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1.
Fine structure of the Deinococcus radiodurans nucleoid revealed by cryoelectron microscopy of vitreous sections
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Transmission electron microscopy revealed that the nucleoid of the extremely radioresistant bacteria Deinococcus radiodurans may adopt an unusual ring shape. This led to the hypothesis that the tight toroidal package of the D. radiodurans genome might contribute to radioresistance by preventing diffusion of ends of double-stranded DNA breaks. The molecular arrangement of DNA in the nucleoid, which must be determined to test this hypothesis, is not discernible by conventional methods of electron microscopy. We have applied cryoelectron microscopy of vitreous sections and found that the DNA arrangement in D. radiodurans differs from toroidal spooling. Diffuse coralline nucleoids of exponentially growing D. radiodurans do not reveal any particular molecular order. Electron-dense granules are generally observed in the centers of nucleoids. In stationary-phase cells, the nucleoid segregates from cytoplasm and DNA filaments show locally parallel arrangements, with increasing aspects of cholesteric liquid crystalline phase upon prolonged starvation. The relevance of the observed nucleoid organization to the radiation resistance of D. radiodurans is discussed. 相似文献
2.
Summary The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa
et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication.The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed. 相似文献
3.
This review compares the results of different methods of investigating the morphology of nucleoids of bacteria grown under conditions favoring short generation times. We consider the evidence from fixed and stained specimens, from phase-contrast and fluorescence microscopy of growing bacteria, and from electron microscopy of whole as well as thinly sectioned ones. It is concluded that the nucleoid of growing cells is in a dynamic state: part of the chromatin is "pulled out" of the bulk of the nucleoid in order to be transcribed. This activity is performed by excrescences which extend far into the cytoplasm so as to reach the maximum of available ribosomes. Different means of fixation provide markedly different views of the texture of the DNA-containing plasm of the bulk of the nucleoid. Conventional chemical fixatives stabilize the cytoplasm of bacteria but not their protein-low chromatin. Uranyl acetate does cross-link the latter well but only if the cytoplasm has first been fixed conventionally. In the interval between the two fixations, the DNA arranges itself in liquid-crystalline form, supposedly because of loss of supercoiling. In stark contrast, cryofixation preserves bacterial chromatin in a finely granular form, believed to reflect its native strongly negatively supercoiled state. In dinoflagellates the DNA of their permanently visible chromosomes (also low in histone-like protein) is natively present as a liquid crystal. The arrangement of chromatin in Epulocystis fishelsoni, one of the largest known prokaryotes, is briefly described. 相似文献
4.
The bacterial nucleoid visualized by fluorescence microscopy of cells lysed within agarose: comparison of Escherichia coli and spirochetes of the genus Borrelia.
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The nucleoids of Escherichia coli and the spirochetes Borrelia burgdorferi and Borrelia hermsii, agents of Lyme disease and relapsing fever, were examined by epifluorescence microscopy of bacterial cells embedded in agarose and lysed in situ with detergent and protease. The typical E. coli nucleoid was a rosette in which 20 to 50 long loops of DNA emanated from a dense node of DNA. The percentages of cells in a population having nucleoids with zero, one, two, and three nodes varied with growth rate and growth phase. The borrelia nucleoid, in contrast, was a loose network of DNA strands devoid of nodes. This nucleoid structure difference correlates with the unusual genome of Borrelia species, which consists primarily of linear replicons, including a 950-kb linear chromosome and linear plasmids. This method provides a simple, direct means to analyze the structure of the bacterial nucleoid. 相似文献
5.
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. 相似文献
6.
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. 相似文献
7.
A method is described for gently dissociating large DNA-protein complexes and for visualizing and quantitating the substructures by autoradiography. Using this technique, it is shown that nucleoids isolated from exponentially growing Escherichia coli (mean generation time = 35 min) contain on average 2.8 genome equivalents of DNA and that this nucleoid can be dissociated by deproteinization into two substructures having on average 1.4 genome equivalents. This result is correlated with previous sedimentation studies on the unfolded nucleoid DNA to explain prior inconsistencies. Scanning electron microscopy studies demonstrate that the shape and size of the isolated nucleoid is consistent with the proposed subunit structure of the in vivo nucleoid. 相似文献
8.
