Electron Microscope and Autoradiographic Study of Ultrastructural Aspects of Competence and Deoxyribonucleic Acid Absorption in Bacillus subtilis: Localization of Uptake and of Transport of Transforming Deoxyribonucleic Acid in Competent Cells

With the aid of serial-section electron microscopy two types of mesosomes can be distinguished in cells of competent cultures of Bacillus subtilis: (i) mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and (ii) mesosomes which extend from the plasma membrane into the nuclear bodies (nuclear mesosomes). Contrary to plasma membrane mesosomes, nuclear mesosomes are absent from the tip zones. Electron microscopic autoradiography of sections of Bacillus subtilis cells exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid (DNA) for a short period of time shows that the DNA becomes associated with mesosomes. As a function of time the DNA migrates towards the nucleoids. Transport of DNA is completed within 15 to 60 min after termination of DNA uptake. During its migration the DNA continues to be associated with mesosomes, presumably with nuclear mesosomes. DNA initially associated with plasma membrane mesosomes of the tip zones is probably transported first towards the middle zones peripherally and from there towards the nucleoids.     

Nuclear and cell division in Bacillus subtilis: cell development from spore germination.

The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions. Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid. When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm. Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm. Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each. In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest. On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply. Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle.     

Formation and structure of mesosomes in Myxococcus xanthus

Summary Vegetative cells of Myxococcus xanthus, strain FB, were found to contain numerous small mesosomes distributed throughout the cell. They persisted in the cell as long as the cells were maintained on casitone-agar. When these cells were transferred into casitone-broth and grown under aeration large mesosomes were newly formed at the division plane during the first and second cell division after transfer. After four to six more generations when transferred a second time into fresh casitone broth mesosomes were no longer detectable in the cells but reappeared when the cells were retransferred onto casitone-agar.A low oxygen concentration in the medium caused the formation of an unidentified factor found to be responsible for the formation of mesosomes in cells of colonies or in a liquid medium.The shape of the mesosomes seems not to be predetermined but depends upon the inhomogeneity of cytoplasm and nucleoids into which they intrude. In some large mesosomes the infolded membrane consisted of five layers, one dense layer alternating with a translucent one with dense layers limiting the membrane. The width of these membranes was 120 A instead of 160 A as could be expected for two merged triple-layered cytoplasmic membranes each measuring about 80 A. A large poly-phosphate granule was found to be enclosed by a mesosome.     

THE MEMBRANE SYSTEM OF STREPTOMYCES CINNAMONENSIS

The plasma membrane bounding the cytoplasm immediately inside the hyphal wall of Streptomyces cinnamonensis may not retract from the hyphal wall. When it does retract from the wall, it appears as a single dark line in some sections and as 2 dark lines separated by a light zone in others. The membrane system consists of mesosomes and endomembrane structures. The mesosomes are those membrane structures whose derivatives appear to be the plasma membrane. The endomembrane structures, in the present report, are those that appear to have been derived from either the cytoplasm or the limiting membranes of the pre-existing membrane structures. All membranes seem capable of proliferation, a mechanism obviously responsible for the growth of the individual membrane structures and for the origin of many new ones. The mesosomes, according to their limits, are of 2 distinct types, the open mesosomes and the closed mesosomes. The open mesosomes are partially enclosed by limiting membranes, leaving the unenclosed sides limited by the wall. These mesosomes, when old, usually in aerial hyphae, may become attached to the wall and somewhat deleted from their limiting membranes. The individual membranes in their interior may appear disfigured. The closed mesosomes are completely surrounded by the limiting membranes. These mesosomes, as well as endomembrane structures, retain their original positions in the cytoplasm even in the older aerial hyphae, and the membranes in their interior usually remain practically as distinct in the aerial hyphae as they are in the substratal hyphae. New mesosomes and endomembrane structures are being formed continuously as the mycelium develops. The mesosomes, as a rule, occupy more or less the peripheral regions of the cytoplasm, while the endomembrane structures distribute themselves widely in the cytoplasm and also in the nucleoids. The appearance of the unit membranes, being double-layered (2 dark lines separated by a light line), is not consistent. The membranes as a whole are more resistant to degeneration than the cytoplasm and the nucleoids.     

