Isolation of the Escherichia coli nucleoid

Numerous protocols for the isolation of bacterial nucleoids have been descr ibed based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidin e). 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 wi shed to isolate intact nucleoids in the absence of detergents and high conc entrations of counterions. Here, we compare the general lysis method using detergents with a procedure involving osmotic shock of Escherichia coil sph eroplasts that resulted in nucleoids free of envelope fragments. After stai ning the DNA with DAPI (4',6-diamidino-2-phenylindole) and cell lysis by ei ther isolation procedure: free-floating nucleoids could be readily visualiz ed in fluorescence microscope preparations. The detergent-salt and the osmo tic-shock nucleoids appeared as relatively compact structures under the app lied ionic conditions of 1 M and 10 mM, respectively. RNase treatment cause d no dramatic changes in the size of either nucleoid. (C) 2001 Societe fran caise de biochimie et biologie moleculaire / Editions scientifiques et medi cales Elsevier SAS