Relationship between calcium and uroinic acids in the encystment of Azotobacter vinelandii

Encystment of Azotobacter vinelandii (ATCC 12837) in modified Burk nitrogen-free medium (pH 7.0) containing 0.2 percent beta-hydroxybutyrate occurs optimally in 0.37 to 0.44 mM solutions of calcium ions. Suspension of cells in media deficient in calcium results in abortive encystment characterized by the release of viscous cyst coat material. Mature cysts rupture in ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, suggesting that calcium is a structural component of the cyst coat. Maximal stimulation of encystment by calcium ions occurs prior to the completion of the cyst exine or outer coat. The uronic acid composition of cyst components is dependent on calcium levels in the medium. Uronic acids account for 31.7 percent of the intine (inner coat) and 13 percent of the exine dry weight, and only mannuronic and guluronic acids are present in these fractions. These can be extracted as homo- and heteropolymeric sequence "blocks" characteristic of alginic acids. The polyuronic acid fraction of both the cyst coats contain approximately equal amounts of heteropolymeric (mannuronic acid/guluronic acid) blocks. The exine, however, is richer in polyguluronic acid and the intine is richer in polymannuronic acid. As a result, the mannuronic acid/guluronic acid ratio of the exine is lower than that of the intine. Slimes that form in abortive encystment are rich in polymannuronic acid and have a high mannuronic acid/guluronic acid ratio. A polymannuronic acid 5-epimerase is active in the mature cyst central body and the encystment culture fluid.