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.     

Cell length, nucleoid separation, and cell division of rod-shaped and spherical cells of Escherichia coli.

By comparing the dimensions and DNA contents of normal rod-shaped Escherichia coli with those of mutants that grow as spheres or ellipsoids, we have determined that two parameters remain unchanged: the DNA/mass ratio and the average cell length (diameter, for spherical cells). In consequence, the average volumes and DNA contents of the spherical mutant cells are about four to six times greater than those of rod-shaped cells growing at a similar rate. In addition, it was found that cells of both rod and sphere forms had approximately the same number of nucleoids (as seen when the DNA was condensed after inhibition of protein synthesis). The nucleoids of the spherical cells therefore consist of four to six completed chromosomes each (polytene nucleoids). We suggest that the attainment of a minimum cell length is necessary for nucleoid separation after chromosome replication and that such a separation is itself a prerequisite for septum formation.     

Electron Microscope and Autoradiographic Study of Ultrastructural Aspects of Competence and Deoxyribonucleic Acid Absorption in Bacillus subtilis: Ultrastructure of Competent and Noncompetent Cells and Cellular Changes During Development of Competence

By means of electron microscope autoradiography of component cultures of Bacillus subtilis exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid competent and noncompetent cells can be distinguished. Competence is not limited to a specific phase of the cell division cycle. With serial section electron microscopy of competent and noncompetent cells, two types of mesosomal structures are observed: mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and mesosomes which are additionally connected to the nuclear bodies (nuclear mesosomes). The two types show different cellular distributions. Especially the number of nuclear mesosomes is higher in competent than in noncompetent cells. This, and the observation that the increase and decrease of competence is correlated with both the number of cells carrying nuclear mesosomes and the number of nuclear mesosomes per cell, suggests that mesosomes are involved in the acquisition of competence.     

Distribution and dynamics of mitochondrial nucleoids in animal cells in culture

Mitochondrial (mt) nucleoids were visualized in living cells in culture by staining with the fluorochrome picoGreen. The cell types included a line derived from Xenopus heart endothelial cells (XTH-2), 3T3 cells, SV40-transformed 3T3 cells and primary cultures of Xenopus tadpole epidermis cells. In the permanent cell lines 6-60% of the mitochondria were found to be devoid of DNA. The peaks of the frequency distribution of mtDNA content, as revealed by microfluorometry, were not very distinct, indicating the presence of a high amount of aneuploid mt nucleoids. The maximum size of nucleoids (as derived from fluorescence intensity) was 10-12 times that of the minimum peak value in proliferating cell cultures. A linear ratio was found between the volume of the nucleoids and their DNA content, which is interpreted as a uniform package density. In terminally differentiating tadpole epidermis cells mitochondria form large bodies containing giant nucleoids, while in mitotic cells the mt nucleoids are small and of uniform size. Fusion and fission of the nucleoids were observed to occur either for no visible reason or in connection with fusion and fission events of the mitochondria.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

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.     

The effect of light on the number of chloroplast nucleoids in daughter cells of the algaScenedesmus quadricauda

Summary Synchronous cultures of the green algaScenedesmus quadricauda (Turp.) Bréb. grown at mean irradiances 25Wm^–2, 75Wm^–2, and 130Wm^–2 PhAR were exposed to different illumination regimes (ratio of light to dark interval varied from 2:22 to 24:0 hours). The populations of daughter cells released under these conditions differed markedly in their progress in the cell cycle. The cells from these populations were stained with DAPI and the shape, localization and number of chloroplast nucleoids were examined. The nucleoids were of spherical shape, divided asynchronously having dumbbell shape during fission. In the chloroplast, nucleoids were located symmetrically about the transverse axis of the cells. The mean number of nucleoids varied from two in the least developed daughter cells to 16 in the daughter cells of the highest developmental stage. The progress of these cells and thus also the number of nucleoids were proportional to the portion of the light energy amount which these daughter cells shared from the total light energy amount obtained by their mother cells.Abbreviations DAPI 4, 6-diamidino-2-diphenylindole - PhAR photosynthetically active radiation (400–700 nm)     

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.     

