Secretion of alkaline phosphatase and phsopholipase C in Pseudomonas aeruginosa is specific and does not involve an increase in outer membrane permeability

1. INTRODUCTION Under conditions of inorganic phosphate limi- tation, Pseudomonas aeruginosa synthesizes an in- ducible alkaline phosphatase [1] and phospholi- pase C [2,3]. Both enzymes are released into the medium during growth [3,4], with phospholipase C existing mostly in the medium [3], whereas the distribution of alkaline phosphatase between the cell and the medium is variable depending upon culture conditions [5]. Although the mechanism of secretion of these enzymes is as yet unknown, two possibilities exist to account for their extracellular presence. One possibility is that their release is facilitated by a breakdown in the outer membrane permeability barrier, freeing them from a periplasmic location. Alkaline phosphatase is a periplasmic marker in many Gram-negative bacteria and in Pseudomonas aeruginosa some alkaline phosphatase activity is always detectable in the periplasm [as Tris(hy- droxymethyl) aminomethane (Tris)MgC12-released enzyme] [5]. A breakdown in the outer membrane would thus be one mechanism to account for the release of these enzymes. The second possibility is that a mechanism of specific secretion across the outer membrane is responsible for their release, in the absence of any gross permeability changes. In the present study we examined (a) outer membrane permeability as a function of alkaline phosphatase and phosphalipase C secretion (to define any changes in outer membrane permeabil- ity which might be associated with their secretion), and (b) the specificity of the release of alkaline phosphatase and phospholipase C, in an attempt to better define the nature of that release. We now report that secretion of the enzymes is indeed specific and does not involve increased outer mem- brane permeability. 2. MATERIALS AND METHODS Pseudomonas aeruginosa PAO1 strain H103 [6] containing an RPI plasmid was grown under the phosphate-deficient conditions described previ- ously [7] using a medium buffered with sodium N-2-hydroxymethyl piperazine-N'-2-ethane sulfonate (HEPES). Cells were prepared for growth in phosphate-deficient medium by first harvesting by centrifugation overnight cultures grown in phosphate-sufficient medium [7] containing 100 /.tg/ml tetracycline (to maintain the plasmid), washing cells twice in phosphate-sufficient medium'to remove the tetracycline, and resuspend- ing them in phosphate-sufficient medium at an A60 o of 0.05. The cells were then grown at 37°C to mid log phase (A6oo=0.60) and subsequently harvested by centrifugation, washed three times in phosphate-deficient medium and resuspended in 0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies