Mutant of Escherichia coli with Thermosensitive Protein in the Process of Cellular Division

A new thermosensitive mutant of Escherichia coli deficient in cell division was isolated by means of membrane filtration after nitrosoguanidine mutagenesis. The mutant cells grow normally at 30 C but stop dividing immediately after shift to 42 C, resulting in multinucleated filaments lacking septa. The number of colony-forming units does not decrease for at least 6 hr at 42 C. The maximum length of the filaments is 10 to 16 times that of normal cells. Addition of a high concentration of NaCl fails to stimulate cell division at 42 C. The filaments formed at 42 C divide abruptly 30 min after shift to 30 C, and synchronous increase of cell number is shown for 3 hr. The macromolecular synthesis of protein and nucleic acids at 42 C is normal on the whole. The cell division shown after the shift from 42 to 30 C is observed in the absence of thymine, but not in the presence of chloramphenicol or in a medium deficient in amino acids. However, the filament can divide to some extent in the presence of chloramphenicol if some protein synthesis is allowed to proceed at 30 C before the addition of the antibiotic. The elongated cells divide at 42 C provided that they are exposed to 30 C before being shifted to high temperature.     

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments.

Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the premissive temperature, all mutants, particularly the pbpB and ftsQ mutants, showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42 degrees C, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times LO, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. The ftsE mutant continued to constrict and divide at 42 degrees C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42 degree C, chains of cells, which eventually swelled up, were formed. Although the ftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions.     

Rule governing the division pattern in Escherichia coli minB and wild-type filaments.

Escherichia coli minB mutants form anucleate minicells and multinucleate filaments. We show here that the overwhelming majority of nucleate cells contain 2n (n = 0, 1, 2, ...) nucleoids, as determined by 4',6-diamidino-2-phenylindole staining, and 2n (n = 1, 2, 3, ...) copies of the replication origin, as determined by flow cytometry. This shows that division sites are not chosen randomly among the available sites in minB filaments. Similarly, wild-type cells contain 2n nucleoids, both during cell division inhibition and when furazlocillin-induced filaments are allowed to divide. We conclude that the min+ function is only to prevent septation only at polar sites; the placement of internal cell division sites must obey strict rules, which are the same in minB and wild-type cells.     

Sodium dodecyl sulfate-sensitive septation in a mitomycin C-sensitive, mtc, mutant of Escherichia coli.

A mitomycin C-sensitive, mtc, mutant of Escherichia coli has an altered cell surface and is sensitive to sodium dodecyl sulfate (SDS). The mutant, M27, formed multinucleate nonseptated filaments in the presence of a low concentration of SDS (50 microgram/ml). When the culture grown at that concentration of SDS was diluted with an SDS-free medium, the filaments began to divide at a very rapid rate after a lag of about 20 min. Chloramphenicol inhibited this recovery division when added within 10 min after SDS dilution but did not inhibit the division when added 20 min after dilution. Penicillin G at a low concentration, which is enough to cause filamentation, had virtually no effect on the recovery division of SDS-induced filaments. The division of penicillin G-induced filaments was inhibited by SDS.     

Repair of radiation-induced damage to the cell division mechanism of Escherichia coli

Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), William D. Fisher, Alice A. Hardigree, and George E. Stapleton. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. Bacteriol. 91:737-742. 1966.-Microscopic observations of irradiated populations of filamentous Escherichia coli cells indicated that filaments can be induced to divide by a substance donated by neighboring cells. We have made this observation the basis for a quantitative technique in which filaments are incubated in the presence of nongrowing donor cells. The presence of "donor" organisms promotes division and subsequent colony formation in filaments. "Donor" bacteria do not affect nonfilamentous cells. An extract of "donor" cells retains the division-promoting activity. The extract has been partially fractionated, and consists of a heat-stable and a heat-labile component. The heat-stable component is inactive in promoting cell division, but enhances the activity of the heat-labile component. The division-promoting system is discussed as a radiation repair mechanism and as a normal component of the cell division system in E. coli.     

Characterization of osmotically induced filaments of Salmonella enterica

Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.     

Effects of the ccd function of the F plasmid on bacterial growth.

