Overexpression in Escherichia coli and functional analysis of a novel PP(i)-selective porin, OprO, from Pseudomonas aeruginosa

Abstract

Immediately upstream from and adjacent to the oprP gene, which codes for the phosphate-specific porin OprP of Pseudomonas aeruginosa, lies the PR region (oprO), which cross-hybridizes with oprP DNA. To determine the function of this region, the oprO gene was expressed behind the lactose promoter in Escherichia coli, and the resultant OprO protein was purified and reconstituted into planar lipid bilayers. OprO formed sodium dodecyl sulfate-stable trimers, cross-reacted immunologically with OprP, and, like OprP, formed an anion-specific, phosphate-selective porin. However, it demonstrated lower affinity for and higher maximal conductance of both chloride and phosphate than did the OprP channel. Examination by macroscopic conductance inhibition experiments of the affinity of OprO for phosphates of different lengths revealed a preference for PP(i) and tripolyphosphate over P(i), suggesting that OprO functioned as a PP(i)-selective polyphosphate channel, in contrast to OprP, which has a marked preference for P(i).