Abstract Plant-associated bacteria play important regulatory roles in modulating plant hormone auxin levels, affecting the growth and yields of crops. A conserved auxin-degradation (adg) operon was recently identified in the Variovorax genomes, which is responsible for root growth inhibition (RGI) reversion, promoting rhizosphere colonization and root growth. However, the molecular mechanism underlying auxin degradation by Variovorax remains unclear. Here, we systematically screened Variovorax adg operon products and identified two proteins, AdgB and AdgI, that directly associate with auxin indole-3-acetic acid (IAA). Further biochemical and structural studies revealed that AdgB is a highly IAA-specific ABC transporter solute binding protein, likely involved in IAA uptake. AdgI interacts with AdgH to form a functional Rieske non-heme dioxygenase, which works in concert with a FMN-type reductase encoded by gene adgJ to transform IAA into the biologically inactive 2-oxindole-3-acetic acid (oxIAA), representing a new bacterial pathway for IAA inactivation/degradation. Importantly, incorporation of a minimum set of adgH/I/J genes could enable IAA degradation by E. coli , suggesting a promising strategy for repurposing the adg operon for IAA regulation. Together, our study identifies the key components and underlying mechanisms involved in IAA transformation by Variovorax and brings new insights into the bacterial turnover of plant hormones, which would provide the basis for potential applications in rhizosphere optimization and ecological agriculture.

Molecular Mechanism for Bacterial Degradation of Plant Hormone Auxin