Repressible alkaline phosphatase of Staphylococcus aureus.

In our investigations on the kinetics of growth and formation of exoenzymes by staphylococci under various nutritional conditions, a repressible alkaline phosphatase was detected. Search of the literature revealed one report (M. H. Kuo and H. J. Blumenthal, Nature 190:29, 1961) which indicated the absence of repressible alkaline phosphatase in 20 strains of coagulase-positive and coagulase-negative staphylococci. This report describes the formation of a repressible alkaline phosphatase by Staphylococcus aureus. All strains of staphylococci were grown in a medium containing, per liter: acid-hydrolyzed casein, salt free (Serva, Heidelberg, Germany), 3.5 g; (NH4)2SO4, 1.0 g; CaCl2, 0.01 g; MgSO4. 7H20, 0.1 g; nicotinic acid, 0.01 g; thiamine hydrochloride, 0.002 g; K2HPO4, 0.009g; yeast extract (Merck, Darmstadt, Germany), 0.2 g; tris (hydroxymethyl)-aminomethane (Tris), 24.2 g. The medium was adjusted to pH 7.3 with HCl. Inorganic phosphate and phosphate esters in yeast extract were partially removed by treatment with barium acetate at pH 8.2. Analysis of the medium revealed 7 ,ug of inorganic phosphorus and 0.5 ,ug of phosphorus as phosphate esters per ml. When phosphate was the limiting factor for growth, the medium contained, in addition, 0.4% sodium succinate. When the carbon source was the limiting factor for growth, the phosphate concentration was increased to 50 Mg of P/ml, and the sodium succinate concentration was reduced to 0.05 %. After overnight incubation on a rotatory shaker at 37 C in a carbon-limiting excess phosphate medium, the cells were centrifuged, resuspended in a new carbon-limiting excess phosphate medium, and grown for 3 hr. Then, after centrifugation. the cells were washed once with cold water and inoculated in a phosphate-limiting excess carbon medium. At intervals, 4.5-ml samples of the culture were added to 0.5 ml of 1:1,000 Merthiolate solution, and the tubes were placed in an ice bath. Optical density was measured in a Zeiss spectrophotometer at 625 m,. Samples of 2 ml were centrifuged at 4 C; the cells were washed once in cold water and assayed for phosphatases according to the procedure of 0. A. Bessey et al. (J Biol. Chem. 164: 321, 1946). The alkaline phosphatase was assayed at 25 C in 0.1 M Tris buffer (pH 9.1) containing 100 ,umoles of MgCl2/ml, and the acid phospha-