STABILIZATION OF COMPACT SPERMIDINE NUCLEOIDS FROM ESCHERICHIA-COLI UNDER CROWDED CONDITIONS - IMPLICATIONS FOR IN-VIVO NUCLEOID STRUCTURE

Nucleoids from Escherichia coli were isolated in the presence of sperm idine at low salt concentrations. The nucleoids denature at relatively low temperatures or salt concentrations, yielding broad slowly sedime nting zones and/or macroscopic aggregates upon sucrose gradient centri fugation. Denaturation is accompanied by a loss of a characteristicall y compact shape as visualized by light and electron microscopy. Additi on 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 cr owding. Enzymatic digestion of the nucleoid DNA primarily releases thr ee small proteins (H-NS, FIS, and HU) and RNA polymerase, as well as r esidual lysozyme from the cell lysis procedure. If isolated nucleoids are extracted with elevated salt concentrations under crowded, stabili zed conditions, two of the proteins (HU and lysozyme) are efficiently removed and the compact form of the nucleoids is retained. These extra cted nucleoids maintain their compact form upon reisolation into the i nitial uncrowded low-salt medium, indicating that HU, the most common ''histone-like'' protein off. coil, is not a necessary component for m aintaining compaction in these preparations. (C) 1997 Academic Press.