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S B Zimmerman

Publications and source records attributed to S B Zimmerman.

At least 19 recordsLinked to original sources
PubMed2001

A limited loss of DNA compaction accompanying the release of cytoplasm from cells of Escherichia coli.

The DNA of bacteria is compacted into nucleoids. We have lysed cells of Escherichia coli under conditions in which the cell envelope is retained. The extent of DNA compaction was determined by light microscopy, comparing DAPI fluorescence and phase contrast images. The release of cytoplasm upon lysis allowed the nucleoidal DNA to expand to fill the residual cell boundaries, supporting the role of cytoplasmic crowding in nucleoid compaction. The addition of polylysine allowed lysis with retention of DNA compaction. Furthermore, chloramphenicol treatment of cells resulted in nucleoids which were more resistant to decompaction upon lysis.

Artifacts↗
PubMed2001

Release of compact nucleoids with characteristic shapes from Escherichia coli.

The genomic DNA of bacteria is contained in one or a few compact bodies known as nucleoids. We describe a simple procedure that retains the general shape and compaction of nucleoids from Escherichia coli upon cell lysis and nucleoid release from the cell envelope. The procedure is a modification of that used for the preparation of spermidine nucleoids (nucleoids released in the presence of spermidine) (T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. USA 71:3189--3193, 1974). Polylysine is added to prevent the normal decompaction of nucleoids which occurs upon cell lysis. Nucleoids retained their characteristic shapes in lysates of exponential-phase cells or in lysates of cells treated with chloramphenicol or nalidixate to alter nucleoid morphology. The notably unstable nucleoids of rifampin-treated cells were obtained in compact, stable form in such lysates. Nucleoids released in the presence of polylysine were easily processed and provided well-defined DNA fluorescence and phase-contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde fixation and heating with sodium dodecyl sulfate.

DNA, Bacterial↗
PubMed2000

Multiple restraints to the unfolding of spermidine nucleoids from Escherichia coli.

Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at approximately 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged.

Animals↗
PubMed1999

Identification of two new proteins in spermidine nucleoids isolated from Escherichia coli.

The Escherichia coli nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E. coli. Partial amino-terminal sequencing has identified them as the products of rdgC and yejK. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of spermidine nucleoids from Escherichia coli.

Nucleoids isolated from Escherichia coli at low salt concentrations in the presence of spermidine (Kornberg et al., Proc. Natl. Acad. Sci. USA, 71, 3189-3193 (1974)) retain large amounts of protein and RNA and are, thus, potentially useful in structural and other studies. However, these preparations have neither been visualized nor extensively characterized with regard to their protein and other components. We have investigated this type of nucleoid preparation and here supply both light and electron microscope appearances and a description of the DNA-associated proteins. Light microscopy is used to follow the stages of nucleoid release and to demonstrate characteristically rounded nucleoids after chloramphenicol treatment of the cells from which the nucleoids were isolated. The nucleoids are "envelope-associated" particles. Electron microscopy shows an irregular central core that is partially covered with small, membranous vesicles. A significant fraction of the nucleoids have a characteristic doublet/dumbbell-shaped appearance by light microscopy. The nucleoids contain large amounts of protein and RNA in addition to DNA. The DNA and RNA are rendered acid-soluble by very low levels of nucleases, indicating an open structure. A small group of proteins, including H-NS, FIS, HU, and RNA polymerase, is released from the particles upon enzymatic digestion of the DNA.

Bacterial Proteins↗
PubMed1997

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.

Bacterial Proteins↗
PubMed1996

Macromolecular crowding and the mandatory condensation of DNA in bacteria.

Cellular DNA in bacteria is localized into nucleoids enclosed by cytoplasm. The forces which cause condensation of the DNA into nucleoids are poorly understood. We suggest that direct and indirect macromolecular crowding forces from the surrounding cytoplasm are critical factors for nucleoid condensation, and that within a bacterial cell these crowding forces are always present at such high levels that the DNA is maintained in a condensed state. The DNA affected includes not only the preexisting genomic DNA but also DNA that is newly introduced by viral infection, replication or other means.

Bacteria↗
PubMed1996

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

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.

Bacteriophage lambda↗
PubMed1994

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.

Bacterial Proteins↗
PubMed1992

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.

Base Sequence↗
PubMed1991

Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli.

The very high concentration of macromolecules within cells can potentially have an overwhelming effect on the thermodynamic activity of cellular components because of excluded volume effects. To estimate the magnitudes of such effects, we have made an experimental study of the cytoplasm of Escherichia coli. Parameters from cells and cell extracts are used to calculate approximate activity coefficients for cytoplasmic conditions. These calculations require a representation of the sizes, concentrations and effective specific volumes of the macromolecules in the extracts. Macromolecule size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous solution or by using the molecular mass distribution of macromolecules from gel filtration. Macromolecule concentrations in cytoplasm are obtained from analyses of extracts by applying a correction for the dilution that occurs during extraction. That factor is determined from experiments based upon the known impermeability of the cytoplasmic volume to sucrose in intact E. coli. Macromolecule concentrations in the cytoplasm of E. coli in either exponential or stationary growth phase are estimated to be approximately 0.3 to 0.4 g/ml. Macromolecule specific volumes are inferred from the composition of close-packed precipitates induced by polyethylene glycol. Several well-characterized proteins which bind to DNA (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studies in vitro, but are insensitive in studies in vivo. Application of the activity coefficients from the present work indicates that at least part of this discrepancy arises from the difference in excluded volumes in these studies. Applications of the activity coefficients to solubility or to association reactions are also discussed, as are changes associated with cell growth phase and osmotic or other effects. The use of solutions of purified macromolecules that emulate the crowding conditions inferred for cytoplasm is discussed.

Cytoplasm↗
PubMed1990

Quantitation of specific fragments in DNA restriction digests: application to the cohesive fragments in lambda DNA digests.

A procedure for the quantitation of reactions between specific members of a set of DNA restriction fragments is presented. Quantitation of the cohesive fragments in NruI nuclease digests of lambda DNA is used as an example. Restriction fragments are resolved on agarose gels and their amounts are estimated from densitometer scans of photographic negatives of ethidium bromide-stained gels. A linear relationship is found between the peak height of given fragment on the scan and the logarithm of the molecular weight of the fragment, arising in part from the stoichiometry of the digest; this relationship allows simple interpolation between the peak heights of the nonreacting fragments in each gel lane to determine the theoretical maximal amount of each reactive fragment in that gel lane. Similar procedures should be applicable to enzymatic ligation or to site-specific cleavage of specific restriction fragments or to autoradiographic detection of the fragments. Since each lane of the gel is analyzed independently, the method is largely self-correcting for variations in amounts applied to the gel.

Bacteriophage lambda↗