title: Identification of a Chemoreceptor inPseudomonas aeruginosaThat Specifically Mediates Chemotaxis Toward α-Ketoglutarate.
abstract: Pseudomonas aeruginosais an ubiquitous pathogen able to infect humans, animals, and plants. Chemotaxis was found to be associated with the virulence of this and other pathogens. Although established as a model for chemotaxis research, the majority of the 26P. aeruginosachemoreceptors remain functionally un-annotated. We report here the identification of PA5072 (named McpK) as chemoreceptor for α-ketoglutarate (αKG). High-throughput thermal shift assays and isothermal titration calorimetry studies (ITC) of the recombinant McpK ligand binding domain (LBD) showed that it recognizes exclusively α-ketoglutarate. The ITC analysis indicated that the ligand bound with positive cooperativity (Kd1= 301 μM,Kd2= 81 μM). McpK is predicted to possess a helical bimodular (HBM) type of LBD and this and other studies suggest that this domain type may be associated with the recognition of organic acids. Analytical ultracentrifugation (AUC) studies revealed that McpK-LBD is present in monomer-dimer equilibrium. Alpha-KG binding stabilized the dimer and dimer self-dissociation constants of 55 μM and 5.9 μM were derived for ligand-free and αKG-bound forms of McpK-LBD, respectively. Ligand-induced LBD dimer stabilization has been observed for other HBM domain containing receptors and may correspond to a general mechanism of this protein family. Quantitative capillary chemotaxis assays demonstrated thatP. aeruginosashowed chemotaxis to a broad range of αKG concentrations with maximal responses at 500 μM. Deletion of themcpKgene reduced chemotaxis over the entire concentration range to close to background levels and wild type like chemotaxis was recovered following complementation. Real-time PCR studies indicated that the presence of αKG does not modulatemcpKexpression. Since αKG is present in plant root exudates it was investigated whether the deletion ofmcpKaltered maize root colonization. However, no significant changes with respect to the wild type strain were observed. The existence of a chemoreceptor specific for αKG may be due to its central metabolic role as well as to its function as signaling molecule. This work expands the range of known chemoreceptor types and underlines the important physiological role of chemotaxis toward tricarboxylic acid cycle intermediates.
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Martín-Mora
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Ortega
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Reyes-Darias
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Vanina
authors.initials: V
authors.lastname: García
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: López-Farfán
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Matilla
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
authors.forename: Tino
authors.initials: T
authors.lastname: Krell
keywords: Pseudomonas aeruginosa; chemoreceptor; chemotaxis; molecular recognition; signal transduction; α-ketoglutarate