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Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans.

ABSTRACT: 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 (NH3) assimilation by glutamine synthetase (GS), preventing excess release of excess NH3 for plants. Diazotrophic bacteria can be engineered to excrete NH3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target plants. Here, we tested two strategies to control GS regulation and NH3 excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both PII homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH3 derived from N2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH3 excretion specifically under microaerobic conditions, the same cue that initiates N2 fixation, then deleted nifA and transferred a rhizopine nifAL94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N2 fixation and NH3 excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses" where N2 fixation and NH3 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.

SUBMITTER: Haskett TL

PROVIDER: S-EPMC9249168 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies revealed that many Gram-negative bacteria possess reb genes encoding R-body components, and interestingly, many of them are animal and plant pathogens. Azorhizobium caulinodans, a microsymbiont of the tropical legume Sesbania rostrata, also possesses reb genes. In this study, we demonstrate that A. caulinodans has ability to kill the host plant cells by producing R bodies, suggesting that pathogenicity conferred by an R body might be universal in bacteria possessing reb genes. Furthermore, we provide the first insight into the molecular mechanism underlying the expression of R-body production in response to environmental factors, such as temperature and 2-oxoglutarate.

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Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans.

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