The effect of polymyxin B on outer membrane of Serratia marcescens: activation and dissociation of outer membrane associated alkaline phosphatase.

Various reports have indicated that the cationic cyclic-peptide antibiotic, polymyxin B (PB), interacts with a number of outer membrane components of gram-negative bacteria1-5). PB degrades lipopolysaccharide (LPS)1,2) and phospholipids3) and forms complexes with these molecules4,5). Such effects appear to be due to a multiplicity of actions of this antibiotic. More recently, the PB degradation of phospholipids in Pseudomonas aeraginosa and Escherichia coli was suggested to be caused by an in vivo activation of the phospholipase.6) Since phospholipases are outer membrane associated enzymes in gram-negative bacteria7), it follows that one means of studying the effect of PB on the outer membrane is to examine its effect on the activity of enzymes associated with the outer membrane. In this communication, we wish to report the PB induced activation and dissociation of the alkaline phosphatase associated with the outer membrane of Serratia marcescens. Two strains of S. marcescens were used: strain 08 (PB resistant) and strain Bizio (PB sensitive). Cells were grown in an enriched medium with aeration at room temperature and harvested at an optical density between 0.50 and 0.552,4). Outer membranes were isolated by sucrose density gradient centrifugation of sonified lysozymeEDTA treated spheroplasts as previously described". Two types of PB treatment of outer membranes were performed: an in vitro treatment in which isolated outer membranes (1 mg) were treated with 0.05 mg of PB in 1.5 m sodium chloride, and an in vivo treatment in which outer membranes were isolated from cells that had been previously treated with PB2) (20 mg of PB in 1.5 rat sodium chloride for the amount of cells harvested from 1 liter of growth medium for 1 minute at 37°C). Individual active alkaline phosphatase components in the untreated outer membranes, and in those after either in vitro or in vivo PB treatment, were separated by SDSpolyacrylamide gel electrophoresis and their activities quantitatively assayed by incubating the electrophoresed gels in 0.001 m p-nitrophenyl phosphate in 1 t t Tris buffer, pH 89). Gels were scanned at 410 nm after 6 hours, and the areas under the scans measured. Fig. 1 shows the scans of the separated alkaline phosphatase activity associated with the outer membranes before and after PB treatment. The untreated outer membranes from both strain 08 and strain Bizio contained an active component of 190,000 daltons (A') (Fig. 1-a and 1-b). The addition of PB to the isolated outer membranes from the untreated cells resulted in an enhance-