Catabolic pools in Escherichia coli.

Methods are described for measuring soluble pool magnitudes in steady-state or exponentially growing cultures, and for distinguishing between anabolic, catabolic and total metabolic pools within cells. These methods were applied to the measurement of pool magnitudes for several amino acids and other precursors in Escherichia coli THU. Our results support the independence of the magnitudes of total metabolic pools and growth rate in steady-state cultures. Our results also show that the total metabolic pool size is much larger than previously published estimates, which failed to include the contribution of catabolic pools. The average value of the total soluble material in exponential-phase cells is estimated to be 8 to 9% of the cell dry mass; pool values could be almost twice as large during midcycle because of the known increase in the magnitudes of protein and RNA precursor pool during the cell cycle.

The phosphate pool in Escherichia coli THU.

The phosphate pool of Escherichia coli was determined as a fraction of the total cell phosphate. This relative pool size was found to be essentially independent of cell age.

Cell volume increase in Escherichia coli after shifts to richer media.

Synchronous cultures of Escherichia coli 15-THU and WP2s, which were selected by velocity sedimentation from exponential-phase cultures growing in an acetate-minimal salts medium, were shifted to richer media at various times during the cell cycle by the addition of glucose or nutrient broth. Cell numbers and mean cell volumes were measured electronically. The duration of the division cycle of the shifted generation was not altered significantly by the addition of either nutrient. Growth rates, measured as rates of cell volume increase, were constant throughout the cycle in unshifted acetate control cultures. When glucose was added, growth rates also remained unchanged during the remainder of the cell cycle and then increased abruptly at or after cell division. When nutrient broth was added, growth rates remained unchanged from periods of 0.2 to 0.4 generations and then increased abruptly to their final values. In all cases, the cell volume increase was linear both before and after the growth rate transition. The strongest support for a linear cell volume increase during the cell cycle of E. coli in slowly growing acetate cultures, however, was obtained in unshifted cultures, in complete agreement with earlier observations of cell volumes at much more rapid growth rates. Although cell growth and division are under the control of the synthesizing machinery in the cell responsible for RNA and protein synthesis, the results indicate that growth is also regulated by membrane-associated transport systems.

Activation of amino acid transport during steady-state growth of Escherichia coli.

Accumulation of amino acids in exponentially increasing cultures of Escherichia coli was linear, supporting the interpretation that the biphasic response observed when cultures grew without these acids reflects a transient perturbation in accumulation. Rates of accumulation of glutamine, histidine and glycine were compared in steady-state and non-steady-state cultures. Their uptake rates were markedly enhanced in steady-state cultures at low exogenous concentrations, 10 microM or less. The results support the activation of amino acid transport systems by low concentrations of the particular amino acid present during growth. This activation was decreased when exogenous concentrations of the amino acid were markedly increased or when cells were washed free of the amino acid. Upon readdition of the amino acid after washing, recovery of enhanced transport required several generations, supporting a process of recovery other than enzymatic induction. The observation of amino acid enhancement of transport for eight other amino acids examined in steady-state culture suggests that this enhancement is a common process.

Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth.

The magnitudes of several pools of radioactively labeled precursors for RNA and protein synthesis were determined as a function of cell age during the division cycle of Escherichia coli 15 THU. Uracil, histidine, and methionine pools increased from low initial values for cells at birth to maxima during midcycle and then subsided again. These pools were small or nonexistent at the beginning and the end of the cycle, and their average values during the cycle were less than 4% of the total cellular radioactivity. The results are consistent with a linear pattern of growth for cells during the division cycle and provide strong evidence against exponential or bilinear growth of E. coli cells.

Buoyant density variation during the cell cycle in microorganisms.

The behavior of cell buoyant density during the cell cycle has been determined for a number of different cell types, including bacteria, yeast, and mammalian cells. Mean buoyant density was extremely constant and independent of cell age during the cell cycle of the bacterium Escherichia coli, the fission yeast Schizosaccharomyces cerevisiae, the protozoan Amoebae proteus, cells from suspension cell cultures of mouse lymphoma and myeloma, and Chinese hamster ovary cells. In all of these cases, the buoyant densities of these cells were very narrowly distributed, with coefficients of variation of 0.1 to 0.3%. In contrast, buoyant density was variable in cells with thick cell walls and high buoyant densities. Density varied markedly during the cell cycle of the budding yeast Schizosaccharomyces cerevisiae and of the bacterium Streptococcus faecium. The average buoyant densities of cells in exponentially growing cultures of E. coli or Schizosaccharomyces pombe were also independent of growth rate of the cultures. Experiments with E. coli have established that cell buoyant density is controlled by the osmoregulatory system. Although the regulatory mechanisms for this control are unknown, the results suggest that the same or similar mechanisms regulate buoyant density in all of the cells that do not have unduly heavy cell walls and, therefore, these regulatory mechanisms were either conserved during evolution or reflect the convergent evolution found for organic osmolytes.

Determination of bacterial cell volume with the Coulter Counter.

Two methods were used to determine mean volumes of cells of Escherichia coli B/rA in both stationary- and exponential-phase cultures, i.e., electronic measurement with a Coulter Counter-Analyzer system and biophysical measurement of the total volume and number of cells in sedimented cell pellets. Within experimental errors, the methods gave the same mean cell volumes.

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.

A second growth state for Schizosaccharomyces pombe.

