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A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1 as an immunological phosphate-starvation marker

¹Section of Genetics and Microbiology, Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
²Department of Biochemistry and Nutrition, Building 224, The Technical University of Denmark, DK-2800 Lyngby, Denmark

ABSTRACT

A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1, expressed at phosphate concentrations below 0.08—0.13 mM, was purified and characterized. The purification method involved separation of outer-membrane proteins by SDS-PAGE and extraction of the protein from nitrocellulose or PVDF membranes after electrotransfer of proteins to the membranes. The N-terminal amino acid sequence of the purified protein, called Psi1, did not show homology to any known proteins, and in contrast to the phosphate-specific porin OprP of P. aeruginosa its mobility in SDS-PAGE was not affected by solubilization temperature. An antiserum against Psi1 recognized a protein of M_r 55000 in four other P. fluorescens strains among 24 tested strains representing Pseudomonas rRNA homology group I, showing antigenic heterogeneity within this group. A method for immunofluorescence microscopy involving cell permeabilization was adapted to visualize cell-specific expression of Psi1 in P. fluorescens exposed to limiting amounts of phosphate. This approach should be useful for further exploration of Psi1 as a marker to study the availability of phosphate to P. fluorescens in natural environments.

^*Author for correspondence: Ole Nybroe. Tel: + 45 3528 2629. Fax: + 45 3528 2606.

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