Isolation of the Escherichia coli nucleoid.
Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine). Depending on the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate. To investigate the mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of detergents and high concentrations of counterions. Here, we compare the general lysis method using detergents with a procedure involving osmotic shock of Escherichia coli spheroplasts that resulted in nucleoids free of envelope fragments. After staining the DNA with DAPI (4',6-diamidino-2-phenylindole) and cell lysis by either isolation procedure, free-floating nucleoids could be readily visualized in fluorescence microscope preparations. The detergent-salt and the osmotic-shock nucleoids appeared as relatively compact structures under the applied ionic conditions of 1 M and 10 mM, respectively. RNase treatment caused no dramatic changes in the size of either nucleoid.
Cunha
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Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
Odijk
T
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Süleymanoglu
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E
Woldringh
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CL
D001433:Bacteriolysis; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry* / Q000648:ultrastructure; D002876:Chromosomes, Bacterial / Q000737:chemistry*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000648:ultrastructure; D009997:Osmotic Pressure; D012329:RNA, Bacterial / Q000737:chemistry
D004269:DNA, Bacterial; D003902:Detergents; D012329:RNA, Bacterial