Alkaline phosphatase localization and spheroplast formation of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa cells grown in the presence of low levels of inorganic phosphate contain an inducible alkaline phosphatase system. The enzyme has been localized by electron microscopic techniques in the region between the cytoplasmic membrane and the tripartite layer of the cell wall, i.e. the periplasmic space. No deposits of lead salts are observed upon examination of either uninduced cells or cells in which the enzyme has been completely removed by 0.2 M magnesium washing. Samples of cells which were treated with glutaraldehyde before enzyme localization studies show cell wall deposition of lead salts which could give rise to the erroneous conclusion that the alkaline phosphatase was located in the tripartite layer. Cytochemical and biochemical studies are presented which show that discontinuities within the cell wall are insufficient to account for the release of this periplasmic enzyme and that dissociation by a divalent metal, increased pH, or both is required. As a consequence of this study it was possible to prepare true spheroplasts of P. aeruginosa.