Agrobacterium fabrum atu0526-Encoding Protein Is the Only Chemoreceptor That Regulates Chemoattraction toward the Broad Antibacterial Agent Formic Acid

SIMPLE SUMMARY: Soil-borne plant pathogens generally navigate their way to hosts through recognition of the root exudates by chemoreceptors. However, there is still a lack of appropriate identification of chemoreceptors and their ligands in the typical soil-borne plant pathogen Agrobacterium. Here, we characterize Atu0526 as a Cache-type chemoreceptor from Agrobacterium fabrum C58, identify the potential ligands interacting with Atu0526 and analyze the possible signal transduction mechanism after ligand binding. We confirm Atu0526 to be the receptor of the broad antibacterial agent formic acid. The deletion of atu0526 completely abolished the chemotaxis of A. fabrum toward formic acid. Further experiments showed that the residue Arg 115 plays an essential role in the chemotactic function. Molecular modelling suggests that Arg 115 provides “an anchorage” for formic acid to pull the minor loop, thereby forming a conformational change that may be essential for signal transduction. Identifying the first chemoreceptor of antimicrobial agent formic acid in Agrobacterium will provide new perspectives on bacteria navigating their way to the hosts, and the discovered key arginine site can significantly improve our understanding of the signal transduction mechanism of single-Cache-type chemoreceptors. ABSTRACT: Soil-born plant pathogens, especially Agrobacterium, generally navigate their way to hosts through recognition of the root exudates by chemoreceptors. However, there is still a lack of appropriate identification of chemoreceptors and their ligands in Agrobacterium. Here, Atu0526, a sCache-type chemoreceptor from Agrobacterium fabrum C58, was confirmed as the receptor of a broad antibacterial agent, formic acid. The binding of formic acid to Atu0526 was screened using a thermo shift assay and verified using isothermal titration calorimetry. Inconsistent with the previously reported antimicrobial properties, formic acid was confirmed to be a chemoattractant to A. fabrum and could promote its growth. The chemotaxis of A. fabrum C58 toward formic acid was completely lost with the knock-out of atu0526, and regained with the complementation of the gene, indicating that Atu0526 is the only chemoreceptor for formic acid in A. fabrum C58. The affinity of formic acid to Atu0526(LBD) significantly increased after the arginine at position 115 was replaced by alanine. However, in vivo experiments showed that the R115A mutation fully abolished the chemotaxis of A. fabrum toward formic acid. Molecular docking based on a predicted 3D structure of Atu0526 suggested that the arginine may provide “an anchorage” for formic acid to pull the minor loop, thereby forming a conformational change that generates the ligand-binding signal. Collectively, our findings will promote an understanding of sCache-type chemoreceptors and their signal transduction mechanism.