Abstract
Ammonia is a major signal that regulates nitrogen fixation in most diazotrophs. Regulation of nitrogen fixation by ammonia in the Gram-negative diazotrophs is well-characterized. In these bacteria, this regulation occurs mainly at the level of nif (nitrogen fixation) gene transcription, which requires a nif-specific activator, NifA. Although Gram-positive and diazotrophic Paenibacilli have been extensively used as a bacterial fertilizer in agriculture, how nitrogen fixation is regulated in response to nitrogen availability in these bacteria remains unclear. An indigenous GlnR and GlnR/TnrA-binding sites in the promoter region of the nif cluster are conserved in these strains, indicating the role of GlnR as a regulator of nitrogen fixation. In this study, we for the first time reveal that GlnR of Paenibacillus polymyxa WLY78 is essentially required for nif gene transcription under nitrogen limitation, whereas both GlnR and glutamine synthetase (GS) encoded by glnA within glnRA operon are required for repressing nif expression under excess nitrogen. Dimerization of GlnR is necessary for binding of GlnR to DNA. GlnR in P. polymyxa WLY78 exists in a mixture of dimers and monomers. The C-terminal region of GlnR monomer is an autoinhibitory domain that prevents GlnR from binding DNA. Two GlnR-biding sites flank the -35/-10 regions of the nif promoter of the nif operon (nifBHDKENXhesAnifV). The GlnR-binding site Ⅰ (located upstream of -35/-10 regions of the nif promoter) is specially required for activating nif transcription, while GlnR-binding siteⅡ (located downstream of -35/-10 regions of the nif promoter) is for repressing nif expression. Under nitrogen limitation, GlnR dimer binds to GlnR-binding siteⅠ in a weak and transient association way and then activates nif transcription. During excess nitrogen, glutamine binds to and feedback inhibits GS by forming the complex FBI-GS. The FBI-GS interacts with the C-terminal domain of GlnR and stabilizes the binding affinity of GlnR to GlnR-binding site Ⅱ and thus represses nif transcription.
Highlights
Biological nitrogen fixation, the conversion of atmospheric N2 to ammonia (NH3), is carried out by a specialized group of prokaryotes and plays an important role in world agriculture [1]
GlnR generally functions as repressor and inhibits gene transcription under excess nitrogen
Our study for the first time reveals that GlnR simultaneously acted as an activator and a repressor for nitrogen fixation of Paenibacillus by binding to different loci of the single nif promoter region according to nitrogen availability
Summary
Biological nitrogen fixation, the conversion of atmospheric N2 to ammonia (NH3), is carried out by a specialized group of prokaryotes and plays an important role in world agriculture [1]. The great demands for nitrogen in modern agriculture far outstrip this source of fixed nitrogen, and chemical nitrogen (N) fertilizer is used extensively in agriculture. Overuses of N fertilizer in many parts of the world have led to soil, water, and air pollution [2]. Ammonia is a major signal that regulates nitrogen fixation in most diazotrophs [3, 4]. Regulation of nitrogen fixation in the Gram-negative diazotrophs is well-characterized. In these bacteria, this regulation occurs mainly at the level of nif gene transcription, which requires a nif-specific activator, NifA [5]. NifA acts as an enhancer binding protein (EBP) that recognizes sequences (TGT-N10-ACA), located upstream of the -24/-12 region of the promoters controlled by RNA polymerase containing the alternative σ54 factor [3,6,7,8]
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