Fine Structure of Bacillus megaterium During Synchronous Growth

A fine-structure study of synchronously dividing Bacillus megaterium revealed the sequence of events involved in the division of the cell. First, a mesosome develops as a concentric fold of the plasma membrane at the site of septum formation. The mesosome contains membrane-bound vesicular structures, 300 to 500 A in diameter, plus a large membrane-bound structure, 2,000 A in diameter. These larger vesicles are peculiar to mesosomes in this stage of division and are not observed in the mesosomes involved in spore septum formation. The transverse septum originates within the mesosome and remains enclosed during its subsequent growth across the cell. An intimate association is observed between mesosome vesicles, mesosome membrane, and the growing edge of the transverse septum. Prior to completion of the septum, the membranes bounding the mesosome fuse, and further wall thickening occurs within the structure formed by this fusion. At this time, the septum only equals the parent cell wall in thickness. The doubling in thickness of the septum, which is required for the production of two normal daughter cell walls, occurs during a second phase of wall thickening, which is characterized by the appearance of a constriction at the base of the septum. As the constriction widens, the wall in this region thickens, forming the typical rounded poles of the daughter cells. Capsular synthesis at the poles occurs during this second phase of wall thickening. Throughout the division process, the nuclear material appears to be associated at one end with a mesosome at or near the pole of the cell and at the other end to the mesosome involved in septum formation. This association frequently takes the form of a stalklike extension of the mesosome penetrating into the chromatin fibrils.     

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.     

Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium

Ellar, D. J. (Syracuse University, Syracuse, N.Y.), and D. G. Lundgren. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium. J. Bacteriol. 92:1748-1764. 1966.-A study was made of the fine structure of sporulating cells of Bacillus cereus grown in a chemically defined medium. The developmental stages of sporulation occurred in a fairly synchronous manner and were complete by 14 hr. This time period was shortened when spore wall peptide components were added to the medium, but the addition had no effect upon fine structure except to thicken the cell wall. Sporulation could be separated into six morphological stages which generally agreed with those published for other sporulating bacteria. The initiation of the spore (forespore) septum takes the form of an inward folding of the cytoplasmic membrane toward the pole of the cell. The inward folding forms a characteristic Y-shaped membrane structure enclosing an area within which vesicles are found. These vesicles comprise the perisporal mesosome of the cell. The membranes on opposite sides of the cell progress toward the cell center where they fuse to form the double unit membrane of the spore septum. As the proliferation of the spore septum continues, the vesicular areas move towards the pole. The end result is a double forespore membrane which completely encloses a part of the vegetative cell's chromatin. Sporal mesosomes, as well as membrane vesicles, are involved in the proliferation of the forespore. Vesicles are generally bounded by a single unit membrane, whereas in the sporal mesosomes several unit membranes are arranged concentrically. The latter become associated with the segregation of a portion of the nuclear material into the forespore region of the cell.     

Fine Structure of Strictly Anaerobic,Non-Spore-Forming,Gram-Negative Bacilli

The cell division of a strain of Bacteroides convexus was examined by the ultrathin sectioning and the electron microscopy. The cell division was initiated by the invagination of the cytoplasmic membrane from the opposite sites at the middle of the cell. The constriction of the cell wall also occurred simultaneously or soon after the initiation of the invagination of the cytoplasmic membrane. A short septum structure similar to those of gram positive bacteria originated within the base of mesosome. The two mesosomes arising from the opposite sites fused at the center of the cell. After the tips of invaginating outer membrane reached to the middle between cell center and original outer membrane, the mesosomes were reduced gradually and finally disappeared. In this stage of the cell division, a transverse septum was usually completed. The invagination of the outer membrane proceeded progressively and finally fused at the center of the division plane.     

Fine structure of mesosomal involvement during Bacillus macerans sporulation.

The ultrastructure of endospore formation in Bacillus macerans ATCC 8244 is characterized by the examination of thin sections of cells grown synchronously in a defined medium. For the most part, sporulation in this organism proceeds as described in other Bacillus species. However, unusually extensive mesosomal involvement occurs during the early stages of sporulation, through the completion of engulfment. A large mesosome is associated with spore septum formation and a portion of this mesosome is included in the developing forespore. As engulfment continues, the forespore mesosome moves to the apex of the cell and participates in the completion of the double forespore membrane. This participation is morphologically similar to mesosome involvement in division and spore septation and seems to comprise a second sporal septation process. Based on this study, it is suggested that the mesosome functions to facilitate the "fusion" of membranes thought to occur during cell division and sporulation.     

