The effect of rifampicin on the developmental phases of germinating spores of Clostridum sp., MSp+.

The effect of rifampicin on the developmental phases of germinating spores of Clostridium botulinum, MSp+, has been studied. At sublethal concentrations of rifampicin (0.05 ng/ml) the time periods required for outgrowth and vegetative growth was significantly prolonged because of the inhibition of RNA and protein synthesis. However, rifampicin had essentially no effect on DNA synthesis or on subsequent spore formation. Chemical analyses showed that the amount of protein present in vegetative cells of the rifampicin-treated cultures was twice as great as in the untreated cultures but the total protein content of endospores was the same in both cases. It was revealed in ultrastructural studies of rifampicin (0.1 ng/ml) treated cultures, examined after 22 h, that septum formation and normal cell division of the emerging cell was blocked and a few cells showed constriction which produced one normal and one protoplast-like daughter cell.     

Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site

The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.     

Co-ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly

Spores of a thymine-requiring strain of Bacillus subtilis 168, which is also temperature sensitive for the initiation of chromosome replication, were germinated and allowed to grow out at the permissive temperature in a minimal medium containing no added thymine. Under these conditions, there was no or very limited progression into the elongation phase of the first round of replication. In a significant proportion of the outgrown cells, a Z ring formed precisely at mid-cell and over the centrally positioned nucleoid, leading eventually to the formation of a mature division septum. When initiation of the first round of replication was blocked through a temperature shift and with thymine present, the Z ring was positioned acentrally. The central Z ring that formed in the absence of thymine was blocked by the presence of a DNA polymerase III inhibitor. It is concluded that the very early stages of a round of replication (initiation plus possibly limited progression into the elongation phase) play a key role in the precise positioning of the Z ring at mid-cell and between replicating daughter chromosomes.     

Division septation in the absence of chromosome termination in Bacillus subtilis.

Germinating spores of the temperature-sensitive DNA initiation mutants of Bacillus subtilis, TsB134 and dna-1(Ts), were allowed to undergo a single round of replication by shifting to the restrictive temperature shortly after its initiation. To monitor the progress of the round 5-bromouracil was added at various times and DNA extracted after a further time, sufficient to allow completion of the chromosome. Average replication was measured from the relative amounts of LL and LH material in Cs₂SO₄ gradients. The replication state of origin (purA), intermediate (leuA) and terminus (metB) markers at the times of 5-bromouracil addition were obtained from genetic analysis of the density species fractionated in gradients of CsCl.The DNA replication inhibitor, 6-(p-hydroxyphenylazo)-uracil (HPUra), was added at various stages of the single round and the outgrown cells examined at later times for the frequency and type of septation. Under the conditions of the experiment, central division septation was blocked if HPUra (20 μm) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrations of HPUra, 40 and 100 μm, it was shown that central division septation would occur at about its normal time if replication was blocked after this 70% stage but before termination. In these circumstances there was a distinct tendency for the DNA to remain close to the septum on both sides of it. The B. subtilis spore contains a single chromosome, which means that the central septum that forms in the absence of termination must pass through a partially completed chromosome. Electron microscopic evidence for such a situation has already been described (Van Iterson &; Aten, 1976). It is concluded that, at least under the restrictive conditions of the present experiments, termination of chromosome replication is not obligatory for the formation of the division septum with which it is normally coupled.     

Sporulation in Bacillus subtilis. Effect of medium on the form of chromosome replication and on initiation to sporulation in Bacillus subtilis

Thymine-requiring mutants of Bacillus subtilis and mutants that are temperature-sensitive for initiation of chromosome replication have been used to study the relationship between sporulation and chromosome formation. The DNA synthesis that normally occurs when cells are transferred to sporulation medium is essential for spore induction. This is shown by the fact that thymine-starved cells are unable to form spores and are unable to perform even the earlier steps of sporulation, such as septum formation or synthesis of alkaline phosphatase. The nature of the medium in which the cells are growing while the DNA is being completed is also important because it determines both the shape and the position of the daughter chromosomes. If the cells are in a rich medium, the newly synthesized chromosomes are discrete and compact bodies: the cells are primed for growth, and sporulation cannot be induced by transferring them at this stage to a spore-inducing medium. If DNA synthesis was completed with the cells in a poor medium the daughter chromosomes, by the time DNA synthesis has ceased, are spread in a single filamentous band and the cells are morphologically already in stage I of sporulation.     

