Format

Send to

Choose Destination
Mol Microbiol. 2015 May;96(4):694-707. doi: 10.1111/mmi.12964. Epub 2015 Mar 6.

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

Author information

1
Department of Biochemistry, University of Otago, PO Box 56, Dunedin, 9054, New Zealand.

Abstract

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. aeruginosa PAO1 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 (Psa chemoreceptors A, B and C) bound 3, 10 and 3 amino acids respectively. In each case, their binding profiles were distinct from their P. aeruginosa PAO1 homologs. Notably, Psa 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 Psa PscA-LBD (Ala146) that is critically important for determining its narrow specificity.

PMID:
25656450
DOI:
10.1111/mmi.12964
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center