Characterization of Escherichia coli nucleoids released by osmotic shock

S. Wegner, S.V. Alexeeva, T. Odijk, C.L. Woldringh

Research output: Contribution to journalArticleAcademicpeer-review

13 Citations (Scopus)

Abstract

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50–5 µg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5–10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.
Original languageEnglish
Pages (from-to)260-269
JournalJournal of Structural Biology
Volume178
Issue number3
DOIs
Publication statusPublished - 2012

Keywords

  • folded chromosome
  • salmonella-typhimurium
  • branched polymers
  • dna
  • sedimentation
  • fluorescence
  • chloroquine
  • compaction
  • dynamics
  • domains

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