Functional identification of Agrobacterium tumefaciens chemoreceptor MCP1912 in regulating chemotactic response

Abstract:

[Objective]To identify the chemoeffectors recognized by Agrobacterium tumefaciens C58 chemoreceptor MCP₁₉₁₂ (MCP: methyl-accepting chemotaxis protein), and to verify the function of this MCP in regulating the chemotactic response of A.tumefaciens. [Methods] The ligand binding domain (LBD) of MCP₁₉₁₂ was fused with His tag and expressed as an individual recombinant protein (named LBD₁₉₁₂) through heterologous expression. We employed fluorescence-based thermal shift assay (TSA) to screen the potential ligands of LBD₁₉₁₂, and isothermal titration calorimetry (ITC) to test the binding of the potential ligands to LBD₁₉₁₂ and to determine the equilibrium dissociation constant (K_D) of the ligand-LBD₁₉₁₂ complex. We used homologous recombination-based DNA fragment deletion method to construct MCP₁₉₁₂ deletion mutant (ΔMCP₁₉₁₂). We introduced the plasmid expressing MCP₁₉₁₂ into ΔMCP₁₉₁₂ to construct the complemented strain (ΔMCP₁₉₁₂C). Then, we adopted the capillary chemotaxis assay to test the chemotactic response of various A.tumefaciens strains to the potential MCP₁₉₁₂ ligands and to confirm the function of MCP₁₉₁₂ in regulating the chemotactic response of C58. [Results] Data acquired through TSA showed that 5 chemicals, pyruvate, L-lactate, propionic acid, acetic acid, and glycolate, might be the potential ligands of LBD₁₉₁₂. ITC further confirmed that only pyruvate and propionic acid could specifically bind to LBD₁₉₁₂. The binding of pyruvate to LBD₁₉₁₂ is exothermic with K_D of (17.0±0.9) μmol/L, while the binding of propionic acid to LBD₁₉₁₂ is endothermic with K_D of (31.5±5.4) μmol/L. The capillary chemotaxis assay verified that C58 manifested the attractant response to pyruvate and the repellent response to propionic acid. The deficiency of MCP₁₉₁₂ completely eliminated the chemotactic response of A.tumefaciens to pyruvate and propionic acid. The complementation of MCP₁₉₁₂ could restore the chemotactic response of MCP₁₉₁₂-deficient strain to pyruvate and propionic acid. [Conclusion] The chemoeffectors recognized by the chemoreceptor MCP₁₉₁₂ are propionate and propionic acid. MCP₁₉₁₂ mediates the attractant response to pyruvate and the repellent response to propionic acid.