Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa

Abstract

In order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60 +/- 0.64 microM) almost ten times greater than that of the wild type strain (Km = 0.39 microM). There was, however, no change in the Vmax for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium, resulting in an 18%-35% decrease in growth when compared with the wild type.