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.     

A possible role for L24 of Bacillus subtilis in nucleoid organization and segregation

The condensation of DNA in bacterial nucleoids during cell cycle is a complex and dynamic process. Proteins displaying the physico-chemical properties of histones are known to contribute to this process. During a search for B. subtilis nucleoid associated proteins, HBsu and L24 were identified as the most abundant proteins in nucleoid containing fractions. Purified L24 binds and condenses DNA in vitro. In this paper we describe immunofluorescence studies that demonstrated that L24 is located at the poles of the nucleoids in exponentially growing cells. In contrast, the protein is dispersed in the cytoplasm during stationary phase. Moreover, overexpression of the rplX gene encoding L24 disrupts nucleoid segregation and positioning.     

DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins

The volume of a typical Eschericia coli nucleoid is roughly 10⁴ times smaller than the volume of a freely coiling linear DNA molecule with the same length as the E. coli genome. We review the main forces that have been suggested to contribute to this compaction factor: macromolecular crowding (that “pushes” the DNA together), DNA charge neutralization by various polycationic species (that “glues” the DNA together), and finally, DNA deformations due to DNA supercoiling and nucleoid proteins. The direct contributions of DNA supercoiling and nucleoid proteins to the total compaction factor are probably small. Instead, we argue that the formation of the bacterial nucleoid can be described as a consequence of the influence of macromolecular crowding on thick, supercoiled protein-DNA fibers, that have been partly charge neutralized by small multivalent cations.     

Shape and compaction of Escherichia coli nucleoids

The genomic DNA in cells of Escherichia coli is localized in one or two compact, phase-like regions with characteristic shapes. Nucleoids undergo progressive changes in shape and compaction in the presence of drugs such as chloramphenicol or puromycin. Forces which influence nucleoid shape and compaction are reviewed, with particular emphasis on crowding effects of the cytoplasm and confinement effects of the cell envelope. Based in part on the theory of Kornyshev and Leikin for interaction between DNA duplexes, the folding of DNA caused by binding of DNA-associated proteins is suggested to antagonize DNA condensation and, thereby, increase access to DNA sequences. These views are incorporated into a working model for nucleoid organization.     

An elastic rod model to evaluate effects of ionic concentration on equilibrium configuration of DNA in salt solution

As a coarse-gained model, a super-thin elastic rod subjected to interfacial interactions is used to investigate the condensation of DNA in a multivalent salt solution. The interfacial traction between the rod and the solution environment is determined in terms of the Young–Laplace equation. Kirchhoff’s theory of elastic rod is used to analyze the equilibrium configuration of a DNA chain under the action of the interfacial traction. Two models are established to characterize the change of the interfacial traction and elastic modulus of DNA with the ionic concentration of the salt solution, respectively. The influences of the ionic concentration on the equilibrium configuration of DNA are discussed. The results show that the condensation of DNA is mainly determined by competition between the interfacial energy and elastic strain energy of the DNA itself, and the interfacial traction is one of forces that drive DNA condensation. With the change of concentration, the DNA segments will undergo a series of alteration from the original configuration to the condensed configuration, and the spiral-shape appearing in the condensed configuration of DNA is independent of the original configuration.     

Cooperative transitions of isolated Escherichia coli nucleoids: implications for the nucleoid as a cellular phase

The genomic DNA of Escherichia coli occurs in compact bodies known as nucleoids. Organization and structure of nucleoids are poorly understood. Compact, characteristically shaped, nucleoids isolated by the polylysine-spermidine procedure were visualized by DNA fluorescence microscopy. Treatment with urea or trypsin converted compact nucleoids to partially expanded forms. The transition in urea solutions was accompanied by release of most DNA-associated proteins; the transition point between compact and partially expanded forms was not changed by the loss of the proteins nor was it changed in nucleoids isolated from cells after exposure to chloramphenicol or from cells in which Dps, Fis, or H-NS and StpA had been deleted. Partially expanded forms became dispersed upon RNase exposure, indicating a role of RNA in maintaining the partial expansion. Partially expanded forms that had been stripped of most DNA-associated proteins were recompacted by polyethylene glycol 8,000, a macromolecular crowding agent, in a cooperative transition. DNA-associated proteins are suggested to have relatively little effect on the phase-like behavior of the cellular nucleoid. Changes in the urea transition indicate that a previously described procedure for compaction of polylysine-spermidine nucleoids may have an artifactual basis, and raise questions about reports of repetitive local structures involving the DNA of lysed cells.     