The kinetics of volume increase in individual cells of Schizosaccharomyces pombe were determined by phase microscopy at osmolalities lower than those reported in the literature. At the highest osmolality, 550 mmol/kg, all cells followed a biphasic pattern of growth, in which cell volumes increased to their maximum values approximately four-fifths of the way through the growth cycle. At lower osmolalities (400-420 mmol/kg), many or most of the cells followed a different growth pattern, with a linear increase in cell volume throughout the cycle. The following evidence indicates that a different regulatory mechanism is responsible for the linear growth pattern: (1) Regulation of cell length and diameter differed for the two cases. During biphasic growth, cell length also increased biphasically and cell diameters remained essentially constant during the cycle, whereas during linear growth, both cell length and diameter increased linearly until formation of the cell plate very late in cycle. (2) The two different growth states were observed for cells growing on two very different kinds of medium. (3) Frequency distributions of the two growth patterns showed that there were two distinct groups of growing cells, with and without a cell volume plateau; these results rule out a single growth state in which plateaus are graded from large to infinitesimally small. (4) Linear regressions fitted to the data for linear growth did not differ significantly from the theoretical model for linear growth without a terminal plateau. These results reveal the operation of a second regulatory system for cell growth in S. pombe at osmolalities closer to those in liquid medium. The occurrence of transitions between the two growth states in successive generations and the agreement between several growth parameters for the two modes suggest that the growth states are closely related.

Biological effects of background radiation: mutagenicity of 40K.

The naturally occurring radioactive isotope 40K is the single largest contributor to the internal background radiation dose in living organisms. We examined cell growth and mutation rate or frequency in several strains of Escherichia coli in (i) media containing the natural content of 40K, (ii) media containing potassium from which essentially all of the 40K had been removed by isotope separation, and (iii) media highly enriched in 40K. Growth rates (doubling times) were identical in the present or absence of 40K. In more than 40 chemostat experiments, we were unable to detect any significant differences in mutation rate to bacteriophage T5 resistance or in mutation frequency to valine resistance or tryptophan prototrophy attributable to 40K. We conclude that, in the bacterial systems we have studied, 40K does not make a significant contribution to spontaneous mutation.

Buoyant density constancy of Schizosaccharomyces pombe cells.

Buoyant densities of cells from exponentially growing cultures of the fission yeast Schizosaccharomyces pombe 972h- with division rates from 0.14 to 0.5 per h were determined by equilibrium centrifugation in Percoll gradients. Buoyant densities were independent of growth rate, with an average value (+/- standard error) of 1.0945 (+/- 0.00037) g/ml. When cells from these cultures were separated by size, mean cell volumes were independent of buoyant density, indicating that buoyant densities also were independent of cell age during the division cycle. These results support the suggestion that most or all kinds of cells that divide by equatorial fission may have similar, evolutionarily conserved mechanisms for regulation of buoyant density.

Azaserine: further evidence for DNA damage in Escherichia coli.

Azaserine causes DNA damage in stationary-phase cells. In our investigation of this damage, we used strains of Escherichia coli differing in repair capabilities to study azaserine-induced DNA damage, detected as DNA strand breaks by sucrose gradient sedimentation techniques. Reduced sedimentation in alkaline and neutral sucrose gradients indicated the presence of both alkali-labile sites and in situ strand breaks. Azaserine induced DNA single-strand breaks (SSBs) abundantly in all but the recA strain, in which SSBs were greatly reduced. Treatment of purified DNA with azaserine from bacteriophages T4 and PM2 produced no detectable SSBs. Several other studies also failed to detect DNA damage induced directly by azaserine. Increased levels of beta-galactosidase were induced in an E. coli strain possessing a rec::lac fusion, providing further evidence for azaserine induction of the recA gene product. In addition, azaserine induced adaptation against killing but not against mutagenesis in wild-type E. coli strain.

Evidence for osmoregulation of cell growth and buoyant density in Escherichia coli.

The buoyant density of cells of Escherichia coli B/r NC32 increased with the osmolarity of the growth medium. Growth rate and its variability were also dependent upon the osmolarity of the medium. Maximum growth rates and minimum variability of these rates were obtained in Luria broth by addition of NaCl to a concentration of about 0.23 M.

Buoyant density variation during the cell cycle of Saccharomyces cerevisiae.

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.

Independence of buoyant cell density and growth rate in Escherichia coli.

The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.

Constancy of cell buoyant density for cultured murine cells.

The relationship between cell cycle and cell density was determined for three different lines of mouse cells by equilibrium centrifugation of suspension cultures. The mean cell densities of the three lines differed significantly, with values of 1.0622, 1.0678, 1.0540 gm/ml for 70Z/3, S 107, and ABE 8, respectively. However, the density distributions within each of the three lines were indistinguishable, with an average coefficient of variation about 5% of the mean reduced density (i.e., density minus one). Quantitative DNA analysis of the cells separated by density showed that the proportion of cells in G1, S, and G2 + M phase of the cell cycle changed very little or not at all with cell density. In addition, cells separated by size (and therefore by phase of the cell cycle) using velocity sedimentation had the same means and distributions of densities. These results indicate that there is little or no change in cell density as the cells traverse the life cycle and that buoyant density appears to be a constant property of a cell type.

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.

Cell elongation and division probability during the Escherichia coli growth cycle.

A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions. The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history. This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min. The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division. The average value of the coefficient of variation was 13.5% for all strains and growth rates. These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E. coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential.