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

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, Paenibacillus sabinae 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 P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif 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, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 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 nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

Glutamine synthetase and GlnR regulate nitrogen metabolism in Paenibacillus polymyxa WLY78.

Paenibacillus polymyxa WLY78, a N2-fixing bacterium, has great potential use as a biofertilizer in agriculture. Recently, we have revealed that GlnR positively and negatively regulates the transcription of the nif (nitrogen fixation) operon (nifBHDKENXhesAnifV) in P. polymyxa WLY78 by binding to two loci of the nif promoter according to nitrogen availability. However, the regulatory mechanisms of nitrogen metabolism mediated by GlnR in the Paenibacillus genus remain unclear. In this study, we have revealed that glutamine synthetase (GS) and GlnR in P. polymyxa WLY78 play a key role in the regulation of nitrogen metabolism. P. polymyxa GS (encoded by glnA within glnRA) and GS1 (encoded by glnA1) belong to distinct groups: GSI-α and GSI-ß. Both GS and GS1 have the enzyme activity to convert NH4+ and glutamate into glutamine, but only GS is involved in the repression by GlnR. GlnR represses transcription of glnRA under excess nitrogen, while it activates the expression of glnA1 under nitrogen limitation. GlnR simultaneously activates and represses the expression of amtBglnK and gcvH in response to nitrogen availability. Also, GlnR regulates the expression of nasA, nasD1D2, nasT, glnQHMP, and glnS. IMPORTANCE In this study, we have revealed that Paenibacillus polymyxa GlnR uses multiple mechanisms to regulate nitrogen metabolism. GlnR activates or represses or simultaneously activates and inhibits the transcription of nitrogen metabolism genes in response to nitrogen availability. The multiple regulation mechanisms employed by P. polymyxa GlnR are very different from Bacillus subtilis GlnR which represses nitrogen metabolism under excess nitrogen. Both GS encoded by glnA within the glnRA operon and GS1 encoded by glnA1 in P. polymyxa WLY78 are involved in ammonium assimilation, but only GS is required for regulating GlnR activity. The work not only provides significant insight into understanding the interplay of GlnR and GS in nitrogen metabolism but also provides guidance for improving nitrogen fixation efficiency by modulating nitrogen metabolism.