DNA condensation by multivalent cations

In the presence of multivalent cations, high molecular weight DNA undergoes a dramatic condensation to a compact, usually highly ordered toroidal structure. This review begins with an overview of DNA condensation : condensing agents, morphology, kinetics, and reversibility, and the minimum size required to form orderly condensates. It then summarizes the statistical mechanics of the collapse of stiff polymers, which shows why DNA condensation is abrupt and why toroids are favored structures. Various ways to estimate or measure intermolecular forces in DNA condensation are discussed, all of them agreeing that the free energy change per base pair is very small, on the order of 1% of thermal energy. Experimental evidence is surveyed showing that DNA condensation occurs when about 90% of its charge is neutralized by counterions. The various intermolecular forces whose interplay gives rise to DNA condensation are then reviewed. The entropy loss upon collapse of the expanded wormlike coil costs free energy, and stiffness sets limits on tight curvature. However, the dominant contributions seem to come from ions and water. Electrostatic repulsions must be overcome by high salt concentrations or by the correlated fluctuations of territorially bound multivalent cations. Hydration must be adjusted to allow a cooperative accommodation of the water structure surrounding surface groups on the DNA helices as they approach. Undulations of the DNA in its confined surroundings extend the range of the electrostatic forces. The condensing ions may also subtly modify the local structure of the double helix. © 1998 John Wiley & Sons, Inc. Biopoly 44: 269–282, 1997     

Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin-treated oats

Histological and cytological evidence of where and when apoptotic cells occur in Pc-2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been floated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM-TUNEL was positive in the non-collapsed cells but not in the collapsed cells in the treated leaves. However, the EM-TUNEL assay was positive in the nuclei of the non-collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H2O2, suggesting that the victorin-treated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin-treated non-collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM-TUNEL assay indicated that EGTA co-incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.     

Osmotic pressure: resisting or promoting DNA ejection from phage?

Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I in the course of ejection. In this work, we argue by a combination of experimental techniques (osmotic suppression without DNase I monitored by UV absorbance, pulse-field electrophoresis, and cryo-transmission electron microscopy visualization) and simple scaling modeling that intact genome (i.e., undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed.     

Nucleoid Condensation and Cell Division in Escherichia coli MX74T2 ts52 After Inhibition of Protein Synthesis

The reorganization of the bacterial nucleoid of an Escherichia coli mutant, MX74T2 ts52, was studied by electron microscopy after protein synthesis inhibition by using whole mounts of cell ghosts, ultrathin-sectioning, and freeze-etching. The bacterial nucleoid showed two morphological changes after chloramphenicol addition: deoxyribonucleic acid (DNA) localization and DNA condensation. DNA localization was observed 10 min after chloramphenicol addition; the DNA appeared as a compact, solid mass. DNA condensation was observed at 25 min; the nucleoid appeared as a cytoplasm-filled sphere, often opened at one end. Ribosomes were observed in the center. Giant nucleoids present in some mutant filaments showed fused, spherical nucleoids arranged linearly, suggesting that the tertiary structure of the nucleoid reflects the number of replicated genomes. Inhibitors which directly or indirectly blocked protein synthesis and caused DNA condensation were chloramphenicol, puromycin, amino acid starvation, rifampicin, or carbonyl cyanide m-chlorophenyl hydrazone. All inhibitors that caused cell division in the mutant also caused condensation, although some inhibitors caused condensation without cell division. Nucleoid condensation appears to be related to chromosome structure rather than to DNA segregation upon cell division.     

