Cell cycle regulation and the timing of chromosome replication in a marine synechococcus (cyanobacteria) during light- and nitrogen-limited growth

Cell cycle behavior in the marine Synechococcus strain WH8101 was examined in detail over a wide range of light- and nitrogen-limited growth rates. The presence of bimodal DNA frequency distributions under all conditions confirms that the overlapping rounds of DNA replication that characterize E. coli and other fast-growing prokaryotes are not present in this organism. Although chromosome replication time, C , was constrained to a fairly narrow range of values overall, it nevertheless did vary with growth rate and limiting factor. Light-limited cells growing at moderate rates had higher C values than did N-limited cells growing at comparable rates (by as much as a factor of 2). As these cells became light saturated, however, C decreased sharply to the level observed under N limitation. The post-replication period, D , decreased monotonically with growth rate under both light and N limitation, approaching a constant value at moderate to high growth rates. Average cell volume at the time of initiation of DNA replication was calculated from the values of C and D , combined with directly measured mean cell volume, and was found to be constant at all growth rates above ∼0.7 d⁻¹. This pattern was confirmed by estimates of initiation volume based on flow cytometric light scatter measurements, and suggests that as has been found in other prokaryotic systems, cell mass may play an important role in regulating the timing of chromosome replication in cyanobacteria. Furthermore, because the magnitude of C + D influences average cell mass (given a constant mass at initiation), changes in these parameters (particularly C ) may be responsible for the previously reported nonlinear relationship between light-limited growth rate and both RNA cell⁻¹ and average cell volume.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

Changes in cell dimensions during amino acid starvation of Escherichia coli.

Electron microscopic analysis was used to study cells of Escherichia coli B and K-12 during and after amino acid starvation. The results confirmed our previous conclusion that cell division and initiation of DNA replication occur at a smaller cell volume after amino acid starvation. Although during short starvation periods, the number of constricting cells decreased due to residual division, it appears that during prolonged starvation, cells of E. coli B and K-12 were capable of initiating new constrictions. During amino acid starvation, cell diameter decreased significantly. The decrease was reversed only after two generation times after the resumption of protein synthesis and was larger in magnitude than that previously observed before division (F. J. Trueba and C. L. Woldringh, J. Bacteriol. 142:869-878, 1980). This decrease in cell diameter correlates with synchronization of cell division which has been shown to occur after amino acid starvation.     

Recovery from nutrient starvation by a marine Vibrio sp.

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.     

Recovery from nutrient starvation by a marine Vibrio sp

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.     

GROWTH-CONTROLLED OSCILLATION IN ACTIVITY OF HISTONE H1 KINASE DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Synchronous cultures of the cell wall-less mutant Chlamydomonas reinhardtii Dangeard cw 15 were grown under different mean irradiances and different illumination regimes, which produced cell cycles that differed in the number of daughter cells released from one mother cell, in the length of the cell cycle, and in the growth rate. During the cell cycle, the cells reached several commitment points whose number and timing differed according to the particular pattern of the cell cycle. The cell volume was used as a growth parameter and increased in a stepwise manner. Each of the steps consisted of periods of both fast and slow growth. Growth usually stopped when the cells attained a volume twice that of the preceding step. Reaching particular commitment points was coupled with the position of these points in the enlargement of cell volume. Changes in the activity of histone H1 kinase were noted during the cell cycles of all experimental variants, and the activities were compared with the timing of various commitment points. It was found that kinase activity varied markedly within a single cell cycle, attaining maximal values when the cellular volume had doubled. Each peak in kinase activity slightly preceded the commitment to an individual sequence of reproductive events. In addition to the oscillations related to cell growth, a peak of kinase activity always occurred toward the end of the cell cycle when multiple rounds of DNA replication, mitosis, and cell division occurred.     

Cell Growth and Division: I. A Mathematical Model with Applications to Cell Volume Distributions in Mammalian Suspension Cultures

A mathematical model is formulated for the development of a population of cells in which the individual members may grow and divide or die. A given cell is characterized by its age and volume, and these parameters are assumed to determine the rate of volume growth and the probability per unit time of division or death. The initial value problem is formulated, and it is shown that if cell growth rate is proportional to cell volume, then the volume distribution will not converge to a time-invariant shape without an added dispersive mechanism. Mathematical simplications which are possible for the special case of populations in the exponential phase or in the steady state are considered in some detail. Experimental volume distributions of mammalian cells in exponentially growing suspension cultures are analyzed, and growth rates and division probabilities are deduced. It is concluded that the cell volume growth rate is approximately proportional to cell volume and that the division probability increases with volume above a critical threshold. The effects on volume distribution of division into daughter cells of unequal volumes are examined in computer models.     

