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.
 
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. To date we have found that we can assess such biofilms using advanced atomic level analysis of in situ mass spectrometry. We have also developed models to explain the fact that many bacterial cells fail to fix , which appears to reflect cell to cell variability set by the high affinity ammonia transporter AmtB.

It is being explored through additional funding, a BBSRC-funded Impact Acceleration Account award at Imperial College London and two GCRF awards via Imperial College London that alllow close working with colleagues in India , that have access to soil samples and plant growth traits assays that are N fix dependent.

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). To do so, we have set up collaborations in the UK to measure transfer of nitrogen from diazotrophic bacteria into plants, and are analysing the outcomes of this study for evidence of the action of plant growth promoting bacterial biofilms on plant roots.
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 innocula of the bacteria we are studying as core models for our sLoLa award. This work has been supported by two consecutive GCRF awards (from BBSRC-Imperial funds) , and we are measuring directly plant N content as a function of root associated diazotrophs alongside plant growth trials in India.
First Year Of Impact 2018
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 Imaging bacterial chemotaxis to roots: role of the global regulator PTSNtr
Amount £129,428 (GBP)
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2019 
End 10/2021
 
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 Biofertiliser field trials India 
Organisation Gujarat University
Country India 
Sector Academic/University 
PI Contribution Provision of bacterial strains, data sharing, co-working and researcher exchanges, knowledge transfers
Collaborator Contribution Provision of bacterial strains, data sharing, co-working and researcher exchanges, knowledge, plant growth trials
Impact New knowledge and future research avaenues.
Start Year 2016
 
Description "Diverse regulatory control of nitrogen fixation in proteobacteria" International Conference on Nitrogen Fixation 2019, Wuhan, China 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary talk at a major international meeting introducing students to the potential for deriving agronomic benefit from manipulation of associative diazotophs
Year(s) Of Engagement Activity 2019
 
Description #WomenInScience (11defebrero.org) Skype presentation - Carmen Sanchez-Canizares 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Carmen spoke to pupils in a girls secondary school in Spain (Málaga) via Skype. This is part of a programme to inspire girls to seek to study STEM subjects and celebrate women in science (#WomenInScience).
Year(s) Of Engagement Activity 2020
URL https://rhizosphere.org/lab-news/
 
Description 21st Congress on Nitrogen Fixation - 10th-15th Oct 2019, Wuhan, China - Philip Poole 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Phil gave a talk at this international conference. He had many questions on his work and spent time exchanging ideas with colleagues in this research area.
Year(s) Of Engagement Activity 2019
URL http://2019icnf.csp.escience.cn/dct/page/65580
 
Description Algorithms and Models for Single-Cell Genomics - Rowan Brackston - Extrinsic noise in transcription: Implications and examples 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International workshop on single-cell technologies at UC Irvine. Talk communicated recent theoretical work and was well received. Abstract as follows:

Transcription is subject to significant intrinsic and extrinsic noise. Intrinsic noise arises from a processes that involves the interaction of single molecules, while extrinsic noise can be thought of as variation of the parameters governing this stochastic process. In other work (to be discussed in an accompanying talk), we have demonstrated that extrinsic noise can lead to heavy-tailed distributions for the copy number of mRNA. In this talk, we will firstly detail examples of extrinsic noise, such as significant variability from upstream signalling, before detailing the effect of the typical timescales on the validity of our model.
We also examine the implications of extrinsic noise, both biologically and for identifiability in terms of our ability to distinguish between noise sources. Our results indicate that heavy-tailed distributions alone are enough to indicate the presence of extrinsic noise, while the frequently observed scaling of Fano factor with mean expression level is also consistent with our model. Conversely, there can also be situations in which intrinsic noise from bursty transcription may be indistinguishable from cases with significant extrinsic noise.
We will finally look at two applications. The first using large single cell tran- scriptomics data sets to identify genes in which extrinsic noise is demonstrated to be significant. The second involving expression of the tightly regulated nifH genes underlying Nitrogen fixation in diazotrophic bacteria.
Year(s) Of Engagement Activity 2019
 
Description Article in La Revista - Carmen Sanchez-Canizares 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Carmen wrote an article entitled 'Life underground;The basis and future of agriculture'
Year(s) Of Engagement Activity 2019
URL https://philippoole.files.wordpress.com/2019/07/carmen.article.jpg
 
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 Chris Waite, Conference talk: 12th European Nitrogen Fixation Conference (ENFC) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact August 27 2016
Parallel session: Biological nitrogen fixation in non-legume environments
The regulation of nitrogen fixation and assimilation in the associative diazotroph Klebsiella oxytoca M5a1

