Spirochete: Silver Staining for Nucleic Acids

Borrelia burgdorferi the Lyme disease spirochete was grown in modified Kelly medium and characterized by transmission and by scanning electron microscopy. Using silver staining procedures which preferentially bind to nuclear components of eukaryotic cells, signal could be detected by backscattered electron imaging throughout the length of the prokaryotic spirochete. Interestingly, however, the highest levels of backscattered signal were observed in naturally elaborated membrane blebs that were visible attached to cell surfaces and free in the medium. These membrane vesicles could be enriched by filtration through nitrocellulose or Anopore membranes and by differential centrifugation. The possibility of contaminating cellular DNA coating the membrane vesicles was ruled out by exhaustive digestion with pancreatic DNAase I. Intact DNA was demonstrated both by lysing blebs directly on the surface of microscope grids and by extracting molecules from purified bleb preparation with detergents and solvents. Both linear and circular DNA molecules could be identified in purified membrane blebs. A simple, one-step, alternative silver stammg procedure is described which appears to effectively label the protein-nucleic acid complexes contained in the membrane vesicles of the human pathogen fi_. burgdorferi, and may provide an important method to track and to define the biological function of these structures.