Freeze preservation of synchrony in cultures of enterobacteriaceae synchronized by continuous phasing in phosphate-limited media

In order to achieve synchronization of cell division by continuous phasing, the growth of enteric bacteria has been limited by inorganic phosphate. After a short starvation, the culture was automatically diluted twofold so that the limiting nutrient allowed for one doubling exactly. An automatic device was designed to carry out repeated cycles of growth, starvation and dilution with adjustable periodicity. After 12-24 automatic cycles, which were usually achieved largely overnight, synchronous cell divisions could be observed for several generations in nonlimiting culture conditions. When portions of the phased culture were frozen and kept at low temperature for periods up to several months, these freeze-preserved populations exhibited a synchronous growth upon thawing and cultivation. This technique has thus the potential of providing synchronized cultures of a variety of bacterial strains at the desired time.     

Control of cell division by sex factor F in Escherichia coli. III. Participation of the groES (mopB) gene of the host bacteria

Cell division of F+ bacteria is coupled to DNA replication of the F plasmid. Two plasmid coded genes, letA (ccdA) and letD (ccdB) are indispensable for this coupling. To investigate bacterial genes that participate in this coupling, we attempted to identify the target of the division inhibitor (the letD gene product) of the F plasmid. Two temperature-sensitive growth defective mutants were screened from bacterial mutants that escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Phage P1-mediated transduction and complementation analysis indicated that the temperature-sensitive mutations are located in the groES (mopB) gene, which is essential for the morphogenesis of several bacteriophages and also for growth of the bacteria. The nucleotide sequence of the promoter region of the gene in which the temperature-sensitive mutations had occurred was virtually identical with that of the groES gene of Escherichia coli; furthermore the sequence of the first five amino acid residues and the overall amino acid composition predicted from the nucleotide sequence of the gene match those of the purified GroES protein. The temperature-sensitive mutants did not allow the propagation of phage lambda at 28 degrees C and formed long filamentous structures without septa at 41 degrees C, as is observed in the case of groES mutants. Growth of the two groES mutants tested was not inhibited by the F plasmid with the letA mutation. These observations suggest to us that the morphogenesis gene groES plays a key role in coupling between replication of the F plasmid and cell division of the host cells.     

Distribution of cell lengths in cultures of a lexA mutant of Escherichia coli K-12.

Distributions of cell lengths in lexA+ and lexA mutant cultures during normal growth and under thymidine starvation conditions are presented. During normal growth lexA mutant cells were slightly shorter, on the average, than were lexA+ cells. lexA mutant cells were also shorter in comparison with lexA+ cells after a period of thymidine starvation. These results are consistent with the hypothesis that the lexA gene is involved in the coordination of cell division with DNA repair.     

Inhibition and restart of initiation of chromosome replication: effects on exponentially growing Escherichia coli cells.

Escherichia coli strains in which initiation of chromosome replication could be specifically blocked while other cellular processes continued uninhibited were constructed. Inhibition of replication resulted in a reduced growth rate and in inhibition of cell division after a time period roughly corresponding to the sum of the lengths of the C and D periods. The division inhibition was not mediated by the SOS regulon. The cells became elongated, and a majority contained a centrally located nucleoid with a fully replicated chromosome. The replication block was reversible, and restart of chromosome replication allowed cell division and rapid growth to resume after a time delay. After the resumption, the septum positions were nonrandomly distributed along the length axis of the cells, and a majority of the divisions resulted in at least one newborn cell of normal size and DNA content. With a transient temperature shift, a single synchronous round of chromosome replication and cell division could be induced in the population, making the constructed system useful for studies of cell cycle-specific events. The coordination between chromosome replication, nucleoid segregation, and cell division in E. coli is discussed.     

Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.     

Growth and metabolism of inositol-starved Saccharomyces cerevisiae.

Upon starvation for inositol, a phospholipid precursor, an inositol-requiring mutant of Saccharomyces cerevisiae has been shown to die if all other conditions are growth supporting. The growth and metabolism of inositol-starved cells has been investigated in order to determine the physiological state leading to "inositolless death". The synthesis of the major inositol-containing phospholipid ceases within 30 min after the removal of inositol from the growth medium. The cells, however, continue in an apparently normal fashion for one generation (2 h under the growth conditions used in this study). The cessation of cell division is not preceded or accompanied by any detectable change in the rate of macromolecular synthesis. When cell division ceases, the cells remain constant in volume, whereas macromolecular synthesis continues at first at an unchanged rate and eventually at a decreasing rate. Macromolecular synthesis terminates after about 4 h of inositol starvation, at approximately the time when the cells begin to die. Cell death is also accompanied by a decline in cellular potassium and adenosine triphosphate levels. The cells can be protected from inositolless death by several treatments that block cellular metabolism. It is concluded that inositol starvation results in a imbalance between the expansion of cell volume and the accumulation of cytoplasmic constituents. This imbalance is very likely the cause of inositolless death.     

Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis.

