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Dormant cysts of Azotobacter vinelandii germinated at 30 degrees C in Burk nitrogen-free media containing 1% glucose. Samples taken at intervals and examined by electron microscopy revealed that as germination progressed, vesicle-like and fibrillar structures became visible in the intine region. Lamellae associated with the cell membrane appeared in the central body at 6 h post-initiation of germination. Both electron micrographic and chemical analysis showed that the poly-beta-hydroxybutyrate content of cysts decreased significantly after 4 h of germination. Dormant cysts were resistant to sonic oscillation, but this property was lost during their conversion to metabolically active vegetative cells.

Cells of Azotobacter vinelandii encysted in Burk's nitrogen-free liquid media which had been supplemented with n-butyl alcohol, beta-hydroxybutyrate, or crotonate. Butyraldehyde and butyrate did not influence the extent of encystment. In the absence of glucose, beta-hydroxybutyrate enhanced the rate and extent of encystment. In the presence of glucose, it promoted abortive encystment, which was manifested by the disorganization of the exine and the release of a highly viscous material into the medium. The soluble, viscous polymer was separated from the medium by a series of ethyl alcohol precipitations and identified as a mucopeptide. It was cleaved by treatment with lysozyme and lysostaphin with a concomitant increase in reducing power. It contained 13.9% N; 56% amino acids, as alanine (alanine, lysine, and glutamic acids); and 42% hexosamines. The polymer appeared to be similar to a noncross-linked peptidoglycan.

Micrographs of freeze-cleaved Azotobacter vinelandii cysts reveal that the surface is composed of several overlapping layers. This observation is consistent with the previously proposed structure of the outer cyst coat.

Cysts of Azotobacter vinelandii ATCC 12837 were germinated by exposure to 3.0 mm ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane buffer at pH 7.8, and their outer coats (exines) were purified by differential and isopycnic centrifugation. Electron micrographs of exine showed it to consist of multilayers of a three-membered sheet structure whose thickness was 7.0 to 7.5 nm. The inner, less electron-dense layer (intine) was also prepared from cysts by EDTA treatment, centrifugation, concentration, and dialysis. The exine consisted of 32% carbohydrate, 28% protein, 30% lipid, and 3.2% ash, with the ash comprised of 1.62% calcium, 0.02% magnesium, and 0.34% phosphorus. The amino acid composition of exine was similar to that of gram-negative bacterial cell walls. The intine consisted of 44% carbohydrate, 9.1% protein, 37% lipid, and 4.1% ash, with the ash comprised of 2.45% calcium, 0.02% magnesium, and 0.38% phosphorus. The carbohydrates of both exine and intine contained glucose, mannose, xylose, and rhamnose. Glucosamine and galactosamine were found only in the exines. The fatty acids consisted of normal, iso, and anteiso saturated fatty acids with 10 to 18 carbon atoms and mono-unsaturated C(11), C(16), and C(18) fatty acids. The exines contained mostly bound lipid, but intines contained primarily free lipid.

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.