Engineering free-living and associative diazotrophic bacteria for the release of fixed nitrogen (N) into the rhizosphere represents one promising strategy for meeting the global demand for agricultural fertiliser sustainably (1-2). As part of an ongoing collaborative project (BB/N003608/1) aiming to rationally re-engineer the cell signalling and metabolism of model diazotrophs for the supply of surplus reduced N (e.g. ammonium) to plants, we present a preliminary model for the regulatory interplay between N fixation and N assimilation in the associative, soil-dwelling bacterium Klebsiella oxytoca M5a1 (Ko). In most bacteria the rate of N assimilation is coupled to the cellular N status (glutamine/a-ketoglutarate ratio) by a regulatory cascade involving post-translational uridylylation of PII type proteins (GlnB and GlnK) and the s54-type transcriptional activator NtrC (3). In diazatrophs such as Ko, these proteins also regulate the expression of the nif gene cluster, encoding the nitrogenase complex and its associated factors, via a second, downstream s54-type transcriptional activator NifA (4). Nitrogenase expression is coupled tightly to internal N status (via NtrC), anaerobiosis (via NifL, the negative regulator of NifA) and import of exogenous fixed N (via interactions between GlnK and the primary ammonium transporter, AmtB (5)). Ultimately, the co-regulation of the gln (N assimilation) and nif (N fixation) regulons by multifunctional regulatory proteins affords highly economical nitrogen metabolism, according to supply and demand, in which a surplus of fixed N compounds is minimised.

Ko provides a suitably characterised model diazotroph in which to develop an integrated systems-level understanding of N economy management, including identification of key nodes of control and robustness, whether catalytic or regulatory, that prevent excess fixed N production and export. In preparation for omics analyses (RNA-seq, targeted MRM-MS proteomics and LC-MS metabolite profiling) we have characterised multiple key parameters during the diazatrophy transition that follows ammonium run-out including (a) cell growth rate, (b) transcription of key N regulons, (c) nitrogenase activity and (d) critical O2 concentrations. We have developed an O2-independent fluorescent gene reporter system and a library of regulatory mutants (both gene deletions and CRISPR-targeted substitutions) which together reveal novel control mechanisms at play in this organism. We report initial findings employing synthetic transcription factors, for instance a chimera of the O2-sensitive Bradyrhizobium japonicum and native Ko NifA homolgoues, to redirect/tune nif gene expression and thereby uncouple the N demand of the cell from N fixation activity.
Year(s) Of Engagement Activity 2016
 
Description Chris Waite, conference tallk: Allocation of cellular resources and fixed nitrogen during the transition to diazotrophy in Klebsiella oxytoca M5aI 
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 (ENFC)
München Bryggeriet Conference Center, Stockholm, Sweden
August 20 2018
The release of fixed nitrogen (N) compounds into the rhizosphere by free-living diazotrophs has been shown to improve yields of non-leguminous plants (1, 2), highlighting their great potential as sustainable alternatives to chemical fertiliser. However, the remarkable efficiency of N metabolism in diazotrophs, achieved via regulatory coupling between the N fixation and assimilation pathways, hinders the production of surplus fixed N and the effectiveness of natural strains as bio-fertilisers. Using the free-living diazotroph Klebsiella oxytoca M5aI (Ko) as a proof-of-concept model system, we are employing systems and synthetic biology approaches to inform engineering strategies for robust ammonium (NH4) excretion in the rhizosphere.

The 20 Nif proteins that make up the nitrogenase enzyme and associated biosynthetic complexes are highly expressed during N limitation stress; our estimates suggesting they can constitute up to a third of cell protein. Thus, the transition between assimilatory and N-fixing lifestyles presents an apparent paradox - how can diazotrophs afford the large N cost of expressing the Nif proteins when N is limiting? Using 15N isotope labelling and high-resolution targeted mass spectrometry we have developed a method to track the flux of fixed N into specific target proteins, in addition to the free and proteinaceous amino acid pools. The incorporation of fixed N into Nif proteins is very rapid compared to other metabolic proteins, reflecting their relative demand during diazotrophy. Using a non-fixing catalytic nif mutant in which the full complement of Nif proteins are still expressed, we pinpoint when cellular N stores are no longer sufficient to support further adaptation and fixed N becomes essential. With flux analysis and Gaussian process regression, we are able to infer and compare specific protein turnover rates and determine whether protein degradation is important for re-allocation of N for Nif expression. The upregulation of accessory ribosome regulators during N starvation, such as Hpf and RaiA, further suggests that a sizeable pool of ribosomes are inactivated. How nif mRNAs are prioritized for translation in these conditions is an important but unanswered question.
Year(s) Of Engagement Activity 2018
 
