Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans
<p>Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH...
| Main Authors: | , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Public Library of Science
2022
|
| _version_ | 1826308177501618176 |
|---|---|
| author | Haskett, T Karunakaran, R Bueno Batista, M Dixon, R Poole, P |
| author_facet | Haskett, T Karunakaran, R Bueno Batista, M Dixon, R Poole, P |
| author_sort | Haskett, T |
| collection | OXFORD |
| description | <p>Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH<sub>3</sub>) assimilation by glutamine synthetase (GS), preventing excess release of excess NH<sub>3</sub> for plants. Diazotrophic bacteria can be engineered to excrete NH<sub>3</sub> by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH<sub>3</sub> to non-target plants. Here, we tested two strategies to control GS regulation and NH<sub>3</sub> excretion in our model cereal symbiont <em>Azorhizobium caulinodans Ac</em>LP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P<sub>II</sub> homologues <em>glnB</em> and <em>glnK</em> stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a Δ<em>glnE</em> mutant of <em>Ac</em>LP which permitted strong GS shutdown and excretion of NH<sub>3</sub> derived from N<sub>2</sub> fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a <em>uAT</em> allele under control of the NifA-dependent promoter P<em>nifH</em>, permitting GS shutdown and NH<sub>3</sub> excretion specifically under microaerobic conditions, the same cue that initiates N<sub>2</sub> fixation, then deleted <em>nifA</em> and transferred a rhizopine <em>nifA</em><sub><em>L94Q/D95Q</em></sub><em>-rpoN</em> controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N<sub>2</sub> fixation and NH<sub>3</sub> excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N<sub>2</sub> fixation and NH<sub>3</sub> excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.</p> |
| first_indexed | 2024-03-07T07:14:07Z |
| format | Journal article |
| id | oxford-uuid:9dd5daf4-64a3-46c1-993e-c1e2c10f24df |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:14:07Z |
| publishDate | 2022 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | oxford-uuid:9dd5daf4-64a3-46c1-993e-c1e2c10f24df2022-07-28T09:10:31ZControl of nitrogen fixation and ammonia excretion in Azorhizobium caulinodansJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:9dd5daf4-64a3-46c1-993e-c1e2c10f24dfEnglishSymplectic ElementsPublic Library of Science2022Haskett, TKarunakaran, RBueno Batista, MDixon, RPoole, P<p>Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH<sub>3</sub>) assimilation by glutamine synthetase (GS), preventing excess release of excess NH<sub>3</sub> for plants. Diazotrophic bacteria can be engineered to excrete NH<sub>3</sub> by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH<sub>3</sub> to non-target plants. Here, we tested two strategies to control GS regulation and NH<sub>3</sub> excretion in our model cereal symbiont <em>Azorhizobium caulinodans Ac</em>LP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P<sub>II</sub> homologues <em>glnB</em> and <em>glnK</em> stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a Δ<em>glnE</em> mutant of <em>Ac</em>LP which permitted strong GS shutdown and excretion of NH<sub>3</sub> derived from N<sub>2</sub> fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a <em>uAT</em> allele under control of the NifA-dependent promoter P<em>nifH</em>, permitting GS shutdown and NH<sub>3</sub> excretion specifically under microaerobic conditions, the same cue that initiates N<sub>2</sub> fixation, then deleted <em>nifA</em> and transferred a rhizopine <em>nifA</em><sub><em>L94Q/D95Q</em></sub><em>-rpoN</em> controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N<sub>2</sub> fixation and NH<sub>3</sub> excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N<sub>2</sub> fixation and NH<sub>3</sub> excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.</p> |
| spellingShingle | Haskett, T Karunakaran, R Bueno Batista, M Dixon, R Poole, P Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans |
| title | Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans |
| title_full | Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans |
| title_fullStr | Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans |
| title_full_unstemmed | Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans |
| title_short | Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans |
| title_sort | control of nitrogen fixation and ammonia excretion in azorhizobium caulinodans |
| work_keys_str_mv | AT haskettt controlofnitrogenfixationandammoniaexcretioninazorhizobiumcaulinodans AT karunakaranr controlofnitrogenfixationandammoniaexcretioninazorhizobiumcaulinodans AT buenobatistam controlofnitrogenfixationandammoniaexcretioninazorhizobiumcaulinodans AT dixonr controlofnitrogenfixationandammoniaexcretioninazorhizobiumcaulinodans AT poolep controlofnitrogenfixationandammoniaexcretioninazorhizobiumcaulinodans |