Effects of antibiotics on synthesis and persistence of sigma E in sporulating Bacillus subtilis.

A potential regulatory link between the activation of a sporulation-specific sigma factor (sigma E) and forespore septum formation was investigated by treating Bacillus subtilis with inhibitors of protein or peptidoglycan synthesis and monitoring the consequences of these treatments on sigma E activation and septation. Western blot (immunoblot) and electron microscopic analyses revealed that both the formation of sigma E and septation were inhibited to a similar degree when either rifampin or chloramphenicol was added at different times before the second hour into sporulation but that penicillin preferentially blocked septation. We interpret these results as indicating that the syntheses of the gene products for both septation and sigma E activation occur at approximately the same time in development but that synthesis of an intact septum is unlikely to be a prerequisite for the formation of sigma E. We observed that penicillin could not only block septation but, depending on the time of its addition, could also inhibit both the activation of sigma E and the synthesis of its precursor. The basis of this effect is unknown, but it is not due to an overall disruption of protein synthesis. The incorporation of [35S] methionine by the sporulating cultures was unaffected by penicillin treatment. A time course study of the effects of rifampin and chloramphenicol treatments on sigma E levels revealed that both the synthesis of sigma E and its disappearance from sporulating cultures is inhibited by these antibiotics. This suggests that ongoing macromolecular synthesis is required for the turnover of sigma E.     

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.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

Ultrastructural Explanation for Snapping Postfission Movements in Arthrobacter crystallopoietes

The ultrastructure of dividing rod-stage cells of Arthrobacter crystallopoietes was examined by electron microscopy. The cell walls consist of two layers. During cell division, the inner layer invaginates to form the septum. The outer layer does not participate in septum formation. After septum formation is completed, the two daughter cells remain attached by the outer layer of the cell wall. It appears that localized rupture of the outer layer during further wall growth is responsible for the phenomenon known as "snapping division" or "snapping postfission movement."     

Effect of Sodium Nitrite, Sodium Chloride, and Sodium Nitrate on Germination and Outgrowth of Anaerobic Spores

The effects of meat-curing agents on germination and outgrowth of putrefactive anaerobe 3679h (PA 3679h) spores were studied in microcultures. Nitrite concentrations up to 0.06% at pH 6.0 or between 0.8 and 1% at pH 7.0 allowed emergence and elongation of vegetative cells but blocked cell division. The newly emerged cells then lysed. With more than 0.06% nitrite at pH 6.0 or more than 0.8 to 1% at pH 7.0, the spores lost refractility and swelled, but vegetative cells did not emerge. Even as much as 4% nitrite failed to prevent germination (complete loss of refractility) and swelling of the spores. Sodium chloride concentrations above 6% prevented complete germination (i.e., the spores retained a refractile core). In the presence of 3 to 6% sodium chloride, most of the spores germinated and produced vegetative cells, but cell division was often blocked. Sodium nitrate had no apparent effect on germination and outgrowth at concentrations up to 2%.     

Timing and other features of the action of the ts1 division initiation gene product of Bacillus subtilis.

The ts1 division initiation mutation of Bacillus subtilis 160 was transferred into a thymine-requiring strain of B. subtilis 168. Aspects of the role and timing of the action of the ts1 gene product in relation to septum formation were studied by comparing the behavior of this new strain with that of the isogenic wild type after outgrowth of germinated spores. The ts1 gene product was shown to be required for the asymmetric division which occurs in the absence of chromosome replication, in addition to normal division septation. The time interval between completion of the action of the ts1 gene product and initiation of the first central division septum was estimated to be less than 4 min at 34 degrees C, and it is possible that an active ts1 gene product is required until the commencement of septal growth. Recovery of septa after transfer of outgrown spores (filaments) from the nonpermissive to the permissive temperature was also examined. During recovery, septa formed at sites which were discrete fractional lengths of the filaments, with the first septum located at the most polar of these sites. The data have been interpreted in terms of the formation of potential division sites at the nonpermissive temperature and the preferred utilization, upon recovery, of the most recently formed site. Recovery of septa at the permissive temperature occurred in the absence of DNA synthesis but was blocked completely by inhibitors of RNA and protein synthesis. It is possible that the only protein synthesis required for recovery of septa is that of the ts1 gene product itself.     