Structural and ultrastructural changes in Mycobacterium rubrum cells exposed to penicillin

The action of penicillin taken at subbacterioscopic doses on Mycobacterium rubrum cells causes changes in the size and shape of the cells, in the structure of the cell wall, in the intracellular membrane systems and in functions associated with them, and in the structure of nucleoids whose DNA packing becomes more loose. If the antibiotic is added at bacteriostatic doses, the size and shape of the cells do not change, but peptidoglycan precursors being synthesized are not incorporated into the polymer and accumulate in the periplasm. DNA overspiralization in nucleoids is a non-specific reaction, which indicates that DNA is physiologically passive. DNA is isolated with a membrane from the cytoplasm in certain cells. It is possible that the resistance of cells against penicillin is associated with the capability of DNA to become inactive in physiological terms.     

Toroidal nucleoids in Escherichia coli exposed to chloramphenicol

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.     

Cytoplasmic Nucleoids in the Generative Cell,Sperm Cell and Egg Cell of Calystegia heder acea

Cytoplasmic nucleoids in the generative cell of mature pollens, sperm cells of pollens cultured in vitro and egg cell of mature embryo sac in Calystegia bederacea Wall. were studied by means of the DNA fluorochrome DAPI in conjunction with epitluorescence microscopy for in situ detection of cytoplasmic DNA in cells. Results showed that many cytoplasmic DNA nucleoids were present in the generative cell and speim cells. Two types of nucleoids were observed, one with big and strong fluorescent dots, and the other with small and weak fluorescence. Many dot-shaped and a few circle-shaped nucleoids were randomly distributed in the thin layered cytoplasm of the egg cell. It was suggested that different types of nucleoids might represent plastid DNA and mitochondrion DNA respectively. Results provided cytological data that Calystegia hederaeea had the potential of plastid DNA biparental inheritance, and the mode of which merits further study via molecular biological methods.     

Improved gene expression using low molecular weight peptides produced from protamine sulfate

DNA condensation plays a key role in non-viral gene delivery by affecting gene transfection, nuclear targeting, and eventual gene expression efficiency. Theoretically, a DNA condenser with the appropriate DNA condensation ability but without affecting DNA dissociation from DNA condensates inside the cytoplasm should be a perfect carrier for gene delivery. Protamine is a natural DNA condensation agent and has been widely used in gene delivery. In this work, protamine was selectively digested enzymatically to produce low molecular weight protamine fragments (LMWPs) of various lengths and amino acid compositions. The DNA condensation ability and gene transfection efficiency of these LMWP peptides were tested. Compared to protamine, all the LMWP peptides showed lower DNA binding strength. However, some LMWP peptides demonstrated excellent DNA condensation ability and could form very compact DNA condensates with small particle size (∼100 nm). More interestingly, LMWP peptide-mediated in vitro gene delivery showed prolonged (up to 12 days) gene expression. Results from this study suggest that designing DNA condensers with appropriate and tunable DNA binding strengths and condensation abilities would be an effective means to improve gene expression and thus gene therapy efficiency. Since LMWP peptides have low immunogenicity, they would be safer than protamine for use in gene therapies. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 10, pp 1447–1455.     

打碗花生殖细胞,精细胞及卵细胞中的细胞质类核

已有不少超微结构的资料阐明被子植物双亲和单亲母系质体遗传的细胞学基础。近年应用DAPI荧光染色的方法,可快速地从检测质体DNA存在的状况确定被子植物中具双亲遗传潜能的种。从质体的类核存在与否判断质体遗传方式为母系遗传或双亲遗传与已有的遗传分析结论基本一致,只有少数种类是矛盾的。DAPI荧光技术可以认为是研究细胞质遗传机理的一个重要手段。我们曾证明旋花科牵牛属植物生殖细胞、精细胞中存在细胞质类核,确定其具双亲或单亲父系质体遗传的潜能,并用RFLP技术进一步确定其为质体父系遗传型。本研究证明旋花科的打碗花属生殖细胞、精细胞和卵细胞中细胞质类核存在的状况与牵牛属的相似,提供了打碗花可能在质体遗传上与牵牛属 具相同的遗传方式的资料。     

Stabilization of Compact Spermidine Nucleoids fromEscherichia coliunder Crowded Conditions: Implications forin VivoNucleoid Structure

Nucleoids fromEscherichia coliwere 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 occurin 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 ofE. coli,is not a necessary component for maintaining compaction in these preparations.     

