Phosphate transport across the outer membrane of Pseudomonas aeruginosa

When wild-type c e l l s 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 p u r i f i e d to homogeneity and demonstrated to form channels in planar l i p i d bilayer membranes. In agreement with previous data, the channels formed by protein P were anion-specific (due to the presence of a binding s i t e for anions in the channel) and exhibited a marked s e l e c t i v i t y for phosphate 2(HP04 ) over other anions (e.g chloride). These properties were not altered in protein P preparations p u r i f i e d 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 e x t r a c e l l u l a r release was s p e c i f i c and did not involve an increase in outer membrane permeability. The 34K periplasmic protein was p u r i f i e d and demonstrated to bind phosphate in v i t r o (Kd=0.34 uM). S p e c i f i c i t y studies revealed that inorganic phosphate polymers (up to P15) and i i arsenate could i n h i b i t the binding of orthophosphate to the binding protein, although organic phosphates (e.g. glucose6-phosphate) could not. The a b i l i t y of the phosphatebinding protein and protein P to associate was demonstrated in v i t r o , 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 h i g h a f f i n i t y (Km=0.39 uM phosphate), respectively. Mutants de f i c i e n t in the phosphate-binding protein were isolated and shown to lack the h i g h a f f i n i t y phosphate uptake system, confirming the role of the binding protein in h i g h a f f i n i t y phosphate transport in P. aeruginosa. In addition, a role for protein P in h i g h a f f i n i t y phosphate transport was confirmed by the i s o l a t i o n of a Tn501 insertion mutant lacking porin protein P. This mutant exhibited a ten-fold increase in Km for h i g h a f f i n i t y phosphate transport. The loss of these proteins in the respective mutants was correlated with a growth defect in a phosphate-deficient medium. Protein P, l i k e most porins, was isolated as an oligomer (trimer) in i t s native (functional) state, dissociating to non-functional monomers at high temperatures. A polyclonal antiserum s p e c i f i c 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 c r o s s r e a c t i v i t y was observed only with the native, oligomeric forms of these proteins. No cr o s s r e a c t i v i t y was seen with the constitutive porins produced by these strains, indicating that the cr o s s r e a c t i v i t y of phosphatelimitation-inducible oligomeric outer membrane proteins was not due to any homologies r e l a t i n g to porin structure in general. Using a polyclonal antiserum s p e c i f i c for protein P monomers, no r e a c t i v i t y was observed with either the oligomeric or monomeric forms of any of the phosphatelimitation-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 s o l u b i l i t y ) revealed that these proteins could be grouped into two classes, represented by protein P of P. aeruginosa and protein PhoE of Escherichia c o l i . Those proteins resembling protein P were i d e n t i f i e d in members of the fluorescent Pseudomonads, including P. putida, P. fluorescens, P. aureofaciens and P. chlororaphis. The pu r i f i e d proteins formed small, anion/phosphate-selective channels in planar l i p i d bilayers which were quite similar to protein P channels.