Fig. 4: The GS-P95L variant confers temperature-dependent nitrogen feedback regulation of nitrogen fixation and ammonium excretion in K. oxytoca.

a Schematic diagram of the construction of the glutamine synthetase P95L substitution in K. oxytoca. The derivative of K. oxytoca encoding the GS-P95L variant is designated as Ko424. b, c Acetylene reduction activities of K. oxytoca (Ko) (black bars) or the GS-P95L variant Ko424 (green bars), in response to the ammonia concentrations indicated on the x axes. Cultures were grown anaerobically in L medium either at 30 °C (b) or 23 °C (c) for 10 hours prior to assay. Data are normalised to 100% of the nitrogenase activity of Ko in the absence of ammonium (0 mM NH₄⁺) at each temperature. Means and SDs were calculated. n = 3 biologically independent samples. d The ammonia excreted by Ko424 is derived from nitrogen fixation. Cultures were grown in nitrogen-free medium, in which air in the anaerobic flask was evacuated with a vacuum pump and then filled with either ¹⁵N₂ or ¹⁴N₂ as the sole nitrogen source as indicated on each spectrum. A further culture was incubated with the inert gas argon (Ar) in the headspace to provide a negative control. After 72 h of anaerobic cultivation of Ko424 at 23 °C, the supernatant was collected and deuterated dimethyl sulfoxide (DMSO) (10% final concentration) and concentrated hydrochloric acid (5% final concentration) were added. To obtain the peak map of the ¹H-NMR spectrum, samples were analyzed with a selective pulse sequence for ammonia using a high-resolution nuclear magnetic resonance spectrometer (600 MHz Bruker Avance NMR instrument)^27,38 as described in the Methods. Source data are provided as a Source Data file.