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1.
Chlamydia psittaci (6BC) was grown in yolk sac explants and in L cells and fixed by perfusion in situ to provide undamaged material for comparison with gram-negative bacteria. Reticulate, intermediate, and elementary bodies were all seen to lack a well-defined periplasmic space; intermediate and elementary bodies showed condensations of the nucleoid which differ from common bacterial configurations; and the cytoplasm of highly condensed elementary bodies was much more electron dense than that of the gram-negative bacteria, while retaining its basically particulate nature. These important morphological distinctions are interpreted as reflections of a significantly different cellular level of organization in these two groups of organisms. No important morphological differences were noted in comparisons of the chlamydial particles grown in the two different host systems.  相似文献   

2.
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   

3.
Compared to radiation-sensitive bacteria, the nucleoids of radiation-resistant Deinococcus species show a higher degree of compaction. Such a condensed nucleoid may contribute to the extreme radiation resistance of Deinococcus by limiting dispersion of radiation-induced DNA fragments. Architectural proteins may play a role in this high degree of nucleoid compaction, but comparative genomics revealed only a limited number of Deinococcus homologs of known nucleoid-associated proteins (NAPs) from other species such as Escherichia coli. A comparative proteomic approach was used to identify potentially novel proteins from isolated nucleoids of Deinococcus radiodurans and Deinococcus deserti. Proteins in nucleoid enriched fractions were identified and semi-quantified by shotgun proteomics. Based on normalized spectral counts, the histone-like DNA-binding protein HU appeared to be the most abundant among candidate NAPs from both micro-organisms. By immunofluorescence microscopy, D. radiodurans HU and both DNA gyrase subunits were shown to be distributed throughout the nucleoid structure and absent from the cytoplasm. Taken together, our results suggest that D. radiodurans and D. deserti bacteria contain a very low diversity of NAPs, with HU and DNA gyrase being the main proteins involved in the organization of the Deinococcus nucleoids.  相似文献   

4.
Chlamydia-like organisms have been detected in digestive cells and duct cells of the digestive gland of mussels, Mytilus galloprovincialis, collected from the Basque coast. The organisms appeared as basophilic inclusion bodies within digestive cells and consisted of elongate initial reticulate bodies and previously undescribed condensed forms, interpreted as intermediate bodies. Of the 414 mussels examined by light microscopy, 5.31% showed this type of infection. A second type of chlamydia-like organism was found in nonciliated duct cells. The microorganisms were found mostly free in duct cells and large elongate reticulate bodies, intermediate condensing forms, and fully condensed elementary bodies were clearly distinguished. No serious histopathological or ultrastructural changes were observed in host cells but evidences of a possible localized metabolic damage within infected digestive cells is presented.  相似文献   

5.
Methods developed for isolating bacterial nucleoids were applied to bacteria infected with phage T4. The replicating pool of T4 DNA was isolated as a particle composed of condensed T4 DNA and certain RNA and protein components of the cell. The particles have a narrow sedimentation profile (weight-average s=2,500S) and have, on average, a T4 DNA content similar to that of the infected cell. Their dimensions observed via electron and fluorescence microscopy are similar to the dimensions of the intracellular DNA pool. The DNA packaging density is less than that of the isolated bacterial nucleoid but appears to be roughly similar to its state in vivo. Host-cell proteins and T4-specific proteins bound to the DNA were characterized by electrophoresis on polyacrylamide gels. The major host proteins are the RNA polymerase subunits and two envelope proteins (molecular weights, 36,000 and 31,000). Other major proteins of the host cell were absent or barely detectable. Single-strand breaks can be introduced into the DNA with gamma radiation or DNase without affecting its sedimentation rate. This and other studies of the effects of intercalated ethidium molecules have suggested that the average superhelical density of the condensed DNA is small. However, these studies also indicated that there may be a few domains in the DNA that become positively supercoiled in the presence of high concentrations of ethidium bromide. In contrast to the Escherichia coli nucleoid, the T4 DNA structure remains condensed after the RNA and protein components have been removed (although there may be slight relaxation in the state of condensation under these conditions).  相似文献   

