title: Stabilization of compact spermidine nucleoids from Escherichia coli under crowded conditions: implications for in vivo nucleoid structure.
abstract: Nucleoids from Escherichia coli were isolated in the presence of spermidine at low salt concentrations. The nucleoids denature at relatively low temperatures or salt concentrations, yielding broad slowly sedimenting zones and/or macroscopic aggregates upon sucrose gradient centrifugation. Denaturation is accompanied by a loss of a characteristically compact shape as visualized by light and electron microscopy. Addition of polyethylene glycol or dextran prevents these changes, extending the range of stability of the isolated nucleoids to temperatures and ionic conditions like those which commonly occur in vivo. The effects of the polymers are consistent with stabilization by macromolecular crowding. Enzymatic digestion of the nucleoid DNA primarily releases three small proteins (H-NS, FIS, and HU) and RNA polymerase, as well as residual lysozyme from the cell lysis procedure. If isolated nucleoids are extracted with elevated salt concentrations under crowded, stabilized conditions, two of the proteins (HU and lysozyme) are efficiently removed and the compact form of the nucleoids is retained. These extracted nucleoids maintain their compact form upon reisolation into the initial uncrowded low-salt medium, indicating that HU, the most common "histone-like" protein of E. coli, is not a necessary component for maintaining compaction in these preparations.
authors.affiliation: Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0560, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Murphy
authors.forename: S B
authors.initials: SB
authors.lastname: Zimmerman
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification; D002352:Carrier Proteins / Q000302:isolation & purification; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004269:DNA, Bacterial / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D003911:Dextrans; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D009113:Muramidase / Q000302:isolation & purification; D015388:Organelles / Q000187:drug effects / Q000648:ultrastructure*; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D012329:RNA, Bacterial / Q000302:isolation & purification; D013095:Spermidine / Q000494:pharmacology*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003911:Dextrans; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; C031908:H-NS protein, bacteria; D034982:Integration Host Factors; D012329:RNA, Bacterial; C040330:histone-like protein HU, bacteria; C440502:integration host factor, E coli; D011092:Polyethylene Glycols; D009113:Muramidase; D013095:Spermidine