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Phosphate transport across the outer membrane of Pseudomonas aeruginosa Poole, Raymond Keith


When wild-type cells of Pseudomonas aeruginosa were grown in a phosphate-limiting medium (0.2 mM orthophosphate) they were derepressed for the production of an outer membrane protein, designated protein P. This protein was purified to homogeneity and demonstrated to form channels in planar lipid bilayer membranes. In agreement with previous data, the channels formed by protein P were anion-specific (due to the presence of a binding site for anions in the channel) and exhibited a marked selectivity for phosphate 2- (HPO₄²⁻) over other anions (e.g chloride). These properties were not altered in protein P preparations purified free of lipopolysaccharide. Protein P was coinducible with the enzymes alkaline phosphatase and phospholipase C, and with a periplasmic protein of 34,000 molecular weight. Mutants of P. aeruginosa, constitutive or non-inducible for these constituents, were isolated. This suggested that the genes encoding these products were part of a phosphate regulon. Alkaline phosphatase and phospholipase C were demonstrated to be secreted into the external medium upon induction, although this extracellular release was specific and did not involve an increase in outer membrane permeability. The 34K periplasmic protein was purified and demonstrated to bind phosphate in vitro (Kd=0.34 uM). Specificity studies revealed that inorganic phosphate polymers (up to P15) and arsenate could inhibit the binding of orthophosphate to the binding protein, although organic phosphates (e.g. glucose-6-phosphate) could not. The ability of the phosphate-binding protein and protein P to associate was demonstrated in vitro, with implications concerning the means by which phosphate crosses the outer membrane. Two major inorganic phosphate transport systems were identifed, of low (Km=l9.3 uM phosphate) and high-affinity (Km=0.39 uM phosphate), respectively. Mutants deficient in the phosphate-binding protein were isolated and shown to lack the high-affinity phosphate uptake system, confirming the role of the binding protein in high-affinity phosphate transport in P. aeruginosa. In addition, a role for protein P in high-affinity phosphate transport was confirmed by the isolation of a Tn501 insertion mutant lacking porin protein P. This mutant exhibited a ten-fold increase in Km for high-affinity phosphate transport. The loss of these proteins in the respective mutants was correlated with a growth defect in a phosphate-deficient medium. Protein P, like most porins, was isolated as an oligomer (trimer) in its native (functional) state, dissociating to non-functional monomers at high temperatures. A polyclonal antiserum specific for protein P trimers was raised and shown to cross-react with other phosphate-starvation-inducible outer membrane proteins of the families Pseudomonadaceae and Enterobactereaceae. This cross-reactivity was observed only with the native, oligomeric forms of these proteins. No cross-reactivity was seen with the constitutive porins produced by these strains, indicating that the cross-reactivity of phosphate-limitation-inducible oligomeric outer membrane proteins was not due to any homologies relating to porin structure in general. Using a polyclonal antiserum specific for protein P monomers, no reactivity was observed with either the oligomeric or monomeric forms of any of the phosphate-limitation-inducible outer membrane proteins (except for protein P monomers). These data suggested that the common antigenic determinants present in these proteins were conserved in the native functional proteins only. Examination of some of the physical properties of the phosphate-starvation-inducible outer membrane proteins (e.g. molecular weight, peptidoglycan association, detergent solubility) revealed that these proteins could be grouped into two classes, represented by protein P of P. aeruginosa and protein PhoE of Escherichia coli. Those proteins resembling protein P were identified in members of the fluorescent Pseudomonads, including P. putida, P. fluorescens, P. aureofaciens and P. chlororaphis. The purified proteins formed small, anion/phosphate-selective channels in planar lipid bilayers which were quite similar to protein P channels.

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