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.     

Study on Organelle DNA Within the Generative Cell and Sperm Cells in Pharbitis

The organelle DNA in generative cell and its behavior during spermatogenesis in Pharbitis limbata and P. purpurea were observed by epifluorescence microscopy stained with 4',-6-diamidino-2-phenylindole (DAPI). In these two species, the generative cell is long and thin in which a great amount of cytoplasmic DNA is present. Most pairs of sperm cells are isomorphic, in which one end is obtuse and the other is elongate, but in a few pairs dimorphi sperms are present. The nucleus is located at one end of the cell. A lot of cytoplasmic DNA are distributed randomly throughout the cytoplasm. The size of organelle nucleoids and their fluorescence intensity are different in a sperm cell. The features of generative cell and sperm cell, and behavior of cytoplasmic DNA are similar in P. limbata and P. purpurea. The obvious differences between them are that the size and fluorescence intensity of organelle nucleoids in P. purpurea are respectively smaller and weaker than in P. limbata. The results showed that morning glory has potential of biparental or paternal cytoplasmic in heritance. Isomorphism and dimorphism of sperms, and the relationship between the ratio of nucleus and cytoplasm in sperm cell and the plastid biparental inheritance are discussed.     

Cytochemical study of the localization and organization of parental herpes simplex virus type I DNA during initial infection of the cell

Changes in the location and structural organization of parental herpes simplex virus type 1 (HSV-1) DNA during its migration from the extracellular space to the interior of the nucleus of the target cell were examined by in situ hybridization using an HSV-1 DNA probe, specific DNA staining, and autoradiography after infection of cells with tritium-labeled viruses. In situ hybridization was carried out on denatured DNA to reveal as much as possible of the HSV-1 sequence present at the surface of the sections, and also on non-denatured DNA which revealed the presence of single-stranded portions of parental DNA, both prior to and during its intracellular migration. The results from in situ hybridization and autoradiography demonstrated that a short interval of about 15 min separated the initial contact of the viruses with the cells from the entry of parental viral DNA into the nucleus. In transit, morphologically intact nucleoids were released into the cytoplasm, and swollen nucleoids which contained partially decondensed viral DNA became detectable in the juxtanuclear cytoplasm and the periphery of the nucleus among the cell chromatin fibers. Completely decondensed parental viral DNA fibers could not be distinguished structurally from cellular DNA, but their position could be revealed by the in situ hybridization label. The infective DNA became randomly distributed within all compartments of the nucleus except the matrix-associated clusters of interchromatin granules.     

Mutations disturbing the condensation of plastid nucleoids inChlamydomonas reinhardtii

Summary To study the mechanism of condensation of dispersed plastid (pt) nucleoids into a single pt nucleoid with aging of the cells ofChlamydomonas reinhardtii, two mutants, designated cond-1 and cond-2, were isolated. A plastid of a wild type cell, 6.5 m in diameter, contained ten dispersed spherical pt nucleoids within one week of culture on an agar plate. At about one week of culture, the cell number was saturated and pt nucleoids began to associate with each other, condensing into a single pt nucleoid at three weeks of culture. In contrast, cond-1 and cond-2 cells, which had about 20 and 45 pt nucleoids and whose cell diameters were 7.8 and 9.5 m at one week of culture respectively, still had about 10 and 20 pt nucleoids at even 7 weeks of culture. Doubling times of the three cell types were similar. From genetic analysis, each of the two mutants had one gene mutation. The two mutations are probably linked. The measurement of O₂ evolution showed that the two mutations did not affect the photosynthetic system. Lipid contents of the two mutant cells were clearly higher than that of wild type cells. The role of a higher number of pt nucleoids is probably to increase the activity of lipid and/or membrane synthesis for lipid storage.