The ccd segment of the mini F plasmid containing the ccdA and ccdB genes controls the coordination between plasmid proliferation and cell physiology and fate. When the DNA replication of a thermosensitive-replication plasmid carrying the ccd segment of mini F is blocked, plasmid DNA molecules are progressively diluted through cell division until the copy number reaches 1 per cell. From this time on, there is little increase in the number of viable cells, although cells continue to divide, resulting in a mixed population of viable cells (mostly plasmid containing), nonviable but residually dividing cells, and nonviable nondividing cells. Results are presented suggesting that plasmid-containing cells are viable and continue to divide, whereas plasmid-free segregants are nonviable and form filaments after a few residual divisions, with DNA synthesis reduced or arrested in the filaments. Although the ccd functions are known to induce the SOS response when plasmid replication is blocked, the production of nonviable plasmid-free segregants is independent of the SOS cell division inhibition mechanism determined by the sfiA and sfiC genes.     

Mechanism for the Regulation of Cell Division in Agmenellum

We describe a nonlethal temperature-conditional mutant of Agmenellum quadruplicatum which allows dissociation of the processes of growth and cell division. With this system, evidence has been obtained for the regulation of one step in the process of cell division by a small effector molecule. The effector molecule is apparently released into the surrounding medium and can be obtained from lyophilized spent medium by extraction with 80% ethanol. The addition of this extract to serpentine filaments of the SN29 mutant strain stimulates cell division in these filaments and leads to the production of cells approximating normal dimensions within one generation time. The degree of stimulation of cell division is directly related to the amount of extract added. A general hypothesis is presented for the positive regulation of the initiation of cell wall and cell membrane invagination in this organism.     

Actin filaments line up acrossTradescantia epidermal cells,anticipating wound-induced divison planes

Summary This paper describes the role of actin filaments in setting up the phragmosome — the transvacuolar device that anticipates the division plane — and in forming a supracellular system that seems to override cell boundaries. Tradescantia leaf epidermal cells were induced to divide by wounding the leaf. New division planes formed parallel to slits, and encircled puncture wounds — the new division planes lining up across cells, instead of the joints being off-set as in normal, unwounded tissue. Within 30 min after wounding, rhodamine phalloidin staining showed that a belt of fine, cortical actin filaments formed parallel to the wound. In the next stage, migration of nuclei to a wall adjacent to the wound, involved pronounced association of actin filaments with the nucleus. Migration could be inhibited with cytochalasin D, confirming the role of actin in traumatotaxis. Later still, actin strands were seen to line up from cell to cell, parallel to the wound, anticipating the future division plane. Next, actin filaments accumulated in this anticlinal plane, throughout the depth of the cell, thereby contributing to the formation of the phragmosome. The phragmosome has been shown in previous work (Flanders et al. 1990) to contain microtubules that bridge nucleus to cortex, and is now found to contain actin filaments. Actin filaments are therefore involved in the key stages of nuclear migration and division plane alignment. The supracellular basis of actin alignment is discussed.Dedicated to the memory of Professor Oswald Kiermayer     

Cell Division in Escherichia coli: Analysis of Double Mutants for Filamentation and Abnormal Septation of Deoxyribonucleic Acid-Less Cells

The link between chromosome termination, initiation of cell division, and choice of division sites was studied in Escherichia coli by preparing double mutants. Hybrid mutants containing div52-ts, a cell division initiation mutation, and min, mutations which affect the choice of division sites resulting in the septation of minicells, were characterized. The mutants produced minicells and normal cells coordinately under all conditions studied, although the fraction of minicells is half that of the parental minicell strain. The mutant gradually stopped dividing at both the median and minicell septation sites when transferred from 30 to 41 C in rich medium. A synchronous cell division of filaments was induced 15 min after addition of chloramphenicol to the medium, even at 41 C. Divisions were observed at both normal and minicell sites. These results indicate that div52-ts and min functions share a common step in a cell division pathway. A double mutant containing div52-ts and div27-ts, a dnaB mutant which divides in the absence of DNA synthesis, was characterized. The mutant continues to divide after a shift to the high temperature, although at a reduced rate. The behavior of this hybrid mutant suggests a hypothesis that the chromosome termination signal and div52-ts division initiation signal act on a single membrane site which is altered in div27-ts strains.     

