Characterisation of oxygen regulation mechanisms in Rhizobium leguminosarum for repurposing as tools in the engineering of nitrogen fixation
Lead Research Organisation:
University of Oxford
Department Name: Interdisciplinary Bioscience DTP
Abstract
Nitrogen is a key nutrient for plant growth and the use of nitrogen fertilizers has resulted in significant boosts in crop productivity. However, these fertilizers also cause significant
environmental damage and are unaffordable for much of the developing world. Many plant species instead acquire their nitrogen by forming a symbiotic relationship with nitrogen fixing
Rhizobium bacteria. These enter into the plant's roots where they transform into highly specialized quasi-organelles, providing the plant with nitrogen fixed from the atmosphere in
exchange for nutrients and shelter. Oxygen is a key signal in the regulation of this transformation because it is toxic to the nitrogenase enzyme that performs nitrogen fixation.
Two bacterial proteins, NifA and FnrN, are known to be critical for the regulation and activation of nitrogen fixation. We aim to study how these are regulated at the transcriptional and protein
level and to determine the mechanisms by which this occurs. We next aim to use our understanding of these mechanisms to develop synthetic biology tools which can be used in
efforts to introduce this symbiosis into other plants and bacteria. Achieving this could reduce the need for polluting nitrogen fertilizers and provide a sustainable way to boost crop yields.
BBSRC priority areas addressed:
Food, Nutrition and Health
Sustainably enhancing agricultural production
Synthetic biology
Environment and Land Use
Global Food Security
AFS, ENWW
environmental damage and are unaffordable for much of the developing world. Many plant species instead acquire their nitrogen by forming a symbiotic relationship with nitrogen fixing
Rhizobium bacteria. These enter into the plant's roots where they transform into highly specialized quasi-organelles, providing the plant with nitrogen fixed from the atmosphere in
exchange for nutrients and shelter. Oxygen is a key signal in the regulation of this transformation because it is toxic to the nitrogenase enzyme that performs nitrogen fixation.
Two bacterial proteins, NifA and FnrN, are known to be critical for the regulation and activation of nitrogen fixation. We aim to study how these are regulated at the transcriptional and protein
level and to determine the mechanisms by which this occurs. We next aim to use our understanding of these mechanisms to develop synthetic biology tools which can be used in
efforts to introduce this symbiosis into other plants and bacteria. Achieving this could reduce the need for polluting nitrogen fertilizers and provide a sustainable way to boost crop yields.
BBSRC priority areas addressed:
Food, Nutrition and Health
Sustainably enhancing agricultural production
Synthetic biology
Environment and Land Use
Global Food Security
AFS, ENWW
Organisations
People |
ORCID iD |
| Gail Preston (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011224/1 | 01/10/2015 | 31/03/2024 | |||
| 1757775 | Studentship | BB/M011224/1 | 01/10/2016 | 31/03/2021 |
| Description | Nitrogen is a growth limiting factor for many crops. Over the past century the use of nitrogen fertilizers has created large improvements in agricultural yield. However, producing these fertilizers is energy intensive, accounting for 1-2% of global energy demand. They are also environmentally damaging. Only half of the nitrogen they contain enters crops, with the rest often damaging the environment through eutrophication. Our current use of fertilizers is not sustainable, and there is a pressing need to develop alternatives with less environmental side effects which nevertheless allow us to retain high yields. One avenue is the ability of some plants to generate their own nitrogen supply through a partnership called the rhizobium-legume symbiosis. Rhizobia are a group of alpha-proteobacteria able to perform nitrogen fixation. In exchange for nutrients and shelter, rhizobia 'fix' atmospheric nitrogen, converting it into ammonia for their plant host. Transferring such a relationship to plants in which it is not native would reduce their need for nitrogen fertilizers but demands a thorough understanding of the natural relationship. Nitrogen fixation relies on the oxygen-intolerant nitrogenase complex. However, rhizobia are obligate aerobes and must respire to meet the energy demands of fixation. This paradox has led to the evolution of intricate oxygen regulation mechanisms in rhizobia, essential for successful symbiosis. We have studied two common rhizobial oxygen sensors involved in symbiosis, the hFixL and FnrN proteins. Confirming previous work, we found that induction of genes by hFixL is mediated by the FxkR protein and subsequently the FixK transcription factor. FixK and FnrN are close homologs and their similarities were thought to create redundancy, with both proteins known to induce identical or similar sets of genes. Instead, we have demonstrated that hFixL and its downstream effectors FxkR and FixK are hierarchically integrated with FnrN in a single oxygen regulation pathway in Rlv 3841. In vitro experiments show that hFixL and FnrN are derepressed at different oxygen concentrations, creating two stages of activation in this integrated pathway. We also confirmed auto-activation by FnrN, which had been theorized in the literature. Using confocal microscopy, we have been