Compaction of isolated Escherichia coli nucleoids: Polymer and H-NS protein synergetics.

Escherichia coli nucleoids were compacted by the inert polymer polyethylene glycol (PEG) in the presence of the H-NS protein. The protein by itself appears to have little impact on the size of the nucleoids as determined by fluorescent microscopy. However, it has a significant impact on the nucleoidal collapse by PEG. This is quantitatively explained by assuming the H-NS protein enhances the effective diameter of the DNA helix leading to an increase in the depletion forces induced by the PEG. Ultimately, however, the free energy of the nucleoid itself turns out to be independent of the H-NS concentration. This is because the enhancement of the supercoil excluded volume is negligible. The experiments on the nucleoids are corroborated by dynamic light scattering and EMSA analyses performed on DNA plasmids in the presence of PEG and H-NS.

Characterization of Escherichia coli nucleoids released by osmotic shock.

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.