Characterization of an AsnC/Lrp-family transcriptional regulator in Herbaspirillum rubrisubalbicans M1: linking plant interaction, nitrogen response and PHB metabolism

Introduction The mutation of Hrubri_0242 in Herbaspirillum rubrisubalbicans M1 altered the regulation of genes associated with polyhydroxybutyrate (PHB) metabolism, including phaC1 , phaP1, phaP2, phaZ1 , and phaZ2 , disrupting the coordinated balance between biopolymer synthesis, stabilization, and degradation. Methods Quantitative GC-FID analyses, fluorescence microscopy, and gene expression analysis were used to evaluate PHB metabolism, intracellular granule formation, and transcriptional responses under nitrogen-replete and nitrogen-limited conditions. Results Quantitative GC-FID analyses revealed a dynamic accumulation–consumption cycle of PHB in the wild-type strain, whereas the hr0242:Tn5 mutant exhibited reduced production, impaired turnover, and earlier depletion of intracellular reserves. Although both strains synthesized PHB under nitrogen-replete and nitrogen-limited conditions, accumulation was less sustained in the mutant, indicating compromised storage capacity. Fluorescence microscopy confirmed the presence of intracellular granules in both strains, with lower intensity and frequency in the mutant. Gene expression analysis revealed coordinated regulation of genes involved in PHB metabolism in the wild type according to nitrogen availability, whereas the mutant exhibited reduced phaP1 expression, compensatory activation of phaP2 , and increased expression of depolymerases, resulting in enhanced polymer turnover. In addition to metabolic effects, disruption of Hrubri_0242 significantly reduced swarming motility, indicating broader physiological consequences. Discussion Together, these findings demonstrate that Hrubri_0242 functions as an integrative transcriptional regulator linking nitrogen-responsive gene expression to PHB metabolic homeostasis and motility, thereby contributing to physiological adaptation in H. rubrisubalbicans M1.