Linear Cell Growth in Escherichia coli

Growth was studied in synchronous cultures of Escherichia coli, using three strains and several rates of cell division. Synchrony was obtained by the Mitchison-Vincent technique. Controls gave no discernible perturbation in growth or rate of cell division. In all cases, mean cell volumes increased linearly (rather than exponentially) during the cycle except possibly for a small period near the end of the cycle. Linear volume growth occurred in synchronous cultures established from cells of different sizes, and also for the first volume doubling of cells prevented from division by a shift up to a more rapid growth rate. As a model for linear kinetics, it is suggested that linear growth represents constant uptake of all major nutrient factors during the cycle, and that constant uptake in turn is established by the presence of a constant number of functional binding or accumulation sites for each growth factor during linear growth of the cell.     

Growth During the Bacterial Cell Cycle: Analysis of Cell Size Distribution

Cell volume distributions were determined electronically for steady-state cultures of Escherichia coli, Bacillus megaterium, Bacillus subtilis, and Salmonella typhimurium by use of a Coulter transducer-multichannel analyzer system of good resolution. All of the cell volume distributions had the same general shape, even though cultures were grown at widely different rates. Some results were independent of any particular growth model. Both the variability in the volumes of dividing cells and the fraction of constricted and unseparated doublet cells increased with growth rate. The greater separation to single cells at slow growth rates is in agreement with the general finding that filamentous and hyphal forms are greatly reduced in slowly growing chemostat cultures. The distributions were fitted equally well by simple models which assumed that cell growth was either linear or exponential throughout the entire cell cycle. It is concluded that methods of determining growth rate by analysis of distributions of bacterial volumes do not yet have sufficient resolution to distinguish between a variety of alternative models for growth of bacteria.     

Potassium Uptake in Synchronous and Synchronized Cultures of Escherichia coli

Criteria are presented for distinguishing between synchronous and synchronized cultures (natural vs. forced synchrony) on the basis of characteristics of growth and division during a single generation. These criteria were applied in an examination of the uptake of potassium during the cell growth and division cycle in synchronous cultures and in a synchronized culture of Escherichia coli. In the synchronous cultures the uptake of ⁴²K doubled synchronously with cell number, corresponding to a constant rate of uptake per cell throughout the cell cycle. In the synchronized culture, uptake rates also remained constant during most of the cycle, but rates doubled abruptly well within the cycle. This constancy of ⁴²K uptake per cell supports an earlier interpretation for steady-state cultures that uptake is limited in each cell by a constant number of functional sites for binding, transport, or accumulation of compounds from the growth medium, and that the average number of such sites doubles late in each cell cycle. The abrupt doubling of the rate of uptake of potassium per cell in the synchronized culture appears because of partial uncoupling of cell division from activation or synthesis of these uptake sites.     

Increase in cell mass during the division cycle of Escherichia coli B/rA.

Increase in the mean cell mass of undivided cells was determined during the division cycle of Escherichia coli B/rA. Cell buoyant densities during the division cycle were determined after cells from an exponentially growing culture were separated by size. The buoyant densities of these cells were essentially independent of cell age, with a mean value of 1.094 g ml-1. Mean cell volume and buoyant density were also determined during synchronous growth in two different media, which provided doubling times of 40 and 25 min. Cell volume and mass increased linearly at both growth rates, as buoyant density did not vary significantly. The results are consistent with only one of the three major models of cell growth, linear growth, which specifies that the rate of increase in cell mass is constant throughout the division cycle.     

Division competence in Tetrahymena: determination of minimum cell volume and rate of nutrient uptake

Cell volume and doubling time have been determined for exponentially growing Tetrahymena pyriformis cells in broth medium with and without glucose and in media made from these media by dilution with water. The cells tolerate media with dry weights from 105 down to 0.06 g/L. In the diluted media the cells have small volumes and the doubling time is increased. When the cell volume increase per time per cell in a given medium is expressed as a function of the cell volume in this same medium, a direct proportionality is found. From this equation the minimum cell volume of division competence (MVDC) can be found. It is 2,100 microns 3 for T. pyriformis at 28 degrees C. The lag period resulting from an upshift of exponentially growing cells from diluted media to more concentrated media is a function of the initial and resulting cell volumes and MVDC. The increase in cell volume per unit of time for a given cell depends on the dry weight of the medium. This parameter can be transformed to mass increase per cell surface area per time, which represents rate of nutrient uptake. When plotted against the dry weight of the media, a Michaelis-Menten-like curve is obtained with two Km values of 3.8 and 0.08 g/L with corresponding Vmax values of 20 and 4 ng/cm2.s. The low Km value (0.08 g/L) indicates that Tetrahymena is able to take up nutrients from highly diluted media. The high value of Vmax (20 ng/cm2.s) increases the ability of growth in more concentrated media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Constancy of Uptake During the Cell Cycle in Escherichia coli

