Effect of pancreatic DNAse on Bacillus subtilis multiplication depending on the physiological state of the culture

The effect of pancreatic DNAase on the growth of Bacillus subtilis, the synthesis of DNA, RNA and protein in the cells was studied as a function of their physiological state. The cells were found to be sensitive to the action of DNAase at the end of the linear growth phase, which coincided with their transition into the competence state and with their ability to adsorb the enzyme on the cell surface.     

DNA synthesis in vivo in Bacillus subtilis

DNA polymerase III is the enzyme responsible for deoxynucleotide addition to nascent DNA fragments in Bacillus subtilis protoplasts. Nascent single-stranded fragments separated from bulk DNA by hydroxyapatite chromatography cannot self-anneal. Partial inhibition of DNA polymerase III by 6-(hydroxyphenylazo)-uracil, a specific inhibitor, slows the rate of nascent fragment synthesis but has no effect on final size. Neither DNA polymerase I nor II can elongate nascent fragments in protoplasts when DNA polymerase III is completely inhibited.     

Effect of pancreatic DNAse on the nuclear DNA topology of Candida tropicalis yeasts

It has been shown that pancreatic DNAase added to the nutritious medium caused the change in the nuclear DNA topology of asporogenic Candida tropicalis yeast. DNA conformative changes are due to the unwinding of supertwisted molecules as a result of single-strand ruptures formation which induce DNA synthesis acceleration, cell growth and division.     

ATP-independent, DNA synthesis by DNA polymerase II in toluenized Bacillus subtilis

Phleomycin stimulates ATP-independent DNA repair synthesis by polymerase II in toluenized $\text{B. subtilis}$ cells. In the presence of ATP it also increases the synthesis, with BrdUTP, of DNA with a density between that of normal DNA and hybrid DNA, and it enhances replicative DNA synthesis by polymerase III.     

DNA synthesis in toluene-treated bacteriophage-infected minicells or Bacillus subtilis.

Bateriophage (phi29, SPP1, or SPO1)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [3H]dTTP into a trichloroacetic acid-precipitable form. The [3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2 . 10(5)). Short exposure of the DNA molecules containing the incorporated [3H]dTMP to Escherichia coli exonuclease III results in over 90% of the E13H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil.     

Independence of Bacillus subtilis spore outgrowth from DNA synthesis.

The outgrowth of spores of Bacillus subtilis 168 proceeded normally in temperature-sensitive DNA mutants under restrictive conditions and in the absence of DNA synthesis. Two inhibitors of DNA synthesis, nalidoxic acid and 6-(p-hydroxyphenylazo)-uracil, inhibited spore outgrowth under some nutritional conditions; this inhibition of outgrowth however, though not that of DNA synthesis, could be reversed by glucose. The sensitivity of the outgrowing spores to nalidixic acid and 6-(p-hydroxyphenylazo)-uracil inhbition decreased as a function of outgrowth time. The cells became completely resistant to the inhibitors after 90 min. The development of this resistance occurred also in the absence of DNA synthesis. It was concluded that DNA synthesis is not needed for spore outgrowth, and that outgrowing cells and vegetative cells differ in their sensitivity to these inhibitors.     

Effect of decoyinine on peptidoglycan synthesis and turnover in Bacillus subtilis.

The sporulation of Bacillus subtilis can be induced in the presence of amino acids and glucose by partially depriving the cells of guanine nucleotides. This can be achieved, e.g., by the addition of decoyinine, a specific inhibitor of GMP synthetase. To determine the effect of this and other inhibitors on cell wall synthesis, we measured in their presence the incorporation of acetylglucosamine into acid-precipitable material. The rate of wall synthesis decreased by 50% within 5 min after decoyinine addition; this decrease was prevented by the presence of guanosine. A comparison with the effects of other inhibitors of cell wall synthesis indicated that decoyinine inhibited the final portion of the cell wall biosynthetic pathway, i.e., after the steps inhibited by bacitracin or vancomycin. Decoyinine addition also prevented cellular autolysis and cell wall turnover. It is not known whether these two effects of decoyinine on cell wall synthesis are causally related.     

Carbamyl phosphate synthesis in Bacillus subtilis

In vitro and "in situ" assays have been developed to test the carbamyl phosphate synthetase (CPSase) activity of a series of pyrimidine-requiring mutants of Bacillus subtilis. The enzyme has been shown to be highly unstable, and was successfully extracted only in the presence of 10% glycerol and 1 mM dithiothreitol (Cleland's reagent). It loses activity rapidly when sonicated or when treated with lysozyme. Genetic studies, using mutants, indicate that B. subtilis may possess two CPSases. This possibility and its physiological consequences were probed enzymatically. CPSase activity has been shown to undergo inhibition by both uridine triphosphate and dihydroorotate; activation has been demonstrated in response to phosphoribosyl pyrophosphate (PRPP) and (to a lesser extent) ornithine.     

Plasmid DNA transduction in Bacillus subtilis

Transduction of Bacillus subtilis pUB110 plasmid by AR9 phage is described. Some aspects of this process are studied. Plasmid transduction depended on multiplicity of infection similar to cases of chromosomal markers transduction, though optimal multiplicity of infection was achieved using low number of phage particles. No cotransduction of plasmid and chromosomal markers was demonstrated. The transduction frequencies of plasmid and chromosomal markers increased after UV irradiation of phage suspensions within the range of definite doses.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

Effect of UV radiation on the DNA-membrane complex of Bacillus subtilis]

The influence of UV-light on DNA-membrane complex (DMC) of Bacillus subtilis was studied. An increased DNA content in DMC for strains 168 and rec A-, and a degradation of DMC for strain polA- have been registrated. The increase in DNA in DMC of the two former strains is inhibited by caffeine to be correlated with changes in protein content in DMC, determined by a radioactive label, but not with lipid content. Thus, the association of DNA with the membrane is mediated by proteins. DNA increasing capacity seen in DMC after UV-irradiation and after the following incubation of bacteria in the complete medium is correlated with a relative sensitivity of strains. To explain these data, it is supposed that the reparative synthesis is accomplished in cell on their membranes and that for the normal completion of DNA repair the association between DNA and the membrane is necessary.