ABSTRACTSinorhizobium melilotiis a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache (calcium channels andchemotaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpVPRis structurally similar to a sensor domain of a chemoreceptor with unknown function fromAnaeromyxobacter dehalogenans, which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding andS. melilotifor carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpVPR. Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of theS. melilotiwild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion ofmcpVor mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpVPRwere in the micromolar range. An McpVPRvariant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction.IMPORTANCELegumes share a unique association with certain soil-dwelling bacteria known broadly as rhizobia. Through concerted interorganismal communication, a legume allows intracellular infection by its cognate rhizobial species. The plant then forms an organ, the root nodule, dedicated to housing and supplying fixed carbon and nutrients to the bacteria. In return, the engulfed rhizobia, differentiated into bacteroids, fix atmospheric N2into ammonium for the plant host. This interplay is of great benefit to the cultivation of legumes, such as alfalfa and soybeans, and is initiated by chemotaxis to the host plant. This study on carboxylate chemotaxis contributes to the understanding of rhizobial survival and competition in the rhizosphere and aids the development of commercial inoculants.
Comparative study ofSaccharomyces cerevisiaewine strains to identify potential marker genes correlated to desiccation stress tolerance