The envelope of Micrococcus radiodurans: isolation, purification, and preliminary analysis of the wall layers

Abstract

Two methods are presented that separate the complex envelope of Micrococcus radiodurans, strain Sark, into its constituent layers. The first involved treating whole cells with 0.025 M Tris buffer (pH 7.5) containing 2 mM of calcium and 3 mM of magnesium, resulting in the degradation of an intermediate ('compartmentalized') layer and consequent sloughing of the outer subunit and interior layers to form vesicles. This treatment also appears to show that the interior layer may be connected with the peptidoglycan-containing 'holey' layer. The second method involves treating whole cells with benzene followed by sonication; the results suggested that this treatment only released the outer layers from the 'compartmentalized' layer and did not degrade layers. Following benzene treatment, digestion of the 'compartmentalized' layer with cold sodium dodecyl sulfate (SDS) released the 'holey' layer.Electrophoretic analysis of some of the isolated layer preparations suggested that the subunit layer consisted of three major proteins of 90 000, 92 000, and 94 000 molecular weight, one minor protein of 100 000, a small amount of carbohydrate associated with the 94 000 protein, and a small amount of a 55 000 lipoprotein. The interior layer contained at least 10 proteins and may be attached to the peptidoglycan-containing 'holey' layer by means of the 55 000 lipoprotein.