Rates of uptake of several labeled compounds were measured during the cell cycle for three strains of Escherichia coli in balanced growth. Uptake rates were constant during more than the first two-thirds of the cycle, or reasonably so, for all of these compounds: glycine, leucine, glucose, acetate, phosphate, sulfate, and thymidine. When added de novo, uptake of glycine and leucine were not constant, but appeared to be proportional to mean cell volume. These results are in agreement with the finding that cell sizes increase linearly during most of the cell cycle for E. coli. They support the hypothesis, for cultures in balanced growth, that linear growth during the cell cycle is due to constant rates of uptake of all major growth factors. They also support the interpretation that uptake is limited by the presence of a constant number of functional binding or accumulation sites for these growth factors.     

Buoyant density constancy during the cell cycle of Escherichia coli

Cell buoyant densities were determined in exponentially growing cultures of Escherichia coli B/r NC32 and E. coli K-12 PAT84 by equilibrium centrifugation in Percoll gradients. Distributions within density bands were measured as viable cells or total numbers of cells. At all growth rates, buoyant densities had narrow normal distributions with essentially the same value for the coefficient of variation, 0.15%. When the density distributions were determined in Ficoll gradients, they were more than twice as broad, but this increased variability was associated with the binding of Ficoll to the bacteria. Mean cell volumes and cell lengths were independent of cell densities in Percoll bands, within experimental errors, both in slowly and in rapidly growing cultures. Buoyant densities of cells separated by size, and therefore by age, in sucrose gradients also were observed to be independent of age. The results make unlikely any stepwise change in mean buoyant density of 0.1% or more during the cycle. These results also make it unlikely that signaling functions for cell division or for other cell cycle events are provided by density variations.     

INFLUENCE OF ENVIRONMENTAL FACTORS AND POPULATION COMPOSITION ON THE TIMING OF CELL DIVISION IN THALASSIOSIRA FLUVIATILIS (BACILLARIOPHYCEAE) GROWN ON LIGHT/DARK CYCLES1

Cell division patterns in Thalassiosira fluviatilis grown in a cyclostat were analyzed as a function of temperature, photoperiod, nutrient limitation and average cell size of the population. Typical cell division patterns in populations doubling more than once per day had multiple peaks in division rate each day, with the lowest rates always being greater than zero. Division bursts occurred in both light and dark periods with relative intensities depending on growth conditions. Multiple peaks in division rate were also found, when population growth rates were reduced to less than one doubling per day by lowering temperature, nutrients, or photoperiod and the degree of division phasing was not enhanced. Temperature and nutrient limitation shifted the timing of the major division burst relative to the light/dark cycle. Average cell volume of the inoculum was found to be a significant determinant of the average population growth rate and the timing and magnitude of the peaks in division rate. The results are interpreted in the context of a cell cycle model in which generation times are “quantized” into values separated by a constant time interval.     

Control of Cell Growth in Bacteria: Experiments with Thymine Starvation

When cells of Escherichia coli THU were starved for thymine, they continued to grow without division for at least two successive volume doublings at their initial rate. Within experimental error this average rate of volume increase, 0.21 mum(3) per hr, was identical with that observed in control cultures during two generations of growth in the presence of thymine. This growth rate was also independent of the age of the cells at the time of starvation. These results are consistent with the hypothesis, proposed earlier, that growth rates are controlled by uptake sites for binding, transport, or accumulation of compounds into the cell, that the number of these sites is constant throughout most of the cell cycle, and that this number doubles near or at cell division.     

