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 Atu0526LBD 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.

The chemotactic response regulator CheY, when phosphorylated by the phosphoryl group from phosphorylated CheA, can bind to the motor switch complex to control the flagellar motor rotation. Agrobacterium fabrum (previous name: Agrobacterium tumefaciens), a phytopathogen, carries two paralogous cheY genes, cheY1 and cheY2. The functional difference of two paralogous CheYs remains unclear. Three cheY-deletion mutants were constructed to test the effects of two CheYs on the chemotaxis of A. fabrum. Phenotypes of three cheY-deletion mutants show that deletion of each cheY significantly affects the chemotactic response, but cheY2-deletion possesses more prominent effects on the chemotactic migration and swimming pattern of A. fabrum than does cheY1-deletion. CheA-dependent cellular localization of two CheY paralogs and in vitro pull-down of two CheY paralogs by FliM demonstrate that the distinct roles of two CheY paralogs arise mainly from the differentiation of their binding affinities for the motor switch component FliM, agreeing with the divergence of the key residues on the motor-binding surface involved in the interaction with FliM. The single respective replacements of key residues R93 and A109 on the motor-binding surface of CheY2 by alanine (A) and valine (V), the corresponding residues of CheY1, significantly enhanced the function of CheY2 in regulating the chemotactic response of A. fabrum CheY-deficient mutant Δy to nutrient substances and host attractants. These results conclude that the divergence of the key residues in the functional subdomain is the decisive factor of functional differentiation of these two CheY homologs and protein function may be improved by the substitution of the divergent key residues in the functional domain for the corresponding residues of its paralogs. This finding will help us to better understand how paralogous proteins sub-functionalize. In addition, the acquirement of two CheY2 variants, whose chemotactic response functions are significantly improved, will be very useful for us to further explore the mechanism of CheY to bind and regulate the flagellar motor and the role of chemotaxis in the pathogenicity of A. fabrum.