Mesosomes in Escherichia coli

When Escherichia coli was grown in a synthetic medium and fixed with osmium, sections of the cells revealed clearly defined mesosomes. These mesosomes appeared to develop, in dividing cells, as coiled infoldings of the cytoplasmic membrane. Mature mesosomes formed a link between the cytoplasmic membrane and the nucleus of the cell. The arrangement of the mesosomes in dividing cells led to the hypothesis that division of the nucleus in these cells is accomplished by two separate polar mesosomes. One mesosome is derived from the parent cell and is present at one pole of the daughter cell. The other is freshly synthesized at or near the newly forming pole of the daughter cell. While the old mesosome remains attached to the chromosome received from the parent cell, the newly synthesized mesosome becomes attached to and initiates replication of the new chromosome. As the cell grows and elongates, the two mesosomes, attached to their respective chromosomes move apart, thus effecting nuclear division.     

Bacterial mesosomes: Method dependent artifacts

The occurrence of mesosomes was investigated during septum formation of vegetative and sporulating cells of Bacillus cereus. It has been demonstrated that bacterial mesosomes which are considered by numerous microbiologists as an integrated constituent of Gram positive bacteria, are in reality artifacts arising during the preparation for electron microscopy. The conventional fixation methods allowed enough time for the cytoplasmic membrane to react to the changed conditions and to form the typical pocket-like membrane invaginations. With cryofixation followed by freeze-substitution it was shown in ultrathin sections that mesosomes do not occur. The extremely rapid freezing and the substitution of the ice by an organic solvent containing the fixative prevented the formation of membraneous artifacts.Non-standard abbreviations OsO₄ osmium tetroxide - UO₂Ac uranylacetate - PHB poly--hydroxy-butyric acid - M mesosome - CW cell wall - CM cytoplasmic membrane - PF plasmatic fracture of the cytoplasmic membrane     

Mesosomal structures and antimicrobial activity induced by hemin oxidation or porphyrin photodynamic sensitization inStaphylococci

The porphyrin-dependent inactivation ofStaphyloccocus aureus and the induction of mesosomal structures are described. The antimicrobial activity of different photoactivated porphyrins was compared with the dark cytotoxic effect of hemin. Deuteroporphyrin, protoporphyrin, hematoporphyrin, and hematoporphyrin derivative (Hpd) markedly reduced cell growth upon irradiation with light. Photofrin II, the polymerized fraction of Hpd, and other synthetic porphyrins had no effect on staphylococcal growth. Hemin immediately inhibited cell viability in the dark and induced the development of an irregular cell wall, analyzed by scanning electron microscopy (SEM). Inside the cytoplasm a multivesicular mesosome was formed near the septum, as detected by transmission electron microscopy (TEM). The mesosomal volume and its frequency in the cells was increased in a time-dependent manner. The mesosome appearance was not related to fixation by glutaraldehyde or to post-fixation by osmium tetroxide. Glycosyl moieties stained by ruthenium red revealed the formation of membrane-like structures in the mesosome. It is concluded that oxygen-dependent reactions potentiated by porphyrins may induce disturbances in the synthesis of staphylococcal membrane and cell wall, revealed as mesosomes.     

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.     

Morphokinetic Reaction of Cells of Streptococcus faecalis (ATCC 9790) to Specific Inhibition of Macromolecular Synthesis: Dependence of Mesosome Growth on Deoxyribonucleic Acid Synthesis

The application of quantitative electron microscopy to thin sections of cells of Streptococcus faecalis specifically inhibited for deoxyribonucleic acid (DNA), ribonucleic acid, and protein synthesis shows that septal mesosomes (i) increase in size when protein synthesis is inhibited by at least 80% while DNA synthesis proceeds at no less than 50% of the control rate and (ii) decrease in size when DNA synthesis is inhibited 50% or more during the initial 10 min of treatment. This indicates that fluctuations in mesosome size are dependent on the extent of DNA synthesis. The fluctuations in mesosome areas observed on treatment do not correlate with the kinetics of glycerol incorporation per milliliter of a culture. However, when glycerol incorporation is placed on a per cell basis, a strong correlation is observed between increases in (i) the thickness of the electron-transparent layer of the cytoplasmic membrane and (ii) the amount of glycerol incorporated per cell. It seems that the electron-transparent membrane layer may thicken to accommodate changes in lipid content when protein and lipid synthesis are uncoupled.     