The rate and topography of cell wall synthesis during the division cycle of Escherichia coli using N-acetylglucosamine as a peptidoglycan label

The rates of synthesis of peptidoglycan and protein during the division cycle of Escherichia coli were measured by the membrane elution technique using cells differentially labelled with N-acetylglucosamine and leucine. During the first part of the division cycle the ratio of the rates of protein and peptidoglycan synthesis was constant. The rate of peptidoglycan synthesis, relative to the rate of protein synthesis, increased during the latter part of the division cycle. These results support a simple, bipartite model of cell surface increase in rod-shaped cells. Prior to the start of constriction the cell surface increases only by lateral wall extension. After cell constriction starts, the cell surface increases by both lateral wall and pole growth. The increase in surface area is partitioned between the lateral wall and the pole so that the volume of the cell increases exponentially. No variation in cell density occurs, because the increase in surface allows a continuous exponential increase in cell volume that accommodates the exponential increase in cell mass. The results are consistent with the constant density of the growing cell and the surface stress model for the regulation of cell surface synthesis. In addition, the elution pattern suggests that the membrane elution method does work by having the cells effectively bound to the membrane by their poles.     

Bilinear cell growth of Escherichia coli.

Recent electron micrograph measurements of bacterial dimensions in exponentially growing cultures of Escherichia coli support a model of bilinear increase in cell surface area and volume, with a sharp doubling in growth rate at a discrete age during the cell cycle. The results also indicate coordinate regulation of increase of surface area and volume.     

Regulation of the Synthesis of the Lactose Repressor

Measurements of the lactose repressor over a tenfold range of cell growth rates were made on protein extracts from Escherichia coli cultures grown in media with various carbon energy sources. The concentration of lactose repressor varied with the number of genome equivalents per cell over this range in growth rates, suggesting that the number of lactose molecules within the cell is determined by the number of I gene copies present. The timing of repressor synthesis during the cell division cycle and its correlation with deoxyribonucleic acid synthesis was examined by synchronizing the cell division cycle of E. coli ED1039, in which the Lac region has been transposed from 10 to 36 min on the genetic map. Measurements of lactose repressor in the synchronized culture revealed a burst of repressor synthesis at the time of I gene duplication. The concentration of lactose repressor was found to decrease as a function of total cell protein during the division cycle until an increase in synthesis occurred, suggesting that repressor synthesis probably does not occur throughout the division cycle. A model for I gene regulation is proposed.     

Deoxyribonucleic Acid Synthesis During the Division Cycle of Escherichia coli: a Comparison of Strains B/r, K-12, 15, and 15T− Under Conditions of Slow Growth

The rates of deoxyribonucleic acid (DNA) synthesis during the division cycles of the Escherichia coli strains B/r, K-12 3000, 15T(-), and 15 have been measured in synchronous cultures, under several conditions of slow growth. These synchronous cultures were obtained by sucrose gradient centrifugation of exponentially growing cultures, after which the smallest cells were removed from the gradient and allowed to grow. Sucrose gradient centrifugation did not adversely affect the cell cycle, since an experiment in which an exponentially growing culture was pulsed with [(3)H]thymidine prior to the periodic separation and assay of the smallest cells resulted in the same conclusions, as given below. In the strains of E. coli that were studied, a decreased rate of [(3)H]thymidine incorporation was seen late in the cell cycle, prior to cell division. No decrease in the rate of [(3)H]thymidine incorporation was seen at or near the beginning of the cell cycle. Thus, all these strains appear to regulate DNA synthesis in a similar fashion during slow growth. In addition, a correlation between the appearance of cells with visible cross-walls and the start of a new round of DNA synthesis was seen, indicating that these two events might be related.     

Changes in Cell Size and DNA Content in Sulfolobus Cultures during Dilution and Temperature Shift Experiments

Stationary-phase cultures of different hyperthermophilic species of the archaeal genus Sulfolobus were diluted into fresh growth medium and analyzed by flow cytometry and phase-fluorescence microscopy. After dilution, cellular growth started rapidly but no nucleoid partition, cell division, or chromosome replication took place until the cells had been increasing in size for several hours. Initiation of chromosome replication required that the cells first go through partition and cell division, revealing a strong interdependence between these key cell cycle events. The time points at which nucleoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When exponentially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing that the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79 degrees C were shifted to room temperature or to ice-water baths, the cells were found to "freeze" in mid-growth. After a shift back to 79 degrees C, growth, replication, and division rapidly resumed and the mode and kinetics of the resumption differed depending upon the nature and length of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.     

