Diurnal switches in diazotrophic lifestyle increase nitrogen contribution to cereals

The study addresses how to enhance plant-associative biological nitrogen fixation (BNF) for cereals by engineering diazotrophs to excrete ammonia without compromising their growth and stability. Conventional fertilizer use is inefficient and environmentally harmful, while non-symbiotic BNF can significantly contribute nitrogen to cereals. Unlike symbiotic systems (e.g., rhizobium–legume), associative diazotrophs typically assimilate most fixed nitrogen into their own biomass rather than release it. The authors propose decoupling nitrogen fixation from ammonium assimilation by targeting glutamine synthetase (GS), which generates glutamine—a key intracellular signal for nitrogen status that governs Ntr and NifL–NifA regulatory systems. They hypothesize that a specific GS variant can reduce intracellular glutamine, render nitrogen regulation insensitive to ammonium, and, critically, introduce a temperature-dependent switch enabling nocturnal ammonia excretion while preserving daytime growth. This would leverage natural day–night temperature cycles to increase nitrogen transfer to cereals.

Prior work achieved ammonia excretion by perturbing GS activity through inhibitor-resistant glnA mutations in diazotrophs such as Anabaena variabilis and Azospirillum brasilense, which promoted plant growth. Other strategies include placing glnA under tunable promoters (Azotobacter vinelandii) and using truncated GlnE variants to constitutively adenylylate GS (A. brasilense, Azorhizobium caulinodans), sometimes controlled by plant signals to engineer synthetic symbioses. However, these approaches can impose fitness costs, instability, and require redundancy to maintain function. Long-term stability is challenged by selection for revertants under nitrogen starvation and energetic constraints from elevated nitrogenase activity. Studies in Salmonella typhimurium identified glnA mutants with lower glutamine yet near-normal growth on ammonia, suggesting a possible ‘sweet spot’ where glutamine signals nitrogen limitation and activates Ntr without severely impairing growth. This work exploits one such S. typhimurium allele (glnA424; GS-P95L) conserved in γ-proteobacteria to test if it confers ammonium-tolerant nitrogen fixation and conditional ammonia excretion.

• Engineering GS-P95L: The S. typhimurium glnA424 allele encodes GS-P95L. The authors introduced the homologous P95L substitution into E. coli NCM3722 (Ec424) and Klebsiella oxytoca M5al (Ko424) via λ-Red recombination. For K. oxytoca, a double-nucleotide change (CCA→CTG) was used to stabilize the mutation. A ΔglnE derivative (Ko424ΔglnE) was also constructed. A K. oxytoca Δnif strain (KoΔnif) was generated by FnCpf1-mediated deletion of the nif cluster.
• nif gene cluster reconstitution in E. coli: A 26-kb K. oxytoca nif cluster was cloned into pCC1BAC (pKU7824) and introduced into E. coli strains.
• Growth conditions: Aerobic and anaerobic cultures in modified L medium with defined salts, trace metals, 0.6% glucose; nitrogen-free or with specified NH4Cl. Nitrogenase experiments at 30 °C and 23 °C, and a range down to 20 °C for temperature profiling.
• Regulatory assays: Intracellular glutamine and glutamate measured by Glutamine/Glutamate-Glo. Ntr-controlled promoter activation assessed via qRT-PCR and lacZ fusions: E. coli PglnA::lacZYA (pKU101) and K. oxytoca PnifLA::lacZYA (pKU805). Anaerobic conditions at 30 °C with varying NH4+ tested activation hierarchies (glnA, glnH, nac, glnK, serA) and nifLA.
• Nitrogenase activity: Acetylene reduction assays (ARA) normalized to wild-type activity at zero NH4+ for temperature comparisons.
• Ammonia metabolism: External NH4+ consumption measured; ammonia excretion quantified (Sigma AA0100). Time courses correlated nitrogenase activity with NH4+ accumulation.
• Isotopic verification: 1H-NMR on supernatants after anaerobic growth in N-free medium under 15N2 vs 14N2 or Ar headspace to confirm excreted NH4+ originated from N2 fixation.
• Enzyme biochemistry: His-tagged wild-type and P95L GS from K. oxytoca expressed in E. coli BL21(DE3) ΔglnA ΔglnE and purified by Ni-NTA. Biosynthetic activity measured by phosphate release; apparent Km and temperature dependence assessed with saturating substrates; compared 20–37 °C.
• Algal co-culture: K. oxytoca strains streaked with Chlorella sorokiniana on N-free L agar under light, at constant 30 °C, constant 23 °C, or 12 h oscillating 30–23 °C cycles; chlorophyll a quantified.
• Plant studies: Hydroponic rice (japonica) and maize inbred lines (screen of 8; focused on 93-63) grown with diurnal 12 h 30 °C day/23 °C night cycle; no added carbon; inoculation by 1 h root immersion in ~10^8 cells mL−1; growth 9–12 days. Outcomes: dry weight, total N (elemental analyzer). Oxygen in hydroponics monitored; root colonization assessed with GFP-labeled K. oxytoca.
• Isotope-based plant N assays: 15N dilution with 0.5 mM 15NO3− to estimate %N derived from atmosphere (NF%) using KoΔnif as reference, with and without seed 15N subtraction. Direct 15N incorporation measured as fractional 15N in pheophytin after 8 days co-culture in sealed bags with 50% 15N2 and 1% CO2.
• Stability: Stationary-phase supernatant NH4+ tracked up to 144 h; phenotype stability over ~40 generations at 23 °C or 30 °C by screening 400 colonies for ability to support Chlorella growth.

