INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

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The forces which condense the DNA of E. coli into one or a few compact nucleoids per cell are poorly understood, due in large part to difficulties in isolating nucleoids that retain in vivo structural characteristics. A procedure using spermidine to increase the stability of isolated nucleoids under low-salt conditions was briefly described by Kornberg et al. We have further characterized these nucleoids for use in studies of their stabilization and structure. The isolated nucleoids are compact particles by light microscopy, often occurring as doublets. Electron microscopy shows a central core partially coated with membranous vesicles. The isolated nucleoids contain large amounts of protein and RNA. The DNA and RNA of the nucleoids is rapidly degraded by added nucleases, indicating an open, accessible structure that should be amenable for current structural studies. Within the cell, macromolecular crowding effects of the concentrated cytoplasm which surrounds the nucleoids has an enormous potential for nucleoid stabilization. We used the isolated nucleoids in an experimental test of this proposal. Polymers (PEG, dextran) extended the stability of the nucleoids to temperatures and salt concentrations more consistent with those tolerated in vivo, supporting the suggested role of crowding in nucleoid stabilization. These and related studies led us to suggest a hypothesis of mandatory condensation of DNA in bacteria (in press) that proposes that all DNA within a bacterium will adopt a condensed form due to the great excess of condensing forces present. Mandatory condensation has many implications in bacterial and viral replication, with potential application to eukaryotic cells.