FIG 2.

Accumulation of 7,10 DiHOME oxylipin in culture supernatants of PAO1 and its isogenic mutant ΔxcpQ. A) The ΔxcpQ mutant failed to produce 7,10-DiHOME when cultured on LB agar + OA (1%). The production of 7,10-DiHOME was restored by ΔxcpQ when it was complemented with a WT xcpQ gene expressed from a plasmid. B) Thin layer chromatography (TLC) analysis of supernatants from PAO1, ΔxcpQ, and ΔxcpQ complemented in trans detected the oxylipins 10-HOME and 7,10-DiHOME in the supernatant of PAO1 and ΔxcpQ complemented in trans. C) Immunoblot for OdsA in PAO1 and ΔxcpQ supernatants only detects the enzyme in PAO1 media. D) DS enzyme activity, as determined by the ability of cell-free supernatant to convert OA into oxylipins, was only observed for PAO1 and not ΔxcpQ when cultured in the presence of OA or E) oxylipins. F) DS enzymes accumulate in the in the periplasm of ΔxcpQ versus WT as determined by the ability of isolated periplasm fractions to convert OA into oxylipins. G) Activity of specific subcellular maker enzymes, IDH, isocitrate dehydrogenase (cytosolic), LDH, lactate dehydrogenase (membrane) and AP, alkaline phosphatase (periplasmic) was also measured to confirm the accuracy of cell fractionation. Enzyme activity in the periplasm fraction is expressed as a percentage of the total activity produced by crude cell extracts of the same cultures.