Studies on the cell wall of Spirillum serpens. 1. Isolation and partial purification of the outermost cell wall layer

Abstract

The presence of the hexagonal array of macromolecules on the outer surface of the cell wall of Spirillum serpens VHA required the addition of calcium to an otherwise effective growth medium (vitamin-free casein hydrolysate); slightly improved growth resulted from addition of a complex salts mixture. A means of isolating this layer for chemical and physical study was sought and controlled by electron microscopy of freeze-etched, negatively stained, and sectioned preparations. The structure was destroyed, extracted, or removed by extremes of pH (<4.5, >9), 1 M guanidine hydrochloride (pH 7), 2 M urea, and dimethyl sulfoxide, with varying damage to the cell. Heat (60° for 1 h) removed much of the outer layer from the intact cell as an array of units disposed on a delicate backing layer, leaving the basic Gram-negative triplet wall components. These fragments remained stable through washing and repeated heating in the presence of 0.001 M calcium chloride. Guanidine hydrochloride (1.5 M) dissolved the units from the tubes and vesicles formed by the backing layer. Dialysis against water removed salts and guanidine, caused the precipitation of residual contaminants, and provided a supernatant which, when lyophilized, provided a product containing 98% protein.