Biochimica et Biophysica Acta, 1010 (1989) 325-329 Elsevier 325 BBA 12415 31p-NMR evidence for cytoplasmic acidification and phosphate extrusion in syringornycin-treated cells of Rhodotorula pilimanae Helwig H. Reidl 1,., Thomas A. Grover 2 and Jon Y. Takemoto 1 Molecular Biology~ Biochemistry Program, Departments of s Biology and 2 Chemistry / Biochemistry, Utah State University, Logan, UT (U.$.A.) (Received 25 July 1988) (Revised manuscript received 24 October 1988) Key words: Polyphosphate; NMR, 3~p.; Phosphate efflux: (S. ~yringae) 3t P-NMR spectroscopy was used to investigate the effects of the phytotoxin, syringomycin, on phosphate metabolism and intracellular pH changes in the yeast RhodotorMa pilimanae. Syringomycin, at levels between 20 and 60 units per l0 s cells, caused a cellular efflux of orthophosphate. At 40 and 60 units per l0 s cells, the efflux was accompanied by a decrease in polyphosphate and an acidification of the cytoplasm. At low temperatures (5 o C) and with 75 units per 108 cells, these effects were more rapid and pronounced. The efflux of phosphate was confirmed by chemically assaying extracellular phosphate after syringomycin treatment. Introduction Syringomycin is a potent phytotoxin produced by the bacterium Pseudomonas syringae pv syringae - the causative agent of several plant diseases [1]. The toxin is a broad spectrum antibiotic with antifungal as well as phytotoxic capabilities. Syringomycin is composed of several amino acids, but its complete structure is not yet known [2]. Recent work has shown that the primary site of action for the toxin is the plasma membrane [3-5]. Syringomycin induces changes in the electrical potential and proton gradient of the plasma membrane of the yeast, Rhodotorula pilimanae. These are caused by alter- ations in ion and proton fluxes. One result is charge hyperpolarization of the plasma membrane so that the cell exterior is more positive. The net direction of the proton flux, however, is less certain. Initial observations on the cellular uptake of dimethyloxazolidine suggested * Present address: Edelhoff Staedtereinigung, Postfach 1450, D5860 Iserlohn, F.R.G. Abbreviations: Mes, 4-morpholineethanesulfonic acid; SP, sugar phosophates; Poly-P, polyphosphates; MDP, methylene diphos- phonate. Correspondence: T.A. Grover, Molecular Biology/Biochemistry Pro- gram, Department of Chemistry/Biochemistry, Utah State Univer- sity, Logan, UT 84322-0300, U.S.A. 0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical an intracellular increase in pH with syringomycin treat- ment [3,4]. Also, addition of the toxin to plasma mem- brane fractions of R. pilimanae and red beet storage tissue stimulates an ATPase activity presumably re- sponsible for the active efflux of protons [3,6]. However, pH measurements on yeast cell suspensions showed increases in extracellular pH, which could be due to a net proton influx [5]. In the present study, we used 3~p-NMR spectroscopy to investigate further the effects of syringomycin on cellular proton flux in R. pilimanae and also to observe the effects of this toxin on phosphate metabolites. With 31P-NMR, the intracellular pH can be deduced from the position of the cytoplasmic orthophosphate, and sugar phosphate resonance lines [7-9]. Our results indicate that the intracellular pH decreases and certain phos- phate metabolite pools change dramatically with syrin- gomycin. Materials and Methods Cell suspensions. R. pilimanae (ATCC 26423) was grown at 21-25°C in 200 ml potato dextrose broth medium (Difco Laboratories, Detroit, MI) in 1 1 capac- ity Fernbach flasks on a reciprocal shaker. Cells were also grown in a fermenter (Microferm, New Brunswick) in 5 1 of potato dextrose broth medium supplemented with 10 mM potassium phoshate buffer (pH 6.5). The culture was kept at 25 °C and stirred at 200 rpm with vigorous aeration. Cells were harvested by centrifuga- Division)