Effect of chromosomal breaks induced by x-irradiation on the number of mesosomes and the cytoplasmic organization of Streptococcus faecalis

A model which explains mesosome formation via a contraction of the cytoplasm and nucleoid when bacteria are physiologically disturbed was tested by: (1) X-irradiation of unfixed cells of Streptococcus faecalis to produce chromosomal breaks and to remove DNA attached to the cell membrane; (2) subsequent determination of the number of irradiated cells in which mesosomes (using electron microscopy) and central density changes (using phase-contrast microscopy) could be visualized after fixative was added. Results obtained by exposure of cells to doses up to 1100 krads before fixation indicated that: (1) the number of cells with central mesosomes was reduced proportional to the decrease in the molecular weight of the DNA due to double-strand breaks: (2) the number of cells with total (central plus peripheral) mesosomes and the number of cells with peripheral mesosomes were both reduced proportional to the removal of DNA attached to the cell membrane (M band); (3) the nucleoid became more diffusely organized. Exposure of cells to doses greater than 1100 krads before fixation resulted in: (1) an increase in the number of cells with central and peripheral mesosomes (compared to cells exposed to lower dosages); (2) a return to the centralized, dense nucleoid seen in unirradiated cells.These results suggest that mesosomes are formed when localized sites on the cell membrane are pulled from close contact with the cell wall into the cytoplasm by the action of a cross-linking fixative via the aggregation of intracytoplasmic components such as DNA. This model considers the attachment of DNA and/or other cytoplasmic components to the membrane as an intrinsic part of its mechanism. The formation of central and peripheral mesosomes in unirradiated and X-irradiated cells are contrasted.     

Mesosomes associated with hydrogen peroxide in bacteria

Mesosomes are unique membranous structures in bacteria. It is recognized that the mesosomes should be involved in several fundamental processes. The structure and behaviour of mesosomes have been studied and largely identified, while new evidences of mesosome function have been strikingly obtained. Our previous studies confirmed that hydrogen peroxide is involved in mesosomes formation during cell injury and cell division processes. Mesosome formation is always accompanied by excessive H₂O₂ accumulation. Furthermore, our recent data showed that mesosomes could not only enrich the excess H₂O₂, but also bring the H₂O₂ outside of the cells injured by antibiotics. It is a possibility that the enrichment of H₂O₂ in mesosomes might be a mechanism of drug resistance of bacteria. This article describes the bacterial mesosome and its functions as well as the involvement of hydrogen peroxide in mediating these functions.     

Use of Fluorouracil-Uracil Combinations to Study Growth Accompanied by Insufficient Deoxyribonucleic Acid Synthesis in Bacillus cereus

5-Fluorouracil (FU) at a concentration of 16 muM almost totally inhibited deoxyribonucleic acid (DNA) synthesis and cell division by Bacillus cereus, whereas growth continued at an exponential rate (25% of control for at least 3 h). In cultures simultaneously given 160 muM uracil (U) along with the FU, DNA synthesis still stopped, but cell division continued for one generation at the control rate and at a much slower rate beyond that; in the meantime, cell mass continued to increase at an essentially normal rate. The cells in cultures treated with FU or FU plus U were elongated and contained about half of the control content of DNA, with one nuclear area per cell instead of two. Loss of cloning ability, unlike mass increase, was always correlated with the continuing inhibition of DNA synthesis, in either FU- or U plus FU-treated cultures.     

Ampicillin induced septum formation in Bacillus cereus

Cells of Bacillus cereus grown in the presence of subinhibitory concentrations of ampicillin at either 30 degrees or 45 degrees C exhibited an increase in the numbers of centres of septum formation per unit cell length. Under identical conditions of cultivation, cells of Escherichia coli grew as aseptate filaments. In general, untreated B. cereus cells grown at 45 degrees C were longer than those grown at 30 degrees C. The strain of E. coli used was unaffected in terms of filamentation by elevated growth temperature. Results are discussed in terms of the presence and availability of penicillin binding proteins and autolysins involved in cell growth, division and separation.     

Mesosome Structure in Chromobacterium violaceum

Exponentially growing cells of the gram-negative bacterium Chromobacterium violaceum demonstrate invaginations of the cytoplasmic membrane with a high frequency. These invaginations conform to the ultrastructural appearance of mesosomes of gram-positive bacteria. As many as four mesosomes are observed per cell, each of which may increase the total membrane surface of the cell by 30%. Washing of cells in dilute tris(hydroxymethyl)aminomethane buffer effects a distension of the mesosome "neck" and/or cytoplasmic membrane clarifying the association of the mesosome to the cytoplasmic membrane. Plasmolysis effects an eversion of the mesosome into the plasmolysis vacuole.     

