SummaryChemoreceptors 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.

Chemoreceptors enable bacteria to detect chemical signals in the environment and navigate towards niches that are favourable for survival. The sensor domains of chemoreceptors function as the input modules for chemotaxis systems, and provide sensory specificity by binding specific ligands. Cache-like domains are the most common extracellular sensor module in prokaryotes, however only a handful have been functionally or structurally characterised. Here, we have characterised a chemoreceptor Cache-like sensor domain (PscD-SD) from the plant pathogen Pseudomonas syringae pv. actinidiae (Psa). High-throughput fluorescence thermal shift assays, combined with isothermal thermal titration calorimetry, revealed that PscD-SD binds specifically to C2 (glycolate and acetate) and C3 (propionate and pyruvate) carboxylates. We solved the structure of PscD-SD in complex with propionate using X-ray crystallography. The structure reveals the key residues that comprise the ligand binding pocket and dictate the specificity of this sensor domain for C2 and C3 carboxylates. We also demonstrate that all four carboxylate ligands are chemoattractants for Psa, but only two of these (acetate and pyruvate) are utilisable carbon sources. This result suggests that in addition to guiding the bacteria towards nutrients, another possible role for carboxylate sensing is in locating potential sites of entry into the host plant.

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Nitrate- and nitrite-sensing (NIT) domains are found associated with a wide variety of bacterial receptors, including chemoreceptors. However, the structure of a chemoreceptor-associated NIT domain has not yet been characterized. Recently, a chemoreceptor named PscF was identified from the plant pathogen Pseudomonas syringae pv. actinidiae that is predicted to contain a periplasmic NIT domain. The PscF sensor domain (PscF-SD; residues 42-332) was cloned into an appropriate expression vector, recombinantly produced in Escherichia coli BL21-Gold(DE3) cells and purified via immobilized metal-affinity and size-exclusion chromatography. Purified PscF-SD was screened for crystallization; the best crystal diffracted to a maximum resolution of 1.46 Å in space group P222. However, the data could not be phased using the only available NIT-domain structure (Klebsiella oxytoca NasR; PDB entry 4akk) as the search model. Therefore, a data set from a selenomethionine-labelled protein crystal was also collected. The selenomethionine-labelled protein crystal diffracted to a resolution of 2.46 Å in space group P222. These data will be used to attempt to solve the structure using the single-wavelength anomalous diffraction technique. The structure is expected to provide insights into the ligand specificity of NIT domains and the role of NIT domains in chemotaxis.

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low‐level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF‐G). EF‐G catalyzes ribosomal translocation and mutations in EF‐G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.