6.
7.
Two DNA-binding proteins with similarity to eukaryotic histone H1 have been described in Chlamydia trachomatis. In addition to the 18-kDa histone H1 homolog Hc1, elementary bodies of C. trachomatis possess an antigenically related histone H1 homolog, which we have termed Hc2, that varies in apparent molecular mass among strains. We report the molecular cloning, expression, and nucleotide sequence of the hctB gene encoding Hc2 and present evidence for in vivo DNA-binding activity of the expressed product. Expression of Hc2 in Escherichia coli induces a compaction of bacterial chromatin that is distinct from that observed upon Hc1 expression. Moreover, isolated nucleoids from Hc2-expressing E. coli exhibit markedly reduced sensitivity to DNase I. These properties of Hc2 are consistent with a postulated role in establishing the nucleoid structure of elementary bodies.  相似文献   

8.
The Escherichia coli nucleoid is maintained in its folded highly condensed state by constraints which involve RNA and protein. We have developed a rapid sedimentation assay to determine the state of folding of the membrane-free nucleoid. An approximate measure of the stability of the nucleoids under various conditions can then be estimated by measuring the temperature at which the nucleoids unfold. Using ethidium and gamma irradiation (which removes the negative supercoiling of the native nucleoid) as probes, it can be shown that there are two types of constraint involved in the condensation of the nucleoid. One of these constraints is destabilized by ethidium but stabilized by negative supercoiling; the second constraint is unaffected by both ethidium and negative supercoiling. Several models can be proposed: (i) a DNA . RNA duplex, (ii) a double-strand DNA (dsDNA) . RNA triplex, (iii) DNA-protein interactions, (iv) a topological knot with RNA, and (v) a DNA tetraplex. The topological knot model is not consistent with the data and many combinations of the others can be excluded. If RNA is involved in both constraints then RNA . DNA duplexes and dsDNA . RNA triplexes are involved in stabilizing the nucleoid.  相似文献   

9.
Water-containing biological material cannot withstand the vacuum of the transmission electron microscope. The classical solution to this problem has been to dehydrate chemically fixed biological samples and then embed them in resin. During such treatment, the bacterial nucleoid is especially prone to aggregation, which affects its global shape and fine structure. Initial attempts to deal with aggregation by optimizing chemical fixation yielded contradictory results. Two decades ago, the situation improved with the introduction of freeze-substitution. This method is based on dehydration of unfixed cryo-immobilized samples at low temperature, which substantially reduces aggregation. As a result, the global shape of the nucleoid can be fairly well defined. Overall, in actively growing bacteria, the nucleoids are dispersed and "coralline" but become more confined when growth ceases. However, it is usually impossible to determine the molecular arrangement of DNA in the nucleoids of freeze-substituted bacteria because crystallization and the subsequent removal of water during substitution result in unavoidable distortions at the ultrastructural level. Recently, cryo-electron microscopy of vitreous sections has enabled the fully hydrated bacterial nucleoid to be studied close to the native state. Such studies have revealed aspects of bacterial nucleoid organization that are not preserved by freeze-substitution, including locally parallel or twisted bundles of DNA filaments, which are more frequently observed once bacterial growth has stopped, whereas in actively growing bacteria, the DNA is seen to be in a mostly disordered pattern.  相似文献   

