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

作者：McKellar James L O; Minnell Jordan J; Gerth Monica L^*

来源：Molecular Microbiology, 2015, 96(4): 694-707.

DOI：10.1111/mmi.12964

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, Pseudomonas syringae pv. actinidiae (Psa) strain NZ-V13 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 Psa homologs of the P.aeruginosaPAO1 amino acid chemoreceptors PctA, PctB and PctC. Each recombinant LBD was screened against 95 potential ligands. The three Psa homologs, named pscA, pscB and pscC bound 3, 10 and 3 amino acids respectively. In each case, their binding profiles were distinct from their P.aeruginosaPAO1 homologs. Notably, PsaPscA-LBD only bound the acidic amino acids l-aspartate, d-aspartate and l-glutamate, whereas P.aeruginosaPctA-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 PsaPscA-LBD (Ala146) that is critically important for determining its narrow specificity.

• 出版日期2015-5