17-8-2017-12-52-36
 
Description Closing Ceremony of the Spanish-UK exchange programme UAM/SRUK 2018, Madrid, Spain Feb 2019 (Carmen Sanchez-Canizares) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Selection was made from the UK participants who hosted an exchange student from Spain and Carmen was chosen. Both Carmen and the student she supervised gave presentations at the ceremony to celebrate the success of the exchange programme.
Year(s) Of Engagement Activity 2018
URL https://rhizosphere.org/lab-news/
 
Description Conference talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 13th European Nitrogen Fixation Conference. Stockholm, 18-21 August 2018 (invited speaker).

Talk Jorg Schumacher:
Year(s) Of Engagement Activity 2018
URL https://www.emedevents.com/c/medical-conferences-2018/13th-european-nitrogen-fixation-conference-enf...
 
Description European Nitrogen Fixation Conference, Stockholm Sweden, Aug 2018 (Vinoy Ramachandran, Rachel Wheatley, Carmen Sanchez-Canizares) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Each named researcher presented a poster at this international conference.
Year(s) Of Engagement Activity 2018
URL https://rhizosphere.org/lab-news/
 
Description JIC 50 Open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Open Day to celebrate 50 years of the John Innes Centre in Norwich, where we exhibited a stand to show isolation of microbes from soil and their influence on plant growth.
Year(s) Of Engagement Activity 2017
 
Description Molecular aspects of regulation and metabolism in nitrogen fixation - satellite meeting of European Nitrogen Fixation Conference, Stockholm, Sweden, Aug 2018 (Carmen Sanchez-Canizares) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk given to international scientists working in this field.
Year(s) Of Engagement Activity 2018
URL https://rhizosphere.org/lab-news/
 
Description On-line interaction with Spanish school students (by Carmen Sanchez-Canizares) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Q&A session with Spanish school students from Lab bench in Oxford
Year(s) Of Engagement Activity 2018,2019
 
Description OxBacNet seminar - Feb 2019 (Carmen Sanchez-Canizares) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Carmen gave a seminar at this networking and seminar series held termly.
Year(s) Of Engagement Activity 2018
URL https://rhizosphere.org/lab-news/
 
Description Oxford Botanic Gardens Seminar Series, Nov 2018 (Phil Poole) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Evening seminar to interested members of general public.
Year(s) Of Engagement Activity 2018
URL https://rhizosphere.org/lab-news/
 
Description Proceedings of the 20th International Congress on Nitrogen Fixation. 2-7 September, Granada 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk by Jorg Schmacher
Year(s) Of Engagement Activity 2017
URL https://www.20icnf.congresosgestac.com/es/information/granada
 
Description Selection as divisional (MPLS, University of Oxford) BAME role model for school children (Vinoy Ramanchandran) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Vinoy was selected as the divisional BAME role model. He wrote a short piece about being a scientist and the research he carries out. School children were asked to create an artwork based on his research. He inspired the winner of one of the age categories.
Year(s) Of Engagement Activity 2018
URL https://rhizosphere.org/lab-news/
 
Description The Queen's College, seminar series (Carmen Sanchez-Canizares) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact The Queen's College, seminar series (Carmen Sanchez-Canizares) Title: Life Underground: The basis and future of agriculture
Year(s) Of Engagement Activity 2017
URL https://rhizosphere.org/lab-news/
 
Description Winchester College, Nov 2018 (Phil Poole) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Phil gave a talk to the students of Winchester College.
Year(s) Of Engagement Activity 2018
URL https://rhizosphere.org/lab-news/