Effect of point mutations on Herbaspirillum seropedicae NifA activity

Aquino, Bruno Pinheiro; Stefanello, Adriano Alves; Oliveira, Marco Aurélio Schüler de; Pedrosa, Fábio O.; Souza, Emanuel Maltempi de; Monteiro, Rose A.; Chubatsu, Leda S.

doi:10.1590/1414-431x20154522

Cited by 3 publications

(1 citation statement)

References 38 publications

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“…For example, in Azotobacter vinelandii , the GAF domain binds 2-oxoglutarate, a key metabolic signal of carbon status, the presence of which influences the interaction with the antiactivator protein NifL ( Martinez-Argudo et al, 2004 ). In the same vein, in Herbaspirillum seropedicae , NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK ( Aquino et al, 2015 ). In contrast, to our knowledge, no functional role has been attributed to the N-terminal GAF domain of the rhizobial NifA proteins.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Nitrogen Fixation in Bradyrhizobium sp. Strain DOA9 Involves Two Distinct NifA Regulatory Proteins That Are Functionally Redundant During Symbiosis but Not During Free-Living Growth

et al. 2018

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The Bradyrhizobium sp. DOA9 strain displays the unusual properties to have a symbiotic plasmid and to fix nitrogen during both free-living and symbiotic growth. Sequence genome analysis shows that this strain contains the structural genes of dinitrogenase (nifDK) and the nifA regulatory gene on both the plasmid and chromosome. It was previously shown that both nifDK clusters are differentially expressed depending on growth conditions, suggesting different mechanisms of regulation. In this study, we examined the functional regulatory role of the two nifA genes found on the plasmid (nifAp) and chromosome (nifAc) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using gusA (β-glucuronidase) reporter strains, we showed that both nifA genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with Aeschynomene americana, mutants in only one nifA gene were not altered in their symbiotic properties, while a double nifA mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under in vitro conditions, the nifAc mutant was unable to fix nitrogen, and no effect of the nifAp mutation was detected, indicating that NifAc is essential to activate nif genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of nifAc but not by nifAp or by two chimeric nifA genes encoding hybrid proteins with the N-terminus part of NifAc and the C-terminus of NifAp. Furthermore, transcriptional analysis by RT-qPCR of the WT and two nifA mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural nifDK genes during symbiosis, while only NifAc activated the expression of nifDKc during free-living conditions. In summary, this study provides a better overview of the complex mechanisms of regulation of the nitrogenase genes in the DOA9 strain that involve two distinct NifA proteins, which are exchangeable during symbiosis for the activation of nif genes but not during free-living growth where NifAc is essential for the activation of nifDKc.

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Section: Discussionmentioning

confidence: 99%

Regulation of Nitrogen Fixation in Bradyrhizobium sp. Strain DOA9 Involves Two Distinct NifA Regulatory Proteins That Are Functionally Redundant During Symbiosis but Not During Free-Living Growth

et al. 2018

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Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites

Stefanello

Oliveira

Souza

et al. 2020

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense

Schnabel

Sattely

2021

Appl Environ Microbiol

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Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet’s major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here we design and test a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiontAzospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that post-translationally modifies, and deactivates glutamine synthase, a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly and we leverage the inherent self-contained nature of our post-translational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 μM h−1 OD600−1. When grown in co-culture with the model monocot Setaria viridis, we demonstrate that these strains increases the biomass and chlorophyll content of plants up to 54% and 71% respectively relative to WT. Furthermore, we rigorously demonstrate direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of co-cultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings is derived from diazotrophically fixed dinitrogen, whereas no nitrogen is incorporated in plants co-cultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision could be deployable in a consortium of nitrogen fixing symbiotic diazotrophs for plant fertilization. Importance Statement Nitrogen is the most limiting nutrient in modern agriculture. Free living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here we use a rational engineering approach to generate ammonia excreting strains of Azospirillum. Our design features post-translational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We show that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrate in comparison to WT controls that our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

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Effect of point mutations on Herbaspirillum seropedicae NifA activity

Cited by 3 publications

References 38 publications

Regulation of Nitrogen Fixation in Bradyrhizobium sp. Strain DOA9 Involves Two Distinct NifA Regulatory Proteins That Are Functionally Redundant During Symbiosis but Not During Free-Living Growth

Regulation of Nitrogen Fixation in Bradyrhizobium sp. Strain DOA9 Involves Two Distinct NifA Regulatory Proteins That Are Functionally Redundant During Symbiosis but Not During Free-Living Growth

Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites

Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense

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