Alkaline Phosphatase Suhunits in the Culture Filtrate of Pseudomonas aeruginosa

Abstract

Growth of Pseudomonas aeruginosa at pH 6.8 for 14 h under inorganic phosphate limiting conditions is accompanied by the appearance of cell-associated alkaline phosphatase, and no active phosphatase is detected in the culture filtrate. Analysis of cells and the culture filtrate during the growth cycle showed that active phosphatase accumulated simultaneously in both the cells and the filtrate during the early log phase but as growth proceeded beyond 10 h the activity in the filtrate disappeared and the pH dropped from 6.8 to 4.7. Dialysis against 0.01 M Tris and 0.001 M MgCl2 had no effect upon the phosphatase levels of early growth phase (i.e. up to 10 h) culture filtrates. However, dialysis of filtrates obtained after 10 h of growth restored the decreased phosphatase activity to the level present in 10 h filtrates. Treatment of a 14 h filtrate with either trypsin or pepsin prevented the recovery of activity by dialysis whereas such treatments had no effect upon partially purified phosphatase. Sucrose density gradient centrifugation of the 14 h culture filtrate revealed the presence of activity, after dialysis of each fraction against 0.01 M Tris and 0.001 M MgCl2, which corresponded to a molecular weight of 60 000 as compared to a molecular weight of 125 000 for active culture filtrate or partially purified enzyme. Partially purified phosphatase is inactivated and dissociated after treatment at pH 4.2 and the enzyme is reactivated and reassociated after dialysis or dilution into Tris and MgCl2. The results suggest that active phosphatase, molecular weight 125 000, is secreted to the culture filtrate during the early stages of growth and the activity disappears coincidently with the appearance of an inactive species of molecular weight 60 000. The accumulation of the inactive species in the culture filtrate is the result of acid dissociation of the active species as the initial pH of the filtrate decreases from pH 6.8 to pH 4.7.