A high‐throughput screen for ligand binding reveals the specificities of three amino acid chemoreceptors from Pseudomonas syringae pv. actinidiae. Issue 4 (6th March 2015)

• Record Type: Journal Article
• Authors: McKellar, James L. O.; Minnell, Jordan J.; Gerth, Monica L.
• Is Part Of: Molecular microbiology. Volume 96:Issue 4(2015)
• Page Start: 694
• Page End: 707
• Publication Date: 2015-03-06
• Subjects: Molecular microbiology -- Periodicals; 572.829
• DOI: 10.1111/mmi.12964
• Languages: English
• ISSNs: 0950-382X

Abstract

<abstract abstract-type="main"> <title>Summary</title> <p>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, <italic>P</italic><italic>seudomonas syringae</italic> pv. <italic>actinidiae</italic> (<italic>P</italic><italic>sa</italic>) 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 <italic>P</italic><italic>sa</italic> homologs of the <italic>P</italic><italic>. aeruginosa</italic> PAO1 amino acid chemoreceptors PctA, PctB and PctC. Each recombinant LBD was screened against 95 potential ligands. The three <italic>P</italic><italic>sa</italic> homologs, named <italic>psc</italic><italic>A</italic>, <italic>psc</italic><italic>B</italic> and <italic>psc</italic><italic>C</italic> (<italic><underline>P</underline></italic><italic><underline>s</underline>a</italic><underline>c</underline>hemoreceptors <underline>A</underline>, <underline>B</underline> and <underline>C</underline>) bound 3, 10 and 3 amino acids respectively. In each case, their binding profiles were distinct from their <italic>P</italic><italic>. aeruginosa</italic> PAO1 homologs. Notably, <italic>P</italic><italic>sa</italic> PscA‐LBD only bound the acidic amino acids <sc>l</sc>‐aspartate, <sc>d</sc>‐aspartate and <sc>l</sc>‐glutamate, whereas <italic>P</italic><italic>. aeruginosa</italic> PctA‐LBD binds all of the <sc>l</sc>‐proteinogenic amino acids except for <sc>l</sc>‐aspartate and <sc>l</sc>‐glutamate. A combination of homology modeling, site‐directed mutagenesis and functional screening identified a single amino acid residue in the <italic>P</italic><italic>sa</italic> PscA‐LBD (Ala146) that is critically important for determining its narrow specificity.</p> </abstract>