Biosynthesis of Nucleic Acids in Bacillus megaterium

So-called 'chromatin b)odies' have been observed in whole cells, protoplasts and spores of Bacillus megaterium by cytological and electron-microscopical techniques (Fitzjames, 1953, 1958; Robinow, 1956). In ultra-thin sections of whole cells the chromatin body appears as an area less electron-dense than the surrounding cytoplasm and made up of a network of fine fibrils with no bounding membrane (Piekarski & Giesbrecht, 1956; Giesbrecht & Piekarski, 1958; Giesbrecht, 1958). In protoplasts the morphology of the chromatin body appears to depend on the nature of the stabilizing medium. In media of low ionic strength the bodies are dispersed in an open arrangement of filaments, but in media of high ionic strength the bodies are aggregated into a tight structure with a chromatin material arranged round a non-chromatinic core (Fitzjames, 1958). The investigations of Spiegelman, Aronson & Fitzjames (1958) and of de Lamater (1959) have strongly suggested that a discrete nuclear apparatus ('nucleoids' or 'nuclear material') can be isolated from protoplasts of B. megaterium and that this body can be equated with the chromatin area identifiable in electron micrographs of whole cells. The methods described for the preparation of these nuclear bodies, however, are prolonged and unsuitable for metabolic studies. Also, the procedures employed destroy some of the cellular components in order to liberate the nuclear bodies from the cytoplasm. The methods of Spiegelman et al. (1958) entail dissolving away the cytoplasmic membrane with lipase, and the methods of de Lamater (1959) entail standing the protoplasts in sodium glycocholate for up to 24 hr. and then disrupting the cells by ultrasonic treatment. Several other attempts to isolate a nuclear material have been reported (Ezekiel, 1960), but in most of these cases only a portion of the material has been recovered. The purpose of the present paper is to describe a rapid and convenient method of isolating nuclear material from B. megaterium and fractionating the bacterial cell into three components, namely (a) a nuclear fraction containing almost all of the cellular DNA, together with some RNA and protein, (b) a cytoplasmic fraction containing all of the cellular ribosomes and most of the cellular RNA, and (c) a membrane fractionmade up ofcytoplasmic membranes stripped clean of attached RNA or other material. A preliminary account of some of this work has been given (Godson & Butler, 1962a, b).