Description Chris Waite, poster presentations: In vivo absolute and relative Nif protein abundances of Klebsiella oxytoca. 20th International Congress on Nitrogen Fixation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 1) Optimal nitrogenase assembly and function probably relies on the coordinated expression of nif genes, giving rise to appropriate absolute numbers and stoichiometries of the proteins involved (1). The nif gene cluster of Klebsiella oxytoca (M5a1) comprises 20 genes, organised in seven operons, coding for the nitrogenase subunits (H,D,K), its biosynthesis (Q,B,N,E,V,S,Z,U,X,T), maturation (M,Y,W), electron donation (J,F) and nif gene regulation (L,A). We report the accurate average in vivo protein abundances of 19 Nif proteins under non-diazotrophic, early diazotrophic and diazotrophic growth of Klebsiella oxytoca. To do so, we produced and used Nif protein quantotypic isotopically labelled QconCATs as protein standards in multiple reaction monitoring MS (2). With the possible exception of nifT and nifW, expression of all non-regulatory nif genes was strictly NifA, NifL dependent. Generally, nif genes involved in nitrogenase biosynthesis and maturation are relatively earlier expressed compared with nitrogenase structural genes and electron donors. We found NifH in super-stoichiometry relative to NifD and NifK for the nitrogenase complex, supporting an additional function. Our results now allow discussing various aspects of the nitrogenase biochemistry more quantitatively.

2) Synthetic control of nitrogen fixation and assimilation in thee associative diazotroph Klebsiella oxytoca

The release of fixed nitrogen (N) compounds into the rhizosphere by soil bacteria has been shown to improve yields of non-leguminous plants (1, 2), highlighting the great potential of root-associative diazotrophs for sustainable, biological alternatives to chemical fertilizer. The application of synthetic bacterial inocula optimised for the release of fixed N represents a promising short-term solution for meeting the global demand for food crops, especially in the developing world. However, the remarkable efficiency of N metabolism in diazotrophs, achieved via regulatory coupling between the N fixation and assimilation pathways, hinders the production of surplus fixed N and as such the effectiveness of natural strains as ammonia providers. Using the free-living diazotroph Klebsiella oxytoca M5aI (Ko) as a proof of concept model system, we are employing systems and synthetic biology approaches to inform engineering strategies for robust ammonium (NH4) excretion in the rhizosphere.

In most bacteria the rate of N assimilation is coupled to the cellular N status by a regulatory cascade involving post-translational uridylation of PII type proteins (GlnB and GlnK) and the s54-type transcriptional activator NtrC (3). In diazotrophs such as Ko, these regulators also govern the expression of the nif gene cluster, encoding the nitrogenase complex and its associated factors, indirectly via a second, downstream s54-activator NifA, which is fully active under N limiting and anaerobic culture conditions. The transition between N replete and diazotrophic states has yet to be explored at an integrated systems level. Using RNA-seq, we describe both the global transcription program induced by N run out and, a subset of this, the complete regulon that contributes to N fixation. Concurrently, we are able to monitor the cellular N status and the rapid post-translational modifications that occur in response to it via targeted and quantitative multiple reaction monitoring mass spectrometry (MRM-MS). We have generated functional chimeras of s54 activator proteins. These enable tuneable and orthogonal control of NtrC and NifA transcription control function independently of N status. We demonstrate that these synthetic chimeras are able to drive N fixation and assimilation pathways ectopically and explore their utility for re-directing N metabolism towards tuneable NH4 surplus.
Year(s) Of Engagement Activity 2018
 
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 EMBO plenary lecture "Exploiting fundamental understanding of nitrogen fixation for potential agricultural benefit" Stockholm Sweden 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A major international conference related to biological nitrogen fixation and sustainable agriculture
Year(s) Of Engagement Activity 2018
 
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 GRC Mechanisms of microbial transcription - Rowan Brackston - The route to transcription initiation determines the mode of promoter bursting in bacteria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Engagement with other researchers to discuss recent findings and approaches. Abstract as follows:

Transcription by RNA polymerase, the first step during information flow from DNA, is highly regulated. Classical ensemble averaging of transcription events across cell populations serve to relate the average rate of gene expression to its regulatory DNA sequences and masks the differences in expression occurring between individual cells. These differences can have important consequences for a population and a community of bacteria, as well as being of major significance in multicellular organisms. Studying single cells reveals whether global constraints influence gene expression dynamics. Knowledge of expression dynamics is therefore essential for working out origins of cell-to-cell variability in protein content and hence the basis of vital phenotypic variation between cells. Here we determined single cell mRNA levels from two paradigmatic stress responses. Each require a specific class of bacterial sigma factors, s70 and s54, that drive different modes of transcription initiation. Outcomes reveal evidence for a universal sigma factor independent adaptive strategy employed by bacterial stress responses that combines bet hedging and environmental sensing. The mechanism underpinning transcriptional bursting however is sigma factor specific. Variability of the s70 controlled stress response, where transcription initiation occurs spontaneously, is regulated via the burst size and correlates with the level of expression, similar to other s70 dependent systems. Variability of the s54 controlled stress response, where transcription initiation requires ATP hydrolysis by a bacterial enhancer binding protein, is regulated via the burst frequency and is independent of the level of expression. Transcription initiation involving the s54 class of sigma factors yields bursting kinetics characteristic of yeast promoters and enhancer dependent gene expression in mammalian cells, thus in stark contrast to bacterial housekeeping transcription systems. Our study therefore implies, (i) that the current view of a uniform mode of transcriptional bursting amongst bacteria is not strictly true and (ii) that the mode of transcriptional bursting observed in complex higher organisms is rooted in the simpler bacterial kingdom.
Year(s) Of Engagement Activity 2019
 
Description GRC Stochastic Physics in Biology - Rowan Brackston - Extrinsic noise and variability in nif gene expression 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International conference on stochastic physics in biology. Current work and new ideas were discussed with other researchers in the field. Abstract as follows:

Free living diazotrophic bacteria can provide their own source of Nitrogen by fixing atmospheric N2 into Ammonia. This process requires synthesizing the nitrogenase enzyme in large quantities and significant energy. Due to this large expense, expression of the nif genes is tightly regulated.
Here we examine expression of genes in the regulatory cascade at the single cell mRNA level, uncovering the significant heterogeneity between cells. By fitting stochastic master equation models of gene ex- pression, we examine the regulatory mechanism and the contributions of extrinsic noise. We also consider the extent to which variability between cells may be advantageous.
Year(s) Of Engagement Activity 2019
 
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 Plenary Lecture "Engineering Nitrogen fixation for Agricultural Benefit" at the XVII International Meeting of the Spanish Society of Nitrogen Fixation, Madrid ,Spain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This was an opportunity to introduce students to the benefits of sustainable agriculture . Subsequently at the meeting I was invited to write a book chapter on this subject
Year(s) Of Engagement Activity 2019
 
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 Rothamsted Research invited seminar - Phil Poole 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Phil gave a lunch-time research seminar.
Year(s) Of Engagement Activity 2019
URL https://rhizosphere.org/lab-news/
 
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 Single Cell Workshop - Rowan Brackston - Using mRNA-FISH to probe the sources of heterogeneity in Nitrogenase expression 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at workshop on single-cell methodologies and biological applications. Abstract as follows:

Free living diazotrophic bacteria have the ability to provide their own source of Nitrogen by fixing atmospheric N2 into Ammonia. This process involves the Nitrogenase enzyme which must be synthesised in large quantities, and subsequently consumes significant energy. Due to the large expense associated with the synthesis and activity of the Nitrogenase enzyme, expression of the nif genes is tightly regulated to reduce costs under conditions of plenty. In this work we use single cell mRNA-FISH data to examine the expression of several genes in the regulatory cascade, uncovering the significant heterogeneity between cells. By fitting stochastic master equation models of gene expression, we examine regulatory mechanisms and the relative contributions of intrinsic and extrinsic noise. This allows us to investigate contributions to heterogeneity from upstream sources in the regulatory pathway and the implications this has for a lower bound in variability. We also consider the extent to which variability between cells is advantageous from a bet-hedging perspective.
Year(s) Of Engagement Activity 2019
 
Description The Cellular World, exhibition and series of talks at the Natural History Museum Oxford (Dec 2019) - Carmen Sanchez-Canizares 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Carmen gave a talk entitled ' Bacteria and the Future of Food' to A level students.
Year(s) Of Engagement Activity 2019
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 Visit of Counsellor for Agriculture and Fisheries, Food and the Environment from the Embassy of Spain (Nov 2019) - Carmen Sanchez-Canizares 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact In her role as Vice-Chair of Society fro Spanish Researchers in the UK (SRUK) Carmen spoke to the Counsellor from the Embassy of Spain and her team.
Year(s) Of Engagement Activity 2019
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/
 
Description XVII National Meeting of the Spanish Society of Nitrogen Fixation, Spain (July 2019) Carmen Sanchez-Canizares 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Carmen gave a talk entitled ' Control of bacterial nitrogen and carbon metabolism by a PTSNtr-regulated switch'.
Year(s) Of Engagement Activity 2019
URL https://eventos.uam.es/32212/detail/xvii-reunion-nacional-de-la-sociedad-espanola-de-fijacion-de-nit...