• GS-P95L lowers intracellular glutamine and activates Ntr under ammonium: In E. coli Ec424, glutamine decreased >2-fold aerobically at 37 °C; under anaerobic 30 °C conditions, glutamine fell ~4.4-fold vs wild type, correlating with increased PglnA activity and activation of NtrC-dependent promoters despite 1–5 mM NH4+.
• Ammonium-tolerant nif regulation: In Ec424 carrying pKU7824, nitrogenase activity persisted with NH4+ at 30 °C (declining with more NH4+) and was markedly more tolerant at 23 °C, retaining ~70% of wild-type no-NH4+ activity even at 5 mM NH4+.
• Temperature-dependent ammonia excretion: Ec424(pKU7824) excreted NH4+ at ≤27 °C with maximal excretion at 20 °C (−1.1 mM), coinciding with stronger growth penalties during N2-dependent growth.
• Translation to K. oxytoca (Ko424): At 30 °C, Ko424 showed partial ammonium tolerance; at 23 °C, nitrogenase activity was substantially more tolerant to NH4+ and external NH4+ consumption was minimal. Temperature-dependent NH4+ excretion reached ~3.32 ± 0.15 mM at 20 °C; detectable excretion began at 30 °C (0.21 ± 0.03 mM) and increased as temperature decreased (Table 1). Intracellular glutamine in Ko424 declined with temperature (1.6 mM at 33 °C to 0.5 mM at 20 °C), while glutamate pools were only modestly reduced (<2-fold), indicating signaling of N limitation without collapse of glutamate homeostasis.
• Source of excreted NH4+: 1H-NMR showed NH4+ in Ko424 supernatants matched 15NH4Cl standard when grown under 15N2, confirming derivation from N2 fixation; no signal under Ar control.
• GlnE not required for excretion: Deleting glnE in Ko424 did not alter NH4+ excretion at 23 °C, suggesting P95L either resists adenylylation or low glutamine fails to activate feedback.
• Biochemistry: Purified K. oxytoca GS-P95L exhibited lower activity and heightened temperature sensitivity compared with wild-type GS; Km(glutamate) for wild-type varied with temperature; variant showed low activity preventing precise Km determination, consistent with in vivo cold-sensitive GS activity.
• Algal beneficiary and diurnal advantage: Chlorella growth and chlorophyll a increased adjacent to Ko424 at 23 °C, but not at 30 °C; 12 h 30–23 °C oscillations enhanced chlorophyll a beyond constant 23 °C, indicating that daytime recovery improves net N donation.
• Plant growth promotion under diurnal cycles:
  • Rice (japonica): Under 30–23 °C cycles, Ko424 increased biomass by ~12% and total N by 18% vs wild-type K. oxytoca; no added carbon or nitrogen.
  • Maize screens: Among 8 inbred lines, three responded to Ko424 with dry weight increases of 14% (B73), 20% (Fu8701), and 29% (93-63) vs wild type.
  • Maize 93-63 detailed tests: Under 30–23 °C cycles without added N, Ko424 increased dry weight by ~30–42% and N content by ~34–38% vs wild type. At constant 23 °C, gains were smaller; at constant 30 °C, no significant improvement.
• Isotope evidence in maize 93-63:
  • 15N dilution: NF% = 14.1 ± 1.2% (Ko424) vs 0.0 ± 1.1% (KoΔnif) and 1.8 ± 0.9% (Ko). After subtracting seed 15N, NF% = 25.6 ± 1.4% (Ko424) vs 0.0 ± 4.2% (KoΔnif) and 2.1 ± 2.7% (Ko).
  • Direct incorporation: 1.4% of total N in pheophytin was 15N in Ko424-inoculated maize; 0.4% in rice; both significant vs controls.
• Stability: Ammonia excretion plateaued and remained stable up to 144 h in stationary phase; no detectable reversion in ~40 generations (400 colonies screened) at either 23 °C or 30 °C. Overall, a single GS substitution creates a temperature-controlled switch enabling nocturnal ammonia excretion with daytime recovery, boosting nitrogen delivery and growth in cereals, particularly maize line 93-63.

