Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis.
Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.
D001407:Bacillus / Q000032:analysis* / Q000648:ultrastructure; D001426:Bacterial Proteins; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000302:isolation & purification; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D012329:RNA, Bacterial; D012260:Ribonucleases / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D012329:RNA, Bacterial; D012260:Ribonucleases