able to visualise in planta how these systems operate at a very high spatial resolution. This has let us see how the two stages of activation occur in different locations in root nodules. hFixL is active very early in establishment of the symbiosis, at the tip of nodules, where it induces fnrN expression through FixK. This early induction of fnrN primes it for auto-activation deeper in the nodule, where oxygen concentration decreases and FnrN is derepressed. Integration of both proteins is required for full nitrogen fixation activity, with knockouts of hfixL and fnrN reduced to 75% and 15% of wild-type activity, respectively. It is common for multiple oxygen sensors to be used by rhizobia. Combining and integrating these sensors likely allow rhizobia to achieve oxygen regulation capabilities which cannot be provided by any single system. Our work has helped elucidate a new layer of nuance in the oxygen regulation of rhizobia during symbiosis with legumes. |
| Exploitation Route | Our work has demonstrated a hierarchical relationship between two commonly studied oxygen regulation systems in rhizobia, and how these operate at different oxygen concentrations in planta. It sheds light on how the multiple oxygen regulation systems used by many other rhizobia may cooperate to tune their response to the low oxygen conditions of symbiosis. It also helps shed light on why the use of multiple systems is common in rhizobia. These findings suggest that legume sanctioning of nodules, known to operate based on how efficiently rhizobia can convert their carbon supply into a nitrogen supply for the plant, also discriminates rhizobia based on how quickly they begin fixing nitrogen. The hierarchical system in Rlv 3841 likely speeds up the bacteria's transition from its free-living lifestyle to adaptation to life inside the nodule. This may give the bacteria a competitive advantage over other nitrogen fixers who take longer to begin fixing after the start of symbiosis. The project has also demonstrated the importance of studying oxygen regulation in planta. Many studies to date have studied the oxygen sensors of rhizobia under free-living microaerobic conditions, assuming these adequately mimic in planta oxygen concentrations. We have shown that key sensors may not be active outside of nodules. This can mask their importance and miss critical regulators which are only active inside nodules. Finally, given the key role of oxygen regulation in symbiotic nitrogen fixation, the work could also have implications for current efforts to re-engineer the rhizobium-legume symbiosis into new bacteria and plants. We show that a single oxygen regulator may be insufficient to achieve high levels of nitrogen fixation, and a more complex system may be needed. Our results with Rlv 3841 and peas also show that rhizobia have adapted to the presence of oxygen gradients inside nodules. Thus work to induce artificial nodules on the roots of plants which do not naturally produce these may need to recapitulate these gradients for rhizobia to effectively fix nitrogen inside these artificial nodules. |
| Sectors | Agriculture, Food and Drink,Environment,Manufacturing, including Industrial Biotechology |
| Description | Inquiry into the UK government's approach to infectious diseases and bioweapons |
| Geographic Reach | National |
| Policy Influence Type | Contribution to a national consultation/review |
| URL | https://www.parliament.uk/business/committees/committees-a-z/joint-select/national-security-strategy... |
| Description | Travel grant |
| Amount | £750 (GBP) |
| Organisation | University of Oxford |
| Department | Wolfson College |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 08/2017 |
| End | 09/2017 |
| 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 | Development of Poole Lab website (Rhizosphere.org) |
| Form Of Engagement Activity | Engagement focused website, blog or social media channel |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | The Rhizosphere website is used to describe work performed, profiles lab members, lists outreach activities and celebrates achievements. Also includes a list of Lab publications. |
| Year(s) Of Engagement Activity | 2014,2015,2016,2017,2018,2019,2020 |
| URL | https://rhizosphere.org |
| Description | OxBacNet seminar - Oct 2018 (Paul Rutten) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Paul gave a seminar at this networking and seminar series held termly. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://rhizosphere.org/oxbacnet/ |
| 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 | Talk at OxBacNet 2018 October Meeting - How does oxygen regulation prepare Rhizobium for life in nodules? |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | I gave at talk at the OxBacNet 2018 October meeting, which brought together microbiologists and life scientists across Oxford. My talk was entitled "How does oxygen regulation prepare Rhizobium for life in nodules?". The talk was well received and I was approached by several people for very good discussions in a subsequent networking session. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://rhizosphere.org/oxbacnet/ |
| Description | Twitter account Rhizosphere @PooleLabOxford |
| Form Of Engagement Activity | Engagement focused website, blog or social media channel |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | Twitter account to publicise the work of the Poole Lab |
| Year(s) Of Engagement Activity | 2017,2018,2019,2020 |
| URL | https://twitter.com |