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.
Pseudomonas aeruginosa
ATCC 9027 contains an inducible alkaline phosphatase. The enzyme is readily removed from 14-hr cells by washes in 0.2
m
MgCl
2
,
p
H 8.4. Similar washes in tris(hydroxymethyl)aminomethane buffer, 20% sucrose, monovalent ions, or water partially release enzyme from the cells. The release of alkaline phosphatase is correlated with an increased release of protein and retention of internal enzymes. The effect of 0.2
m
MgCl
2
washing upon the cells is minimal since both viability and growth rates remain unchanged as compared to water washing. Although cells are plasmolyzed in both 0.2
m
MgCl
2
and 20% sucrose, it is evident that plasmolysis alone is unable to account for total enzyme release and that a divalent metal, i.e. Mg
2+
, augments the release pattern. Growing cells in the presence of increasing concentrations of MgCl
2
or at increased
p
H values results in an almost total secretion of the enzyme to the culture filtrate. The findings suggest that
P. aeruginosa
alkaline phosphatase is linked to the exocytoplasmic region through divalent metal ion, presumably Mg
2+
, bridges.
Spheroplasts prepared by lysozyme treatment of cells of
Pseudomonas aeruginosa
, suspended in 20% sucrose or 0.2
m
MgCl
2
, were examined in detail. Preparation of spheroplasts in the presence of 0.2
m
Mg
2+
released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of
P. aeruginosa
alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.
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