Morphological effect of cerulenin treatment on Streptococcus faecalis as studied by ultrastructure reconstruction.

Exponential-phase cells of Streptococcus faecalis ATCC 9790 were treated with a concentration of cerulenin (5 micrograms/ml) that has been shown to block both lipoteichoic acid and lipid synthesis and cell division within 10 min. The morphological effect of this treatment was studied by making three-dimensional reconstructions of cells based on measurements taken from axial thin sections. This analysis indicated that cerulenin interferes with cell division by inhibiting normal constriction of the division furrow and centripetal growth of the cross wall in envelope growth sites. Rather than dividing, many of the sites in treated cells apparently continue to elongate and produce abnormally large amounts of peripheral wall surface. These observations were interpreted in terms of a previously proposed model in which cerulenin would prevent the synthesis of a lipid-containing inhibitor of autolytic enzyme activity needed for division. In addition, measurements showed that the average number of envelope growth sites per cell increased during treatment, suggesting that although cerulenin treatment blocks division, it does not interfere with the formation of new envelope growth sites. It was also observed that the size and frequency of mesosomes did not decline during the 60-min period of drug treatment. This tends to decrease the likelihood that mesosomes are formed from a pool of intracellular membrane precursors that would be depleted during a period of restricted lipid biosynthesis.     

An essential enzyme for phospholipid synthesis associates with the Bacillus subtilis divisome

The mechanism by which the membrane synthetic machinery might be co‐organized with the cell‐division architecture during the bacterial cell cycle remains to be investigated. We characterized a key enzyme of phospholipid and fatty acid synthesis in Bacillus subtilis, the acyl–acyl carrier protein phosphate acyltransferase (PlsX), and identified it as a component of the cell‐division machinery. Comprehensive interaction analysis revealed that PlsX interacts with FtsA, the FtsZ‐anchoring protein. PlsX mainly localized at the potential division site independent of FtsA and FtsZ and then colocalized with FtsA. By multidirectional approaches, we revealed that the Z‐ring stabilizes the association of PlsX at the septum and pole. The localization of PlsX is also affected by the progression of DNA replication. PlsX is needed for cell division and its inactivation leads to aberrant Z‐ring formation. We propose that PlsX localization is prior to Z‐ring formation in the hierarchy of septum formation events and that PlsX is important for co‐ordinating membrane synthesis with cell division in order to properly complete septum formation.     

Revisiting the Mesosome as a Novel Site of Hydrogen Peroxide Accumulation in Escherichia coli

The major source of endogenous hydrogen peroxide is generally thought to be the respiratory chain of bacteria and mitochondria. In our previous works, mesosome structure was induced in cells during rifampicin effect, and the mesosome formation is always accompanied by excess hydrogen peroxide accumulation in bacterial cells. However, the underlying mechanisms of hydrogen peroxide production and the rationale behind it remain still unknown. Here we report that hydrogen peroxide can specifically accumulate in the mesosome in vitro. Mesosomes were interpreted earlier as artifacts of specific cells under stress through TEM preparation, while, in the current study, mesosomes were shown as intracellular compartments with specific roles and features by using quickly freezing preparation of TEM. Formation of hydrogen peroxide was observed in suspension of mesosomal vesicles by using either a fluorescence-based reporter assay or a histochemical method, respectively. Our investigation provides experimental evidence that mesosomes can be a novel site of hydrogen peroxide accumulation.     

Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil

Reich, Melvin (The George Washington University School of Medicine, Washington, D.C.), and H. George Mandel. Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil. J. Bacteriol. 91:517-523. 1966.-5-Fluorouracil (FU) produced a marked inhibition of growth and deoxyribonucleic acid (DNA) synthesis in Bacillus cereus 569H. Protein and ribonucleic acid (RNA) synthesis were not specifically inhibited, and proceeded at the rate of turbidometric increase of the cells. Cell-wall synthesis, respiration, and penicillinase production continued in the presence of FU at essentially the control rate. The addition of equimolar concentrations of uracil and FU prevented growth inhibition but did not restore DNA synthesis. The addition of thymidine with FU did not relieve growth inhibition but did restore the DNA content to normal. Thymidine supplementation also increased the quantity of FU, but not uracil, incorporated into RNA and the acid-soluble fraction. The data indicate that inhibition of growth can be dissociated from inhibition of DNA synthesis and that more DNA is present in normal cells than is needed for growth and reproduction.