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.     

Multiplication and Differentiation of Plastid Nucleoids during Development of Chloroplasts and Etioplasts from Proplastids in Triticum aestivum

The morphological changes of plastid nucleoids (pt nucleoids)in the shoot apex and along the axis of the leaf blade in Triticumaestivum L. cv. Asakaze were followed with fluorescence microscopyafter staining with 4'6-diamidino-2-phenylindole (DAPI) andquantified with supersensitive microspectrophotometry. Proplastidsin the shoot apex contained 1–10 spherical pt nucleoids.These pt nucleoids changed to a row of spherical and cup-shapedpt nucleoids in sausage-shaped plastids at the leaf base inboth dark and light conditions, in which active cell divisionwas observed. These structures have a higher copy number ofplastid DNA (pt DNA) (72–78 copies) compared to proplastidsin the shoot apex (32–45 copies) and, therefore, may reflectthat active pt DNA synthesis is in progression. In the dark,the cup-shaped pt nucleoids in the spherical etioplasts, whichoriginated from the sausage-shaped plastids, grew to form ring-shapedpt nucleoids. Each ring-shaped pt nucleoid is sub-divided intosmaller pt nucleoids. Under continuous illumination, similarmorphological changes of pt nucleoids occurred except for distributionof small pt nucleoids into young chloroplasts as well as inmature chloroplasts. However, pt nucleotids of leucoplasts inepidermal and vascular bundle sheath cells did not show conspicuouschanges along the axis of the leaf blade. The significance ofthese observations is discussed in relation to plastid differentiationand to the plastid division cycle. ⁴ Present address: Faculty of Science, University of Tokyo,Hongo, Bunkyo-ku, Tokyo, 113 (Received August 15, 1989; Accepted April 13, 1990)     

哈氏噬纤维菌生活史中形态的变化

【目的】研究哈氏噬纤维菌Cytophaga hutchinsonii 在生活史中细胞形态的变化。【方法】利用光学显微镜、荧光显微镜和电子扫描显微镜对哈氏噬纤维菌生活状态进行详细观察。【结果】发现在饥饿状态下,长杆状菌体开始逐渐弯曲,菌体两端靠近成环形,环形菌体又进一步盘绕收缩成微小球形体,微小球形体在一定条件下能像生孢噬纤维菌的小孢囊一样萌发形成长杆状菌。另外还观察到哈氏噬纤维菌特殊的类核分裂现象。【结论】首次对哈氏噬纤维形成环形菌体和类似小孢囊的微小球形体的过程进行详细描述,为进一步揭示其形态变化与纤维素降解能力之间的关系提供依据。     

Division of chloroplast nucleoids and replication of chloroplast DNA during the cell cycle ofDunaliella salina grown under blue and red light

Summary Synchronous cultures of the algaDunaliella salina were grown in blue or red light. The relationships between replication of chloroplast DNA, cell size, cell age and the number of chloroplast nucleoids were studied. The replication of chloroplast DNA and the division of chloroplast nucleoids occurred in two separate periods of the chloroplast cycle. DNA replication was concomitant with that in the nucleocytoplasmic compartment but nucleoid division occurred several hours earlier than nuclear division. Red-light-grown cells were bigger and grew more rapidly than those grown in blue light. In newly formed daughter cells, the chloroplast nucleoids were small and spherical and they were localized around the pyrenoid. During the cell cycle they spread to other parts of the chloroplast. The number of DNA molecules per nucleoid doubled during DNA replication in the first third of the cell cycle but decreased several hours later when the nucleoids divided. Their number was fairly constant independent of the different light quality. Cells grown in red light replicated their chl-DNA and divided their nucleoids before those grown in blue light and their daughter cells possessed about 25 nucleoids as opposed to 15.Abbreviations DAPI 4,6-diamidino-2-phenylindole - chl-DNA chloroplast DNA - PAR photosynthetically active radiation     

Underlying regularity in the shapes of nucleoids of Escherichia coli: implications for nucleoid organization and partition

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).     

Studies on the compaction of isolated nucleoids from Escherichia coli

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.     

