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.
Bacillus subtilis sporulating cells at stage III were fractionated into mother cell and forespore fractions by means of a lysozyme-detergent method. Three forms of DNA-dependent RNA polymerase enzymes, termed M sigma, F sigma, and F delta, in addition to core enzyme (alpha 2, beta', and beta) have been purified from the cell fractions. Enzymes M sigma and F sigma are present in the mother cell and forespore, respectively, and contain sigma factor of 55,000 daltons in addition to the core subunits. On the other hand, enzyme F delta is present specifically in the forespore and contains delta 1 factor of 28,000 daltons instead of the sigma factor. The amount of RNA polymerase in the forespore is about twice that in the mother cell. The enzymes M sigma and F sigma also differed in their elution profiled from DEAE-cellulose columns and in their heat stabilities indicating that the two sigma-containing holoenzyme forms may be different in their structural properties. The enzyme F delta transcribed B. subtilis DNA about 1.6 times more actively than enzyme F sigma, and the enzymes M sigma and F sigma transcribed the DNA about 2.2 times more actively than did core enzyme.
Folded chromosomes were prepared as membrane-associated complexes from vegetative cells of Bacillus subtilis by stepwise sucrose gradient centrifugation. From nucleoids, a deoxyribonucleic acid-bound polypeptide with a molecular weight of 6,000 (P6) was purified by KCl-(NH4)2SO4 salting out, diethylaminoethyl cellulose column chromatography, and deoxyribonucleic acid cellulose column chromatography. The amino acid composition of polypeptide P6 was determined.
Actinomycin D allowed 3.6% of spore formation and 4.9% of dipicolinic acid synthesis of control cultures when added at 0.4 microM at late stage (T6.5) during sporulation process of Bacillus subtilis although the drug inhibited sporulation almost completely when added at log phase and early stages. Daunorubin added at log phase, on the other hand, did not inhibit either growth and sporulation of the bacteria up to a concentration of 2.9 microM. The joint use of both antibiotics at late stages decreased the levels of spore formation and the synthesis of dipicolinic acid allowed by the use of actinomycin D alone to 0.9% and 2.1% respectively, and also inhibited the synthesis of spore coat protein in a cooperative manner.
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