Strain-specific difference that affects the inhibition of division of Escherichia coli filaments by chloramphenicol.

The Escherichia coli mutations ts1882 and ts2158 cause temperature-sensitive septum formation and result in growth of cells as long, multinucleate, nonseptate filaments at 42 C. When filaments are transferred to 28 C, they divide into short cells. Chloramphenicol, when added to cultures of filaments at the time of temperature reduction. Inhibited division of filaments when these temperature-sensitive mutations were present in the K-12 strain AB1157. However, when the ts1882 and ts2158 mutations were present in another k-12 strain, UTH4113, filaments of these strains divided in the presence of chloramphenicol.     

Transition of G1 to early S phase may be required for zinnia mesophyll cells to trans-differentiate to tracheary elements

We have used the Zinnia elegans mesophyll cell system, in which single isolated leaf mesophyll cells can be induced to trans-differentiate into tracheary elements in vitro, to study the relationship between the cell division cycle and cell differentiation. Almost all cells go through several rounds of division before characteristic features of tracheary element formation are observed. The addition of aphidicolin, a DNA synthesis inhibitor, blocks cell division but not cell differentiation in the zinnia system. Low concentrations of aphidicolin, which possibly delay cells in the early S phase, can significantly enhance levels of tracheary element formation. In contrast, roscovitine, an inhibitor of cyclin-dependent kinase activity, decelerates the cell division cycle and inhibits tracheary element formation with similar dose responses. Cells blocked in S phase and then transferred to roscovitine-containing medium can divide once, indicating that roscovitine may target the G1/S transition, but do not differentiate. Cells inhibited in G1/S in roscovitine-containing medium that are subsequently blocked in S phase by transfer to aphidicolin-containing medium, do not divide but do differentiate. Taken together, our results indicate that cells may be required to transit the G1/S checkpoint and enter early S phase to acquire competence to trans-differentiate to tracheary elements.     

Defective cell division in thermosensitive mutants of Salmonella typhimurium

Summary Two temperature-sensitive mutants ofSalmonella typhimurium defective in cell division (divA anddivC) have been isolated. Cell division in these mutants is arrested at elevated temperature while DNA and protein synthesis are unaffected. This results in formation of long filaments. Filaments returned to permissive temperature divide after a short lag. Inhibition of DNA synthesis by nalidixic acid does not block these divisions. This suggests that the thermosensitive step is required late in the division cycle. Chloramphenicol prevents the division of filaments shifted back to permissive temperature in one of these mutants (divA) and allows limited division to take place in the other mutant (divC). The arrest of cell division at elevated temperature may be phenotypically cured by high osmolarity of the medium. The mutationdivA has been mapped betweenrha andmetB and the mutationdivC betweenleu andaziA.If the filaments ofdivA are starved for thymine and then returned to permissive temperature with the simultaneous restoration of thymine the start of their division is delayed in comparison with the division of the control (unstarved) filaments. The argument is raised that a proper ratio of terminated chromosomes to cell mass must be attained to allow division.     

神经营养因子诱导分化的神经元样PC12细胞分裂的研究

神经营养因子（nerve growth factor,NGF）诱导PC12细胞分化产生的神经元样细胞一直被认为属于分裂后的细胞,没有分裂能力。然而在本研究中,我们观察了一些已经发生分化的PC12细胞,这些细胞长有很长的神经突起,在形态上属于神经元样细胞。在这些细胞中,我们不仅检测到DNA合成,而且观察到这些细胞的分裂现象。更令人感兴趣的是,除了胞体发生分裂外,位于胞体分裂位置的突起也一分为二,分别分配给两个子细胞。这些结果说明,形态发生分化的神经元样PC12细胞仍有分裂能力。本研究首次报道神经元样PC12细胞及其突起能发生分裂。     

Charakterisierung des Regenerationsprozesses im Caulonema eines Mooses durch autoradiographische Bestimmung der DNS-Synthese

Summary Ten-cell-long filaments of the caulonema of Funaria hygrometrica were isolated and labeled with ³H-thymidine. During the process of regeneration this precursor is incorporated into the nucleus and the chloroplasts. The nuclei of aged cells are preferentially labeled, even the nuclei of such cells which probably will no longer divide.From these facts it is concluded that DNA synthesis can occur during the process of regeneration irrespectively of a following cell division.     

