ISOLATION AND PARTIAL CHARACTERIZATION OF BORRELIA-BURGDORFERI INNER AND OUTER MEMBRANES BY USING ISOPYCNIC CENTRIFUGATION

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes b y using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2 %, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F-1/F-0 ATPa se (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane o r flagellar components. The outer membrane fractions (rho = 1.19 g/cm( 3)) contained 0.15 mg (dry weight) of protein per mg. Inner membrane s amples (rho = 1.12 g/cm(3)) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer me mbrane vesicles contained about 1,700 intramembranous particles per mu m(2) while inner membrane vesicles contained about 6,600 intramembran ous particles per mu m(2), similarly to previously observed particle d ensities for inner and outer membranes. Sodium dodecyl sulfate-polyacr ylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel elect rophoresis-SDS-PAGE analyses of inner and outer membrane samples revea led several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly s hows that the inner and outer membranes isolated by this technique are unique structures.