Abstract
A lysozyme-detergent method was developed for the fractionation of sporulating cells of B. subtilis 168 wild type into mother cell and forespore fractions. The method is very mild and is reproducible with optimum concentrations of Brij-58, deoxycholic acid and sucrose. The results were confirmed by application of the method to temperature sensitive mutants, Ts-1 and Ts-3.
The amounts of proteins, and the activities of protease, alkaline phosphatase and glucose dehydrogenase were about 55, 56, 91, and 40%, respectively, in the mother cell fraction, and about 45, 44, 9, and 60%, respectively, in the forespore fraction, taking the totals for the combined fractions as 100%.
Slab gel electrophoretic patterns indicated that many species of proteins with different molecular weights were present in the two fractions.
Pulse-labeling with [3H]UTP was carried out in vivo at stage III, and 35.2 and 64.8% of the [3H]UMP incorporated into RNAs were distributed in the mother cell and forespore fractions, respectively. The results indicate that more RNA synthesis occurs in the forespores than in the mother cells of sporulating cells.