Replication through the terminus region of the Bacillus subtilis chromosome is not essential for the formation of a division septum that partitions the DNA.

Germinated and outgrowing spores of a temperature-sensitive DNA initiation mutant of Bacillus subtilis were allowed to initiate a single round of replication by being shifted from 34 to 47 degrees C at the appropriate time. The DNA replication inhibitor 6-(parahydroxyphenylazo)-uracil was added to separate portions of the culture at various times during the round. Samples were collected from each around the time of the first division septation for measurements of the extent of the round completed, the level of division septation, the position of the septum within the outgrown cell, and the distribution of DNA (nucleoid) in relation to the septum. The extent of replication was measured directly through a hybridization approach. The results show clearly that a central division septum can close down onto a chromosome that is only partially replicated (to a minimum extent of about 60% of the round) such that DNA appears on both sides of the septum and frequently very close to it. It is concluded, as claimed previously on the basis of a less direct approach (T. McGinness and R.G. Wake, J. Mol. Biol. 134:251-264, 1979), that replication through the terminus region of the chromosome is not essential for the formation of a division septum that partitions the DNA.     

SpoIIIE and a novel type of DNA translocase, SftA, couple chromosome segregation with cell division in Bacillus subtilis

Cell division must only occur once daughter chromosomes have been fully separated. However, the initiating event of bacterial cell division, assembly of the FtsZ ring, occurs while chromosome segregation is still ongoing. We show that a two-step DNA translocase system exists in Bacillus subtilis that couples chromosome segregation and cell division. The membrane-bound DNA translocase SpoIIIE assembled very late at the division septum, and only upon entrapment of DNA, while its orthologue, SftA (YtpST), assembled at each septum in B. subtilis soon after FtsZ. Lack of SftA resulted in a moderate segregation defect at a late stage in the cell cycle. Like the loss of SpoIIIE, the absence of SftA was deleterious for the cells during conditions of defective chromosome segregation, or after induction of DNA damage. Lack of both proteins exacerbated all phenotypes. SftA forms soluble hexamers in solution, binds to DNA and has DNA-dependent ATPase activity, which is essential for its function in vivo . Our data suggest that SftA aids in moving DNA away from the closing septum, while SpoIIIE translocates septum-entrapped DNA only when septum closure precedes complete segregation of chromosomes.     

THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation.     

Ultrastructure of the Cell Wall and Cell Division of Unicellular Blue-green Algae

The fine structure of the cell wall and the process of cell division were examined in thin sections of two unicellular blue-green algae grown under defined conditions. Unilateral invagination of the photosynthetic lamellae is the first sign of cell division in the rod-shaped organism, Anacystis nidulans. Symmetrical invagination of the cytoplasmic membrane and inner wall layers follows. One wall layer, which appears to be the mucopolymer layer, is then differentially synthesized to form the septum; the outer wall layers are not involved in septum formation. Centripetal splitting of the inner layer separates the two daughter cells. A second division, in a plane parallel to the first, usually occurs before the first daughter cells are separated. In the coccoid organism, Gleocapsa alpicola, the features of cell division are broadly similar; however, unilateral invagination of the lamellae is not observed and the second division takes place in a plane perpendicular to the plane of the previous division.     

The "Point of no Return" Concept in Cell Division. The Effects of Some Metabolic Inhibitors on Synchronized Chlorella pyrenoidosa

The coarse of growth and cell division in synchronized cultures of Chlorella pyrenoidosa was studied after the addition of metabolic inhibitors at differing times during the cell cycle (14 h light - 10 h darkness with nitrate as nitrogen source. 12 h light: 12 h darkness with urea as nitrogen source). Dinitrophenol (DNP) added to a final concentration of 0.3 mM at any time in the synchronization cycle, the compound remaining in the suspension from the time of addition to the end of the dark period, inhibited spore formation completely. Growth measured as increase in cell volume was less sensitive to the action of the inhibitor. Chloramphenicol (CAP) added dining the 0–5 h interval to a final concentration of 0.1 mM resulted in 80 per cent inhibition of cell division. Similar treatment started at successive times thereafter resulted in a gradual decrease of the inhibition. Treatment at the 14th hour and during the dark period did not affect the sporulation. Similar experiments with 0.9 mM puromycin added at various times during the illumination period gave almost complete inhibition of cell division, while the growth was reduced by only 25 per cent. para-Fluorophenylalanine (p-FPhe) at 3.3 × 10^?2 mM stopped cell division nearly completely irrespective of addition time in the light period. Addition during the dark period also prevented an increase in the number of tree cells. In this case about half of the cells produced spores which were not released. It is concluded that DNP inhibits all stages of preparation for cell division, as well as the division process itself. With CAP a genuine transition point of preparation for cell division was observed, although its interpretation as related to protein synthesis is somewhat uncertain. With puromycin and p-FPhe no transitions were observed.     

