Isolation and characterization of the Legionella pneumophila outer membrane

Abstract

A whole cell lysate of Legionella pneumophila was fractionated into five membrane fractions by sucrose gradient centrifugation. Membranes were characterized by enzymatic, chemical, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Two forms of cytoplasmic membrane (CM-1, CM-2), a band of intermediate density (IM), and two forms of outer membrane (OM-1, OM-2) were detected. The CM-1 fraction was the purest form of cytoplasmic membrane, and fraction CM-2 was primarily cytoplasmic membrane associated with small amounts of peptidoglycan. The IM, CM-1, and CM-2 fractions were enriched in peptidoglycan, and the amount of carbohydrate and 2-keto-3-deoxyoctonic acid was not appreciably greater in outer membrane relative to cytoplasmic membrane. Phosphatidylethanolamine and phosphatidylcholine were found to be the major phospholipids in the membrane fractions. The major outer membrane proteins had molecular sizes of 29,000 and 33,000 daltons and were both modified by heating. The 29,000-dalton protein was tightly associated with the peptidoglycan and was equally distributed in the IM, OM-1, and OM-2.