Targeting glutamine synthetase with the P95L substitution lowered intracellular glutamine to levels that mimic nitrogen limitation, thereby sustaining Ntr activation and nif expression even with external ammonium. Crucially, the variant introduced cold-sensitive GS activity, which uncouples nitrogen fixation from assimilation at lower temperatures to cause ammonia excretion, while permitting coupling and growth at higher temperatures. Leveraging natural diurnal temperature ranges (30 °C day/23 °C night), engineered K. oxytoca (Ko424) alternates between daytime growth and nighttime ammonia donation, enhancing net nitrogen transfer to plants compared with constant conditions. This thermally driven oscillator avoids complex synthetic regulatory circuits, appears genetically stable, and fits environmental constraints. The results address the central goal of increasing BNF contributions to cereals by providing a controllable, environment-responsive mechanism for nitrogen release. The findings highlight genotype dependence in plant–microbe interactions (strongest response in maize 93-63) and suggest that temporal separation of nitrogenase activity (night) from higher oxygen periods may favor microaerobic conditions suitable for BNF. Together, these insights provide a practical pathway to improving associative nitrogen fixation with minimal genetic modification.

This study demonstrates that a single amino acid substitution in glutamine synthetase (GS-P95L) creates an environmentally driven, diurnal switch in diazotrophic lifestyle: ammonium-tolerant nitrogen fixation with nocturnal ammonia excretion at cooler temperatures and daytime recovery at warmer temperatures. Implemented in E. coli and particularly in the plant-associated K. oxytoca (Ko424), this strategy resulted in significant nitrogen donation and growth promotion in cereal crops under diurnal temperature cycles, supported by isotope-based evidence of N transfer. The approach offers a simple, genetically stable, and externally actuated alternative to complex synthetic circuits for enhancing associative BNF. Future work could: (1) tune GS thermal sensitivity to match diverse agroecological temperature profiles; (2) explore additional environmental or optogenetic oscillators; (3) integrate plant-controlled signaling tied to the plant circadian clock; (4) expand host range and assess broader cereal genotypes; and (5) validate performance and stability in field soils with realistic microbial competition and environmental dynamics.

• Experiments were conducted under controlled hydroponic or agar co-culture conditions; performance in field soils with complex microbiomes and gradual temperature changes remains to be validated.
• Nitrogen transfer quantification has methodological constraints: 15N dilution relies on appropriate references and seed 15N accounting; direct 15N incorporation in pheophytin under sealed-bag conditions likely underestimates transfer due to compromised plant physiology and exudation.
• The beneficial effect was plant genotype dependent (e.g., strongest in maize 93-63); generalizability across varieties and crops requires broader testing.
• Growth penalties for the donor at low temperatures reflect severe nitrogen starvation during excretion; although diurnal recovery mitigates this, long-term ecological fitness and persistence need field assessment.
• Stability testing detected no reversion over ~40 generations, but longer-term evolutionary stability and potential for suppressor mutations in diverse environments remain open questions.
• Oxygen dynamics favorable for nitrogenase were achieved in hydroponics; consistency of microaerobic niches around roots in soils with fluctuating aeration and moisture is uncertain.