Ultrastructural studies of the nucleoids of the pleomorphic forms of Chlamydia psittaci 6BC: a comparison with bacteria. 总被引:6,自引:0,他引:6
The nucleoids of the various pleomorphic forms of Chlamydia psittaci have been examined by direct observation of infected cells and by observations on isolated particles. The fixation and staining methods used were the same as those routinely used for the examination of bacteria to facilitate the comparison of chlamydial fine structure with that of bacteria. The nucleoids of reticulate bodies were composed of fine fibrils which extended throughout these particles. The nucleoids of intermediate bodies are characterized by an electron-dense mass with which the fibrous elements are associated in a structurally coherent manner. As condensation of the intermediate bodies proceeds, the electron-dense mass becomes eccentrically located and the fibers form a distinct radiating structure. Large elementary bodies have a few fibers associated with their condensed electron-dense nucleoids but the more condensed mature elementary bodies have a very discrete and homogeneous electron-dense nucleoid which is separated from the cytoplasmic elements of these particles by a very distinct electron-transparent space. These highly condensed elementary body nucleoids are usually ovoid, but may be elongated or irregular, and a small number of these structures react very strongly with ruthenium red. While the nucleoid structure of reticulate bodies resembles that of the bacterial cell, both the condensation process and the nucleoid morphologies which result from it in intermediate and elementary bodies have no parallels among the bacteria. Thus we conclude that major differences in nucleoid organization exist between the chlamydia and the bacteria. 相似文献
9.
Escherichia coli nucleoids were visualized after the DNA of OsO4-fixed but hydrated cells was stained with the fluorochrome DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). In slowly growing cells, the nucleoids are rod shaped and seem to move along the major cell axis, whereas in rapidly growing, wider cells they consist of two- to four-lobed structures that often appear to advance along axes lying perpendicular or oblique to the major axis of the cell. To test the idea that the increase in cell diameter following nutritional shift-up is caused by the increased amount of DNA in the nucleoid, the cells were subjected to DNA synthesis inhibition. In the absence of DNA replication, the nucleoids continued to move in the growing filaments and were pulled apart into small domains along the length of the cell. When these cells were then transferred to a richer medium, their diameters increased, especially in the region enclosing the nucleoid. It thus appears that the nucleoid motive force does not depend on DNA synthesis and that cell diameter is determined not by the amount of DNA per chromosome but rather by the synthetic activity surrounding the nucleoid. Under the non-steady-state but balanced growth conditions induced by thymine limitation, nucleoids become separated into small lobules, often lying in asymmetric configurations along the cell periphery, and oblique and asymmetric division planes occur in more than half of the constricting cells. We suggest that such irregular DNA movement affects both the angle of the division plane and its position. 相似文献
10.
Zimmerman SB 《Journal of structural biology》2003,142(2):256-265
The genomic DNA of Escherichia coli is localized in one or a few compact nucleoids. Nucleoids in rapidly grown cells appear in complex shapes; the relationship of these shapes to underlying arrangements of the DNA is of structural interest and of potential importance in gene localization and nucleoid partition studies. To help assess this variation in shape, limited three-dimensional information on individual nucleoids was obtained by DNA fluorescence microscopy of cells as they reoriented in solution or by optical sectioning. These techniques were also applied to enlarged nucleoids within swollen cells or spheroplasts. The resulting images indicated that much of the apparent variation was due to imaging from different directions and at different focal planes of more regular underlying nucleoid shapes. Nucleoid images could be transformed into compact doublet shapes by exposure of cells to chloramphenicol or puromycin, consistent with a preexisting bipartite nucleoid structure. Isolated nucleoids and nucleoids in stationary-phase cells also assumed a doublet shape, supporting such a structure. The underlying structure is suggested to be two subunits joined by a linker. Both the subunits and the linker appear to deform to accommodate the space available within cells or spheroplasts ("flexible doublet" model). 相似文献
11.
Analysis of nucleoid morphology during germination and outgrowth of spores of Bacillus species
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After a few minutes of germination, nucleoids in the great majority of spores of Bacillus subtilis and Bacillus megaterium were ring shaped. The major spore DNA binding proteins, the alpha/beta-type small, acid-soluble proteins (SASP), colocalized to these nucleoid rings early in spore germination, as did the B. megaterium homolog of the major B. subtilis chromosomal protein HBsu. The percentage of ring-shaped nucleoids was decreased in germinated spores with lower levels of alpha/beta-type SASP. As spore outgrowth proceeded, the ring-shaped nucleoids disappeared and the nucleoid became more compact. This change took place after degradation of most of the spores' pool of major alpha/beta-type SASP and was delayed when alpha/beta-type SASP degradation was delayed. Later in spore outgrowth, the shape of the nucleoid reverted to the diffuse lobular shape seen in growing cells. 相似文献
12.