Reactivation of stationary Tetrahymena : A contribution to the question of G0 state

Stationary cells of Tetrahymena were reactivated to exponential growth phase by transfer to fresh medium. The sequence of resuming cell cycle events was analysed by scoring the division index, the labelling index for macro- and micronuclei and the increase in cell number. By long-term labelling it was found that all cells replicate in stationary phase cultures. They also divide eventually. Upon transfer to fresh medium a small fraction of cells (about 3%) divide immediately, whereas the rest divide 3 h later after having replicated their macronuclear DNA. The kinetics of entry into the S phase indicates that these cells have a lag period of about 2 h before they resume progress through the cell cycle. It takes more than 1 h until all cells have begun replication. These data show that in stationary cultures all cells proceed through the events of the cell cycle. The cell cycle phases are extended differentially, G1 taking the largest part. During G2 cells pass very slowly through a certain stage close to division. Under the present conditions there is no indication for cells being in a resting state that is not part of the cell cycle, from which they can be restimulated and which has been called the G0 state. The criteria to demonstrate a resting state of this nature are discussed.     

Changes in calcium and magnesium levels during heat-shock synchronized cell division in Tetrahymena

Calcium and magnesium contents were measured in cells of Tetrahymena pyriformis induced to divide synchronously by a multi-heat-shock procedure. During free-running synchronized cell division in complex proteose peptone medium, significant peaks of both calcium and magnesium were observed at points in the cell cycle just prior to division. No such peaks were detected in cells dividing asynchronously in proteose peptone. When synchronized cell division was followed after transfer to an inorganic medium, cell calcium and magnesium levels were observed to decrease in relation to the corresponding cell number increase, indicating that in concentration terms, calcium and magnesium remain fairly constant. This latter result suggests that neither calcium nor magnesium influxes act as triggers for cell division in Tetrahymena and that the fluctuations of these metals seen during the synchronized division cycle in complex medium represent an effect rather than a cause.     

A cell length‐dependent transition in MinD‐dynamics promotes a switch in division‐site placement and preservation of proliferating elongated Vibrio parahaemolyticus swarmer cells

Vibrio parahaemolyticus exists as swimmer and swarmer cells, specialized for growth in liquid and on solid environments respectively. Swarmer cells are characteristically highly elongated due to an inhibition of cell division, but still need to divide in order to proliferate and expand the colony. It is unknown how long swarmer cells divide without diminishing the population of long cells required for swarming behavior. Here we show that swarmer cells divide but the placement of the division site is cell length‐dependent; short swarmers divide at mid‐cell, while long swarmers switch to a specific non‐mid‐cell placement of the division site. Transition to non‐mid‐cell positioning of the Z‐ring is promoted by a cell length‐dependent switch in the localization‐dynamics of the division regulator MinD from a pole‐to‐pole oscillation in short swarmers to a multi‐node standing‐wave oscillation in long swarmers. Regulation of FtsZ levels restricts the number of divisions to one and SlmA ensures sufficient free FtsZ to sustain Z‐ring formation by preventing sequestration of FtsZ into division deficient clusters. By limiting the number of division‐events to one per cell at a specific non‐mid‐cell position, V. parahaemolyticus promotes the preservation of long swarmer cells and permits swarmer cell division without the need for dedifferentiation.     

Variations on a theme: the many modes of cytokinesis

Animal cell division is believed to be mediated primarily by the 'purse-string' mechanism, which entails furrowing of the equatorial region, driven by the interaction of actin and myosin II filaments within contractile rings. However, myosin II-null Dictyostelium cells on substrates divide efficiently in a cell cycle-coupled manner. This process, termed cytokinesis B, appears to be driven by polar traction forces. Data in the literature can be interpreted as suggesting that adherent higher animal cells also use a cytokinesis B-like mechanism for cytokinesis. An additional chemotaxis-based cytokinesis that involves a 'midwife' cell has also been reported. Collectively, these findings demonstrate an unexpected diversity of mechanisms by which animal cells carry out cytokinesis.