Effects of microtubule-inhibitors on nuclear migration and rhizoid differentiation in germinating fern spores (Onoclea sensibilis)

Summary Germinating spores of the sensitive fern,Onoclea sensibilis L., undergo premitotic nuclear migration before a highly asymmetric cell division partitions each spore into a large protonemal cell and a small rhizoid initial. Nuclear movement and subsequent rhizoid formation were inhibited by the microtubule (MT) inhibitors, colchicine, isopropyl-N-3-chlorophenyl carbamate (CIPC) and griseofulvin. Colchicine prevented polar nuclear movement and cell division so that spores developed into enlarged, uninucleate single cells. CIPC and griseofulvin prevented nuclear migration, but not cell division, so that spores divided into daughter cells of approximately equal size. In colchicine-treated spores, MT were not observed at any time during germination. CIPC prevented MT formation at a time coincident with nuclear movement in the control and caused a disorientation of the spindle MT. Both colchicine and CIPC appeared to act at a time prior to the onset of normal nuclear movement. The effects of colchicine were reversible but those of CIPC were not. Cytochalasin b had no effect upon nuclear movement or rhizoid differentiation. These results suggests that MT mediate nuclear movement and that a highly asymmetric cell division is essential for rhizoid differentiation.     

Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif, xerC and xerD) of Escherichia coli

We have studied the growth and division of xerC, xerD and dif mutants of Escherichia coli, which are unable to resolve dimer chromosomes. These mutants express the Dif phenotype, which includes reduced viability, SOS induction and filamentation, and abnormal nucleoid morphology. Growth was studied in synchronous cultures and in microcolonies derived from single cells. SOS induction and filamentation commenced after an apparently normal cell division, which sheared unresolved dimer chromosomes. This has been called guillotining. Microcolony analysis demonstrated that cell division in the two daughter cells was inhibited after guillotining, and microcolonies formed that consisted of two filaments lying side by side. Growth of these filaments was severely reduced in hipA+ strains. We propose that guillotining at dif destroys the expression of the adjacent hipBA genes and, in the absence of continued formation of HipB, HipA inhibits growth. The length of the filaments was also affected by SfiA: sfiA dif hipA mutants initially formed filaments, but cell division at the ends of the filaments ultimately produced a number of DNA-negative cells. If SOS induction was blocked by lexA3 (Ind-), filaments did not form, and cell division was not inhibited. However, pedigree analysis of cells in microcolonies demonstrated that lethal sectoring occurred as a result of limited growth and division of dead cells produced by guillotining.     

The price of independence: cell separation in fission yeast

The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise.     

Prospore Membrane Formation Defines a Developmentally Regulated Branch of the Secretory Pathway in Yeast

Spore formation in yeast is an unusual form of cell division in which the daughter cells are formed within the mother cell cytoplasm. This division requires the de novo synthesis of a membrane compartment, termed the prospore membrane, which engulfs the daughter nuclei. The effect of mutations in late-acting genes on sporulation was investigated. Mutation of SEC1, SEC4, or SEC8 blocked spore formation, and electron microscopic analysis of the sec4-8 mutant indicated that this inability to produce spores was caused by a failure to form the prospore membrane. The soluble NSF attachment protein 25 (SNAP-25) homologue SEC9, by contrast, was not required for sporulation. The absence of a requirement for SEC9 was shown to be due to the sporulation-specific induction of a second, previously undescribed, SNAP-25 homologue, termed SPO20. These results define a developmentally regulated branch of the secretory pathway and suggest that spore morphogenesis in yeast proceeds by the targeting and fusion of secretory vesicles to form new plasma membranes in the interior of the mother cell. Consistent with this model, the extracellular proteins Gas1p and Cts1p were localized to an internal compartment in sporulating cells. Spore formation in yeast may be a useful model for understanding secretion-driven cell division events in a variety of plant and animal systems.