Factors affecting the release of outer membrane from a gram-negative marine bacterium

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Abstract
Suspending cells in 0.5 M sucrose was shown to be an essential step in the wash procedure used to remove the outer membrane from Alteromonas haloplanktis 214. Reducing the length of time the cells were suspended in 0.5 M sucrose and lowering the temperature of the sucrose solution from 25 to 4 °C reduced the extent of release of or damage to the outer membrane as determined by observation of thin sections of the cells by electron microscopy, the extent of conversion of rod-shaped cells to spheres by lysozyme, and the percentage release of alkaline phosphatase. The protease inhibitor phenylmethylsulfonyl fluoride, at a concentration which inhibited over 90% of the protease activity in a sonicated cell preparation and 79% of the activity in cells pretreated with the inhibitor, failed to affect the extent of release of outer membrane from the cells as determined by electron microscopy. The findings are consistent with previous conclusions that interaction of salts with polyanionic macromolecules are required to maintain the integrity and permeability properties of the outer membrane of this organism.