10.
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   

11.
Reticulate bodies of the meningopneumonitis (MP) microorganism were purified from L cells 18 hr after infection by the combination of differential centrifugation in 30% sucrose solution and potassium tartrate density gradient centrifugation. It was ascertained by electron microscopy that purified preparations of reticulate bodies obtained were almost entirely free of host-cell components and of infectious elementary bodies of MP microorganisms. Purified reticulate bodies were easily disrupted by mechanical agitation, and it was observed in shadowed preparation that ribosome-like particles 15 mmu in diameter were scattered from broken reticulate bodies. In shadowed preparations, reticulate bodies were found to range in size from 1.0 to 1.6 mu in diameter, but in cross-section the range was 0.5 to 1.0 mu. In these preparations, the purified reticulate bodies were irregular in shape, round or oval, and were composed of rather homogenous, amorphous, or reticulate material with moderate density. Some particles exhibited a less-dense internal structure, in which a coarse fibrous reticulum was seen. Chemical fractionation of (32)P-labeled purified reticulate bodies showed that they contained three times more ribonucleic acid (RNA) than deoxyribonucleic acid, with the RNA being composed primarily of 21S, 16S, and 4S RNA. No infectivity of purified reticulate bodies could be demonstrated.  相似文献   

12.
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   

13.
14.
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.  相似文献   

15.
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   

16.
The ultrastructure of Mycoplasma pneumoniae FH was examined by a mild fixation method, the freeze-substitution technique, for thin-section transmission electron microscopy and the following new findings were obtained. In the cytoplasm, no nuclear region could be clearly identified. The cytoplasm was filled with many ribosome-like particles, fine fibers and electron-dense particles. The electron-dense particles appeared to be similar to the particles found in the nucleoid region of Pseudomonas aeruginosa and might therefore possibly be a kind of DNA binding protein. The cell surface was completely enveloped with a thin opaque layer. The presence of this surface layer prevented any direct contact of the cell surface with that of the two M. pneumoniae cells.  相似文献   

17.
The fine structure of mitochondria and mitochondrial nucleoids in exponentially growing Physarum polycephalum was studied at various periods throughout the mitochondrial division cycle by light and electron microscopy. The mitochondrial nucleoid elongates lingitudinally while the mitochondrion increases in size. When the nucleoid reaches a length of approximately 1.5 mum the mitochondrial membrane invaginates at the center of the mitochondrion and separates the mitochondrial contents. However, the nucleoid does not divide even when the mitochondrial sections are connected by a very narrow bridge. Just before division of the mitochondrion, the nucleoid divides by constriction of the limiting membrane of the dividing mitochondrion. After division, one end of the nucleoid appears to be associated with the inner mitochondrial membrane. The nucleoid then again becomes situated in the center of the mitochondrion before repeating these same processes.  相似文献   

18.
19.
我们实验室与上海细胞生物学研究所的有关同志多年来在特殊涡鞭毛虫──尖尾虫(Oxyrrhismarina)的细胞生物学和生物化学上作了一系列的研究,本文所报道的是这些工作中的一部分。对尖尾虫(Oxyrrhismarina)的永久浓聚染色体的精细形象以及不同固定方法对其精细形象的影响作了观察,并与人肠道细菌的类核体、典型涡鞭毛虫原甲藻(Prorocentrummicans)和鞭毛虫眼虫(Englenasp.)的永久浓聚染色体作了比较。结果表明,细菌的类核体的精细形象受固定方法的影响极大。反之,不同的固定方法对于眼虫染色体的精细形象则看不出有任何显著的影响。至于典型涡鞭毛虫类的染色体,单用OsO4或用戊二醛-OsO4双固定法固定,都会得到典型涡鞭毛虫类染色体的横带样结构,然而单纯用戊二醛固定,却会得到大不相同的形象。在这方面尖角虫的染色体与一般的涡鞭毛虫类的染色体相距甚远,其染色体的精细形象本身也与眼虫类的染色体较为相似,而与一般涡鞭毛虫类的大不相同。本工作所得到的结果与以前我们在不同方面所得到的结果一致。研究结果全都表明,失足虫与典型涡鞭毛虫有着一系列重大的差异,实际上代表着一个介于一般鞭毛虫类与典型涡鞭毛虫类  相似文献   

20.
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   

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