Regulation of the Ald gene encoding alanine dehydrogenase and its induction of ammonium-tolerant nitrogen fixation in Paenibacillus polymyxa WLY78.

Background: Paenibacillus polymyxa WLY78, a Gram-positive diazotroph with plant growth promotion and phytopathogen suppression, represents a promising candidate for agricultural biofertilizers. However, its nitrogen fixation capacity is inherently limited by ammonium-mediated repression. Recent studies revealed that ammonium-tolerant nitrogen fixation in certain Paenibacillus species correlates with alanine overproduction mediated by alanine dehydrogenase (ADH) encoded by the ald gene.

Results: This study establishes a dual regulatory mechanism governing ald expression in P. polymyxa WLY78. The transcription activator AdeR positively regulates ald expression, while the global nitrogen regulator GlnR exerts repression on both ald and its activator gene adeR. Under high ammonium conditions, GlnR-mediated suppression maintains basal ald expression levels, preventing alanine biosynthesis. Upregulation of ald expression through high-copy plasmid or mutagenesis of GlnR-binding sites in the adeR-ald regulatory region significantly enhanced alanine concentration. Both endogenous overproduction and exogenous supplementation of alanine suppressed glutamine synthetase (GS) activity, thereby reducing intracellular glutamine levels. This prevents the formation of glutamine-feedback-inhibited GS complexes (FBI-GS), disrupting the GlnR-FBI-GS interaction required for nif gene repression. Consequently, GlnR transitions to its activated state, enabling nif gene expression even under elevated ammonium concentrations.

Conclusions: Our findings elucidate a conserved regulatory paradigm in Paenibacillus species where alanine metabolism modulates nitrogen fixation through GS-mediated metabolic signaling. The ald overexpression or exogenous alanine supplementation can bypass ammonium inhibition provides practical strategies for enhancing biofertilizer performance in nitrogen-rich agricultural soils.