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.

Chemoreceptor (also called methyl-accepting chemotaxis protein, MCP) is the leading signal protein in the chemotaxis signaling pathway. MCP senses and binds chemoeffectors, specifically, and transmits the sensed signal to downstream proteins of the chemotaxis signaling system. The genome of Agrobacterium fabrum (previously, tumefaciens) C58 predicts that a total of 20 genes can encode MCP, but only the MCP-encoding gene atu0514 is located inside the che operon. Hence, the identification of the exact function of atu0514-encoding chemoreceptor (here, named as MCP₅₁₄) will be very important for us to understand more deeply the chemotaxis signal transduction mechanism of A. fabrum. The deletion of atu0514 significantly decreased the chemotactic migration of A. fabrum in a swim plate. The test of atu0514-deletion mutant (Δ514) chemotaxis toward single chemicals showed that the deficiency of MCP₅₁₄ significantly weakened the chemotactic response of A. fabrum to four various chemicals, sucrose, valine, citric acid and acetosyringone (AS), but did not completely abolish the chemotactic response. MCP₅₁₄ was localized at cell poles although it lacks a transmembrane (TM) region and is predicted to be a cytoplasmic chemoreceptor. The replacement of residue Phe328 showed that the helical structure in the hairpin subdomain of MCP₅₁₄ is a direct determinant for the cellular localization of MCP₅₁₄. Single respective replacements of key residues indicated that residues Asn336 and Val353 play a key role in maintaining the chemotactic function of MCP₅₁₄.

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.

Bacteria utilize chemotaxis to sense the surrounding chemical signals to seek a more favorable survival environment. The chemotaxis process includes signal sensing, signal transduction, and signal response (i.e., regulating flagellar rotation to control motility). Agrobacterium fabrum, as a soil-born facultative phytopathogen, can survive in diverse environments from bulk soil, the rhizosphere, to the plant-associated niches, and needs to cope with diverse challenges from various survival environments. It must recognize a variety of environmental signals and thus has evolved a chemosensory signaling system more complicated than the prototypical chemotaxis system. The chemosensory system of A. fabrum possesses one histidine kinase, but more chemoreceptors, coupling proteins (2 CheWs), and response regulators (2 CheYs and 1 CheB) than the well-studied prototypical system in the model bacterium Escherichia coli, which has only one CheW, one CheY, and fewer chemoreceptors. More response regulators imply that the chemosensory system may involve other physiological functions beyond chemotaxis. In this review, we outline the recent advances in the prototypical chemosensory signaling system and discuss the functions of protein components in A. fabrum’s chemosensory system by comparing those proteins with the homologous proteins observed in the paradigm and other closely related species. Meanwhile, we place particular emphasis on reviewing the data about the chemosensory system of A. fabrum, propose a “one-system two-pathways” model depicting that A. fabrum possibly utilizes one histidine kinase to assemble two chemosensory signaling pathways, and envision future directions for studying this system. The insights provided will aid in understanding the diversity of chemosensory signaling pathways in other organisms and the molecular mechanism mediating the signal crosstalk among chemosensory signaling pathways.

Soil salinization poses a significant threat to global agriculture, necessitating the development of sustainable strategies to increase crop resilience. Pl

Methyl-accepting chemotaxis proteins (MCPs, or chemoreceptors) function as sensors and signal transmitters of the chemotaxis signaling system. As a soil-borne facultative phytopathogen, Agrobacterium tumefaciens (or fabrum) C58 encodes 20 MCPs, but only seven have been experimentally verified. Here, we characterized Atu0646, predicted as a transmembrane chemoreceptor with an sCache_2-type periplasmic ligand-binding domain (LBD₀₆₄₆). Differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC) results confirmed LBD₀₆₄₆ directly binds L-malate and succinate. Gene deletion assays validated Atu0646 as the chemoreceptor for these two ligands in A. tumefaciens C58. The chemotactic attraction of A. tumefaciens toward two plant-derived C4-dicarboxylates provides reasonable support for the critical role of chemotaxis in rhizosphere colonization and susceptible wound site localization of this phytopathogen. AlphaFold structural modeling, molecular docking, and site-directed mutagenesis identified key binding residues: five substitutions (Y90A, H103A, K155A, R125Y, Y174K) resulted in loss of function due to the alteration of its LBD structure, two (R108Y, N151Y) attenuated it, and one (S119Y) enhanced its binding capacity to its cognate ligand, while also broadening its ligand spectrum to recognize Asp. Ligand-binding mechanism analysis of Atu0646 improves our understanding of the structure–function relationship of chemoreceptors and provides a feasible strategy for the rational protein engineering of chemoreceptors.