The Pseudomonas syringae pv. actinidiae chemoreceptor protein F (PscF) periplasmic sensor domain: cloning, purification and X-ray crystallographic analysis.

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 P212121. 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 P212121. 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.