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.     

Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790.

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.     

Origin and sequence of chromosome replication in Escherichia coli B-r

The initial rates of induced synthesis of tryptophanase, beta-galactosidase, and d-serine deaminase were measured in relation to the chromosome replication cycle of Escherichia coli B/r. Exponentially growing cultures were exposed briefly to (14)C-thymidine or the appropriate inducers (or both), and the amount of label or enzyme (or both) in cells of different ages was found by measuring these quantities in their progeny. The rates of induced synthesis of the three enzymes increased abruptly at about 4, 20, and 34 min, respectively, after the start of a round of replication lasting 40 min. By matching this sequence to the ind, lac, and Dsd loci on the genetic map of E. coli K-12, it was estimated that replication began at about 8 o'clock (60 min) and proceeded clockwise. In rapidly growing cells, the sequence during the division cycle was consistent with the concept that rounds of replication overlapped.     

Control of F′lac Replication in Escherichia coli B/r

The timing of replication of an F'lac plasmid during the division cycle of Escherichia coli B/r lac(-)/F'lac was examined in relation to the timing of initiation of chromosome replication. This was accomplished by measuring the induction of beta-galactosidase and the incorporation of radioactive thymidine into cells at different ages in cultures growing exponentially at various rates. In cells growing with interdivision times of 27, 36, and 55 min, the F'lac replicated at various stages in the division cycle but always at approximately the same time as initiation of chromosome replication. In cells growing with an interdivision time of 85 min, the F'lac episome replicated midway through the division cycle, whereas chromosome replication initiated at the start of the cycle. Measurements of absorbance at 450 nm per cell suggested that the F'lac replicated when the cells reached a mass which was a constant multiple of the number of episomes per cell at each growth rate. In contrast, the mass per cell at initiation of chromosome replication in cells with an 85-min interdivision time was significantly lower than this constant value. A possible explanation for the apparent coupling between F'lac replication and initiation of chromosome replication at the higher growth rates, and the lack of coupling at the lowest growth rate, is discussed.     

Cell cycle analysis of F''lac replication in Escherichia coli B/r.

The timing and control of replication of an F'lac plasmid was investigated in two substrains of Escherichia coli B/r lac/F'lac growing at a variety of rates. The cellular content of covalently closed circular F'lac deoxyribonucleic acid and the cellular mass at the time of F'lac replication both increased as a function of growth rate. The timing of plasmid replication during the division cycle was determined by measuring the inducibility of beta-galactosidase in cells of different ages in exponentially growing cultures. At all growth rates, the rate of induced beta-galactosidase synthesis increased in a step-wise fashion during the division cycle, indicating that the F'lac plasmid replicated at a discrete time in the cycle. At growth rates greater than one doubling per h, the cell age at F'lac replication was indistinguishable from the cell age at chromosomal lac+ replication in an isogenic F- parent. The ratio of plasmids to chromosomal origins decreased from about 0.7 to 0.4 between growth rates of 1.0 to 2.5 doublings per h. These observations are all consistent with replication of F'lac at about the same time in the division cycle as replication of the homologous chromosomal region at these growth rates. This similarity in timing of replication of homologous deoxyribonucleic acid regions was not evident in slower-growing cells.     

A simple model for cell volume and developmental compartments in nutrient limited cyclostat cultures of algae

Daily light-dark cycles can entrain cell growth and division cycles in populations of algae growing in nutrient limited continuous cultures, or cyclostats. In this study a simple model for the flux of cells between discrete developmental stages is formulated for periodic cyclostat cultures of algae. Cell growth, in terms of volume, was set as being constant within a given developmental compartment, but variable between compartments. Growth within a given compartment or transition between compartments was restricted to specific intervals of the subjective day. The model was calibrated to phosphate limited cyclostat growth of Euglena gracilis, with the intervals for transition between compartments fixed at the times relative to the subjective dawn corresponding to critical transition points in the phased cell cycle of this organism. The model output for mean population volume per cell agreed well with experimental data. Although greatly simplified, the periodic behavior of the model volume frequency distributions for the discrete compartments provide reasonable approximation of experimentally determined distributions.