Macromolecular crowding effects on the interaction of DNA with Escherichia coli DNA-binding proteins: a model for bacterial nucleoid stabilization.

DNA-binding protein fractions from exponential and stationary phase cell extracts of E. coli were isolated by affinity chromatography on native DNA-cellulose. The ability of these fractions to convert DNA into a readily-sedimented form was compared in the absence or presence of added polymers. In the absence of polymers, large amounts of the proteins were required. In the presence of polyethylene glycol or polyvinylpyrrolidone, much smaller amounts of the DNA-binding proteins were required, indicating a macromolecular crowding effect from these polymers. The enhanced binding under crowded conditions appears to resolve a paradox between the cellular abundance of the DNA-binding proteins and the amounts required in earlier in vitro studies. The 'histone-like' protein HU from the DNA-binding protein fraction was preferentially incorporated into the pelleted DNA in the presence of polymers. Purified HU at roughly similar amounts caused a similar conversion of DNA to a readily-sedimentable ('condensed') form. Crowding-enhancement of DNA condensation by promoting the binding of proteins to the DNA provides a model for the stabilization of systems such as the bacterial nucleoid or kinetoplast DNA.

Condensation and cohesion of lambda DNA in cell extracts and other media: implications for the structure and function of DNA in prokaryotes.

DNA added to concentrated extracts of Escherichia coli undergoes a reversible transition to a readily-sedimentable ('condensed') form. The transition occurs over a relatively small increment in extract concentration. The extract appears to play two roles in this transition, supplying both DNA-binding protein(s) and a crowded environment that increases protein binding and favors compact DNA conformations. The two roles of the extract are suggested by properties of fractions prepared by absorption of extracts with DNA-cellulose. The DNA-binding fraction and the DNA-nonbinding fractions from these columns are separately poorer condensing agents than the original extract, but when rejoined are similar to the original extract in the amount required for condensation. The dual role for the extract is supported by model studies of condensation with combinations of purified DNA-binding materials (protein HU or spermidine) and concentrated solutions of crowding agents (albumin or polyethylene glycol 8000); in each case, crowding agents and DNA-binding materials jointly reduce the amounts of each other required for condensation. The condensation reaction as studied in extracts or in the purified systems may be a useful approach to the forces which stabilize the compact form of DNA within the bacterial nucleoid. The effect of condensation on the reactivity of the DNA was measured by changes in the rate of cohesion between duplex DNA molecules bearing the complementary single-strand termini of lambda DNA. Condensation caused large increases in the rates of cohesion of both lambda DNA and of restriction fragments of lambda DNA bearing the cohesive termini. Cohesion products of lambda DNA made in vitro are a mixture of linear and circular aggregates, whereas those made in vivo are cyclic monomers. We suggest a simple mechanism based upon condensation at the site of viral injection which may explain this discrepancy.

Excluded volume effects on the partition of single- and double-stranded oligodeoxynucleotides between two liquid phases.

The distribution coefficients of single- and double-stranded oligodeoxynucleotides in a PEG 8000/phosphate two-phase system are a function of their chain length. Values of the distribution coefficients are in general agreement with a simple extension of a model for excluded volume effects (the "available volume model") which was applied previously to the distribution of proteins in this system. The current results therefore provide a second set of examples for molecules of very different geometry where the distribution added molecules is controlled by excluded volume interactions between those molecules and the PEG 8000 of the two-phase system.

Electrophoresis of polyethylene glycols and related materials as sodium dodecyl sulfate complexes.

Polyethylene glycol (PEG) and polyethylene glycol derivatives are analyzed by a modification of the sodium dodecyl sulfate-polyacrylamide stacking gel electrophoresis procedure of Kurfürst. Gels using a discontinuous buffer system but which do not have a separate stacking gel are used without loss of resolution and with less tendency to form artifactual multiple or distorted bands. Examination of several commercial preparations of PEGs and PEG derivatives on such gels indicates heterogeneity other than the expected unimodal size distributions. SDS-gel electrophoresis of proteins or other materials in samples containing PEGs may yield gels with zones of contamination from the PEGs. Methods of reducing such contamination are suggested.

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 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.