A high‐throughput screen for ligand binding reveals the specificities of three amino acid chemoreceptors from P seudomonas syringae pv. actinidiae

Summary Chemoreceptors play a central role in chemotaxis, allowing bacteria to detect chemical gradients and bias their swimming behavior in order to navigate toward favorable environments. The genome of the kiwifruit pathogen, P seudomonas syringae pv. actinidiae ( P sa ) strain NZ ‐ V 13 encodes 43 predicted chemoreceptors, none of which has been characterized. We developed a high‐throughput fluorescence‐based thermal shift assay for identifying the signal molecules that are recognized by a given chemoreceptor ligand binding domain ( LBD ). Using this assay, we characterized the ligand binding profiles of three P sa homologs of the P . aeruginosa   PAO 1 amino acid chemoreceptors PctA , PctB and PctC . Each recombinant LBD was screened against 95 potential ligands. The three P sa homologs, named psc A , psc B and psc C ( Ps a c hemoreceptors A , B and C ) bound 3, 10 and 3 amino acids respectively. In each case, their binding profiles were distinct from their P . aeruginosa   PAO 1 homologs. Notably, P sa   PscA ‐ LBD only bound the acidic amino acids l ‐aspartate, d ‐aspartate and l ‐glutamate, whereas P . aeruginosa   PctA ‐ LBD binds all of the l ‐proteinogenic amino acids except for l ‐aspartate and l ‐glutamate. A combination of homology modeling, site‐directed mutagenesis and functional screening identified a single amino acid residue in the P sa   PscA ‐ LBD ( A la146) that is critically important for determining its narrow specificity.