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 by 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 ATPase (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 or flagellar components. The outer membrane fractions (rho = 1.19 g/cm(3)) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm(3)) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per mu m(2) while inner membrane vesicles contained about 6,600 intramembranous particles per mu m(2), similarly to previously observed particle densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.