Fluorescence Microscopy of Condensed DNA Conformations of Bacterial Cells

다국어 초록 (Multilingual Abstract)

Cellular DNA in prokaryotes is organized in nucleic acid-protein self-assemblies referred to as the
nucleoid. The physical forces responsible for its stability inside the poor solvent properties of the cytoplasm
and their functional implications are not understood. Studies on the organisation and functioning
of the cytosol of cells largely rely on experimental protocols performed in highly dilute solutions
using biochemically purified molecules, which is not a reliable substitute for the situation existing in
vivo. Our current research interest is focused on the characterization of biological and physical forces
determining the compaction and phase separation of DNA in Escherichia coli cytoplasm. We have
emphasized the effect of excluded volume in solutions with high macromolecular concentrations (macromolecular
crowding) upon self-association patterns of reactions. The prokaryotic cytosol was simulated
by addition of inert polymer polyethylene glycol (PEG) (average molecular weight 20000), as an
agent which afterwards facilitates the self-association of macromolecules. Fluorescence microscopy was
used for direct visualization of nucleoids in intact cells, after staining with DAPI (4',6-diamidino-2-phenylindole
dihydrochloride). Addition of the crowding agent PEG 20,000, in increasing concentrations
generated progressively enhanced nucleoid compaction, the effect being stronger in the presence of 0.2
M NaCl and 5 mM MgCl2. Under these conditions, the nucleoids were compacted to volumes of around
2 μm3 or comparable sizes with that of living cells.