title: Characterization of Escherichia coli nucleoids released by osmotic shock.
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 (<2 s) was on average ∼42 μm(3). Lysozyme at concentrations above 1 μg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 μg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 μM ethidium bromide, but not with chloroquine. While DNase (1 μg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 μg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>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.
authors.affiliation: Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Wegner
authors.forename: Svetlana
authors.initials: S
authors.lastname: Alexeeva
authors.forename: Theo
authors.initials: T
authors.lastname: Odijk
authors.forename: Conrad L
authors.initials: CL
authors.lastname: Woldringh
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry; D009997:Osmotic Pressure; D012697:Serine Endopeptidases / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013104:Spheroplasts / Q000737:chemistry / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D012967:Sodium Dodecyl Sulfate; D003851:Deoxyribonucleases; D012697:Serine Endopeptidases; C028734:protease E; D019286:Endopeptidase K; D004996:Ethidium