Alanine synthesized by alanine dehydrogenase enables ammonium-tolerant nitrogen fixation in <i>Paenibacillus sabinae</i> T27.

Abstract

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH₄⁺ (0 to 2 mM). However, <i>Paenibacillus sabinae</i> T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH₄⁺) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH₄⁺), nitrogenase activity is reestablished when very high levels of NH₄⁺ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of <i>P. sabinae</i> T27 in response to increasing ammonium concentrations. As anticipated, the <i>nif</i> genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH₄⁺ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH₄⁺ (10 mM). Among the differentially expressed genes, <i>ald1</i>, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH₄⁺ (100 mM). Mutation and complementation experiments revealed that <i>ald1</i> is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH₄⁺, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of <i>nif</i> transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.