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

Abstract

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.

Nitric oxide signaling in ctenophores

“…Unique NIT (PF08376) domains ( Oulavallickal et al, 2017 ), containing cyclases, were previously identified in Trichoplax ( Moroz et al, 2020 ) as candidates for nitrite/nitrate sensors, similar to bacteria ( Shu et al, 2003 ; Camargo et al, 2007 ). Mnemiopsis has one sequence (ML02033a) associated with the same cluster ( Figure 5 ) but with a highly derived NIT-like region and unknown function.…”

The American lobster genome reveals insights on longevity, neural, and immune adaptations

“…This pathway frequently works together with the gaseous nitric oxide (NO)-mediated signaling machinery in chemosensory and neuronal processes (30,31). Further genomic deciphering of these NO-dependent pathways and, in particular, the quest for NO-reception complement, led to the discovery and identification of very unusual guanylate cyclases with unique nitrate-and nitrite-sensing (NIT) domains (32) in the lobster genome. These NIT domains were previously characterized in prokaryotes (e.g., bacteria and cyanobacteria) (33).…”

The diversification and lineage-specific expansion of nitric oxide signaling in Placozoa: insights in the evolution of gaseous transmission

“…Unexpectedly, we discovered 12 additional guanylate cyclases with unique NIT domains 76 , which were only previously known from bacteria as nitrate and nitrite sensors 77 , 78 . Nitrate/nitrite sensing type domain in placozoans (NIT: PF08376) is flanked by two transmembrane domains and a C-terminal guanylate cyclase catalytic domain (AC/GC: PF00211).…”