Many electron-dense granules were found in the nucleoid area of Pseudomonas aeruginosa strain K by electron microscopy with the technique of the freeze-substitution method. These granules contained phosphorus and calcium as determined by X-ray microanalysis. The size and the numbers of the granules decreased when the bacteria was cultured in the medium from which phosphate-containing compounds were depleted. From these observations we concluded that the granule was a phosphate-containing granule and possibly a polyphosphate granule. The excellent preservation of the fine structures by the freeze-substitution technique enables us to show very small polyphosphate granules in the nucleoid area of the bacterial cells which cannot be revealed by the conventional chemical fixation method. As we could not see the granules in other bacteria cultured in nutrient medium such as Serratia, Escherichia, Bacillus and Vibrios, the accumulation of the phosphate granules in Ps. aeruginosa might be a unique character of this bacteria and might be related to the growing capability of this bacteria in extremely low nutrient supply. 相似文献
13.
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. 相似文献
14.
15.
Zimmerman SB 《Journal of structural biology》2004,147(2):146-158
The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope. 相似文献
16.
Koit Aasumets Yuliya Basikhina Jaakko L. Pohjoismäki Steffi Goffart Joachim Gerhold 《Biochemistry and Biophysics Reports》2021
The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution. 相似文献
17.
Summary The behavior of nucleoids during the leucoplast division cycle in the epidermis of onion (Allium cepa) bulbs was investigated using DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) staining. The leucoplast was morphologically amoeboid and continuously changed its shape. A dumbbell-shaped leucoplast divided into two spherical daughter ones by constriction in the middle region of the body. Leucoplasts contained 4–10 mostly spherical, oval, partly rodand dumbbell-shaped nucleoids which were dispersed within the bodies. The proportion of one DNA molecule of a T4 phage particle to the small leucoplast nucleoid in the grain density of negative film was 1 to 0.91. Comparison of the present result and another groups' biochemical results suggested that a small leucoplast nucleoid contains one DNA molecule. The dumbbell-shaped leucoplast probably before division contained about twice as many nucleoids as the spherical leucoplast after division, and each half of the dumbbell contained about half the number of nucleoids. Nucleoids increased in number with growth of the leucoplast. The behavior of nucleoids during the leucoplast division cycle in onion bulbs was basically similar to that during the chloroplast division cycle in higher plants and green algae, which was previously reported (Kuroiwa
et al. 1981 b). 相似文献
18.
A simple test has been developed that measures how much DNA damage has occurred in a single mammalian cell. The procedure is based on the microscopic examination of "halos" of nucleoids that adhere to coverslips. Nucleoids are produced by flowing salt solutions containing detergents over the attached cells. The nucleoid halos are thought to be a tangle of loops of free DNA that emanate from the remnants of the nucleus. When visualized by staining with ethidium bromide the nucleoid halos first expand, and then contract as the concentration of ethidium increases. Exposure of nucleoids to very low levels of DNA chain-breaking treatments results in the incremental expansion of the halos to a maximum of 15 microns or more. Our assay is based upon quantitating the degree of halo expansion. If intact cells are exposed to DNA-damaging treatments, then allowed increasing periods of post-treatment growth before forming nucleoids, the DNA repair processes result first in expanded and then in contracted halos. By admixing a supercoiled plasma DNA of known length (38 kb) to nucleoids with contracted halos, the fractional halo expansion and the fraction of surviving plasmid supercoils can be measured from the same solution. Use of photodynamic DNA damage showed that the halo expansion was 11.6 times more sensitive than plasmid relaxation. Use of gamma-irradiation showed that the halo expansion was 3.6 times more sensitive than plasmid relaxation. The latter value demonstrates that one break per 137,000 bp results in the expansion of the halos to 63% of their maximal value. We estimate that this method will detect about 5000 breaks per nucleus containing 5 x 10(9) bp. 相似文献
19.
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. 相似文献
20.
Nucleoid Condensation and Cell Division in Escherichia coli MX74T2 ts52 After Inhibition of Protein Synthesis 总被引:16,自引:16,他引:0
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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. 相似文献