Preparation and ultrastructure of the outer coats of Azotobacter vinelandii cysts

Abstract

Cysts of Azotobacter vinelandii were ruptured with 3.0 mm ethylenediaminetetraacetate in 0.05 m tris(hydroxymethyl)aminomethane buffer (pH 7.8) and fractionated by differential centrifugation in density gradients of sucrose or Ficoll (polysucrose) at 10,000 x g for 2 hr at 4 C. The average densities of the cyst exine, the cyst central body, the vegetative cell, and the intact cyst were found to be 1.13, 1.26, 1.28, and 1.31 g/cm(3) in sucrose, and 1.08, 1.12, 1.13, and 1.17 g/cm(3) in Ficoll, respectively. These data were utilized to devise procedures for the isolation of cyst components. Cyst exine appeared as a multilayered structure in ultrathin sections. This same sheetlike structure was seen when exines were subjected to detergent treatment, metal-shadowed, and examined in an electron microscope. The exine was lysed by trypsin and was resistant to lysozyme, indicating that protein may be the principal structural material of the outer cyst coat.