ULTRASTRUCTURAL CHARACTERIZATION OF THE STAGES OF SPHEROPLAST PREPARATION OF BORRELIA-BURGDORFERI

Spheroplasts were generated from Borrelia burgdorferi by application o f Tris-buffered lysozyme and EDTA. Cultures were examined during prepa ration by scanning electron microscopy (SEM) and by negative staining and/or thin sectioning followed by transmission electron microscopy (T EM). Spirochete and spheroplast proteins were also compared by electro phoresis to determine the effect of outer membrane manipulation during spheroplast preparation on the amounts of two outer surface proteins, OspA and OspB. Periplasmic flagellar release from the confines of the outer membrane began at the earliest steps of the preparation process and increased with continued progress toward the spheroplast conditio n. With the addition of Tris alone, spirochete uncoiling and outer mem brane disruption became apparent. Lysozyme addition resulted in the fo rmation of electron-transparent blebs where the outer membrane separat ed from the cytoplasmic membrane. Despite the permeability of the oute r membrane to the lysozyme and the apparent alterations in its structu re, OspA and B showed no decrease in concentration throughout the sphe roplast preparation process. It is suggested that the flagellar releas e that occurs during spheroplast production also occurs in the host du ring Lyme disease, accounting for early immune responses to flagellin.