BEHAVIOR OF CHLOROPLAST NUCLEOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) IN SYNCHRONIZED CULTURE1

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12:12 h light: dark regimen. They increased in size during the light period, while nuclear division, chloroplast division and cytokinesis occurred during the dark period. Zoospores were liberated toward the end of the dark period. Changes in profile and distribution of chloroplast nucleoids were followed with a fluorescence Microscope after fixation with 0.1%(w/v) glutaraldehyde followed by staining with 4′.6-diamidino-2-phenylidole (DAPI), a DNA fluorochrome. About ten granular nucleoids were dispersed in the chloroplast at the beginning of the light period (0 h). Within 4 h the nucleoids aggregated around the pyrenoid giving a compact profile. The formation of the compact aggregate of cp-nucleoids around the pyrenoid occurred with maximal frequency twice during the light period. Toward the end of the light period the nucleoids were transformed into the form of threads interconnected with fine fibrils spreading throughout the chloroplast. Initially the thread-like nucleoids fluoresced only faintly. The fluorescence of some parts of the threadlike form became brighter over a period of 6 h; these nucleoids were divided into daughter chloroplasts during chloroplast division. Soon after chloroplast division, these thread-like nucleoids were transformed into about 20 granular forms, which were gradually combined to form about ten larger granular bodies in zoospores immediately prior to liberation from mother cells. Fixation of cells with glutaraldehyde at high concentrations or treatment of cells with protease significantly modified the profiles of DAPI-stained nucleoids. The different morphologies of chloroplast nucleoids are discussed in relation to changes in configuration of their protein components.     

Bacteria-shaped Gymnoplasts (Protoplasts) of Bacillus subtilis

Addition of glucose to the medium in which Bacillus subtilis was grown lowered the pH and increased the amount of lysylphosphatidylglycerol relative to the phosphatidylglycerol content of the membrane fraction. This change in phospholipid composition was accompanied by changes in the shape and behavior of the gymnoplasts obtained by cell wall removal with lysozyme. These gymnoplasts appeared to retain most of their original cell shape and internal organization, often with preservation of the mesosomes. Cells harvested from neutral growth medium gave the usual spherical gymnoplasts. In a hypotonic medium, the spherical gymnoplasts lysed rapidly, whereas the rod-like gymnoplasts lost only part of their cell content while showing a tendency to preserve the original shape. This type of gymnoplast could not be produced from cells grown in neutral medium by simply raising the magnesium concentration. When this was done the gymnoplasts assumed bizarre shapes; they became compact and susceptible to the tonicity of the medium. Gymnoplasts or protoplasts, produced from bacilli exposed to low pH values, were found not to conform to the formulations on "protoplasts" given in 1958 by 13 authors. Cells exposed to a low environmental pH during growth seemed to possess a more rigid membrane structure than cells grown at neutral pH.     

Transitional Forms of Corynebacterium acnes in Disease

A clear-cut triad of sequential Corynebacterium acnes transitional forms from disease has been discovered. This entity includes three major forms which are capable of stabilization in culture, the spherical, the intermediate, and the definitive C. acnes. During conversion or reversion among the three forms, a variety of forms with mixed characteristics was observed. The spherical form was gram-negative and osmotically fragile, but it possessed a vestigial cell wall and mesosomes which excluded it from the L forms. In lieu of the L-form designation, the term "transitional" was adopted for all forms leading up to the definitive C. acnes. Culture of the spherical form was successful only on Mycoplasma-type media. The intermediate form was gram-negative, had mixed spherical and filamentous morphology, and bore a striking resemblance to Streptobacillus moniliformis. Like the spherical form, it was nutritionally exacting. The definitive form of C. acnes was preceded by gram-positive transitional forms of C. acnes morphology. It lacked, however, the carbohydrases and proteinases of C. acnes and susceptibility to C. acnes bacteriophages. Reversion was often blocked at this stage. A series of blood cultures from a patient with endocarditis was studied. Postmortem stain sections of the heart-valve lesion included intracellular masses of gram-negative spherical organisms. Indirect fluorescent antibody staining of these masses was strongly positive with antiserum to the spherical form and weakly positive with antiserum to the intermediate form.