At 45 C, in a temperature-sensitive initiation mutant (TsB134) of Bacillus subtilis 168 Thy- tryp-, growing in a glucose-arginine minimal medium, chromosome completion occurred over a period of 80 to 90 min, after which there was no further nuclear division. Normal symmetrical cell divisions continued for a generation afterwards, so that nuclei were segregated into separate cells. During this period asymmetric divisions started to occur. Septa appeared at 25 to 30% from one end of the cell, giving a small anucleate cell and a larger nucleate cell. During inhibition of deoxyribonucleic acid (DNA) synthesis by thymine starvation under the restrictive conditions, asymmetrical division also occurred until there was approximately one nucleus per cell (about one generation time). Asymmetric division, giving anucleate cells, then occurred. Similar results were obtained when DNA synthesis was inhibited by nalidixic acid. After 3 h at 45 C, the rate of anucleate cell production in the presence and absence of thymine was constant at one division per 85 min per chromosome terminus present when DNA synthesis stopped. In the absence of DNA synthesis (during thymine starvation) at 35 C, growth in cell length was linear (i.e., the rate was constant), but at 45 C during thymine starvation the rate gradually increased by more than twofold. It is suggested that this was due to the establishment of new sites of growth associated with anucleate cell production. In the presence of thymine at 45 C, the rate of length extension increased by more than fourfold, which it is suggested was caused by the appearance of new growth zones as a result of chromosome termination and a contribution associated with anucleate cell production. If the mutant was incubated at 45 C for 90 min, both in the presence and absence of thymine, then anucleate cell formation could continue on restoration to 35 C in the absence of thymine...     

Chromosome Replication and Cell Division in Escherichia coli at Various Temperatures of Growth

The effect of temperature on the growth rate and the pattern of chromosome replication during the division cycle of Escherichia coli B/r growing in various media was investigated. The time between divisions, the time for a round of replication (C), and the time between completion of a round and cell division (D) were threefold longer at 21 C than at 37 C. At all temperatures and in all media, D equalled one-half C, suggesting that a common mechanism controls chromosome replication and the progression of the cell toward division after completion of a round of replication.     

Cell Growth and Division: III. Conditions for Balanced Exponential Growth in a Mathematical Model

In a previous paper, we proposed a model in which the volume growth rate and probability of division of a cell were assumed to be determined by the cell's age and volume. Some further mathematical implications of the model are here explored. In particular we seek properties of the growth and division functions which are required for the balanced exponential growth of a cell population. Integral equations are derived which relate the distribution of birth volumes in successive generations and in which the existence of balanced exponential growth can be treated as an eigenvalue problem. The special case in which all cells divide at the same age is treated in some detail and conditions are derived for the existence of a balanced exponential solution and for its stability or instability. The special case of growth rate proportional to cell volume is seen to have neutral stability. More generally when the division probability depends on age only and growth rate is proportional to cell volume, there is no possibility of balanced exponential growth. Some comparisons are made with experimental results. It is noted that the model permits the appearance of differentiated cells. A generalization of the model is formulated in which cells may be described by many state variables instead of just age and volume.     

ppGpp-mediated regulation of DNA replication and cell division in Escherichia coli

ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular processes according to the nutritional potential of the growth medium. This work is interpreted as favoring the view that, in addition to its previously documented role in regulating the rate of ribosome synthesis [4], ppGpp participates in coordinating DNA replication and cell division. We studied the effects of ppGpp on the cell division cycle, using cells containing plasmid pSM11 that codes for the 55-kDa truncated RelA protein under the inducible Ptac promoter. In this system it was found that the rate of initiation of new rounds of DNA replication is inversely correlated with the intracellular level of ppGpp. Furthermore, ppGpp levels similar to those found during the activation of stringent control inhibited replication initiation, in a manner comparable to that resulting from inhibition of protein synthesis by amino acid starvation or by chloramphenicol addition. However, in contrast to chloramphenicol treatment, elevated ppGpp levels did not block septum formation, and, in fact, there is some evidence for enhanced septation. As a result, the residual cell division following elevation in ppGpp levels was higher than after chloramphenicol treatment, resulting in cells with a size similar to that of stationary phase cells.     

Effects of Chromosome Underreplication on Cell Division in Escherichia coli

The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth. We have studied the coordination between replication and cell division by using a temperature-controlled Escherichia coli intR1 strain. In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study the response of cell division to a delayed initiation of replication. The bacteria were grown exponentially at 39°C (normal DNA/mass ratio) and shifted to 38 and 37°C. In the last two cases, new, stable, lower DNA/mass ratios were obtained. The rate of replication elongation was not affected under these conditions. At increasing degrees of underreplication, increasing proportions of the cells became elongated. Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big. The elongated cells often produced one daughter cell lacking a chromosome; this was always the smallest daughter cells, and it was the size of a normal newborn cell. These results favor a model in which cell division takes place at only distinct cell sizes. Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids. This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value. Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion.     

Requirements for DNA replication preceding cell division in Tetrahymena pyriformis

Populations of Tetrahymena pyriformis were synchronized by 30 min heat shocks at 34 °C separated by 160 min intervals at the normal growth temperature. The cells initiate DNA synthesis immediately after the cellular division, and the S period of the population lasts about 80 min. It was found that DNA replication is a prerequisite for the following synchronous division. Inhibition of the DNA synthesis in early S by starvation of the cells for thymidine prevents the forthcoming division. However, inhibition in the latter half of S does not prevent the subsequent division. Thus the cells have synthesized enough DNA to permit cell division before the end of a normal S period. These results are discussed in relation to the organization of the genome replication in the highly polyploid macronucleus.