Buoyant density, growth rate, and the cell cycle in Streptococcus faecium.

The buoyant density in rapidly growing Streptococcus faecium 9790 cells varies over the cell cycle, in contrast to the density in Escherichia coli. Buoyant density in S. faecium was measured by using Percoll (Pharmacia Fine Chemicals, Piscataway, N.J.) density gradients. We found that the mean and coefficient of variation of the population density increased with growth rate; and within a population, the mean cell volume, which was measured electronically, increased with density. These results were compared with electron microscopic measurements of the size distributions of cell wall growth sites within each fraction of the density gradient. As the density increased within a population, the frequency of large cells increased and the frequency of newly initiated cell wall growth sites increased. These effects were more marked as the growth rate increased. Next, these data were regrouped by cell size by using the size of the central growth site as an index of cell cycle stage. Each frequency value was weighted by the proportion of the population represented by that density fraction. Then, the average buoyant density was calculated for each value of cell size. In all cell populations, the density decreased and then increased as the central site enlarged. Peripheral growth sites were initiated as density reached a maximum. At faster growth rates, density increased more steeply, and new peripheral growth sites opened up at a higher frequency. We suggest that the rate at which density increases during the cell cycle correlates with the initiation of new cell wall growth sites.     

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.     

Analysis of initiation of sites of cell wall growth in Streptococcus faecium during a nutritional shift

Three-dimensional reconstruction methods were applied to electron micrographs of Streptococcus faecium to study the initiation of cell wall growth sites during a nutritional shift experiment. Upon lowering the mass doubling time from 76 to 33 min by the addition of excess glutamate, the formation of new cell wall growth sites accelerated above the old steady-state rate at about the same time (10 to 15 min) as did mass, RNA, protein, cell numbers, and autolytic capacity but considerably before DNA (30 min) and peptidoglycan (20 min) synthesis did. During the shift, the average range of cell volumes over which new wall growth sites were introduced did not change significantly. However, upon the shift there was an increase in the frequency of cells having new sites, which was due to the faster-growing cells initiating more new sites in peripheral locations before division. After a transition period, the number of new sites per milliliter of culture increased at a rate that paralleled that of the culture mass. These findings support a model in which new sites are introduced when cells grow to a relatively constant, growth rate-independent size, while the rate at which sites form and grow increases with the growth rate. In this model, chromosome synthesis does not regulate the formation of new sites of cell wall growth, but existing sites cannot be completed until rounds of chromosome synthesis are completed.     

Control of cell length in Bacillus subtilis.

During inhibition of deoxyribonucleic acid synthesis in Bacillus subtilis 168 Thy-minus Tryp-minus, the rate of length extension is constant. A nutritional shift-up during thymine starvation causes an acceleration in the linear rate of length extension. During a nutritional shift-up in the presence of thymine, the rate of length extension gradually increases, reaching a new steady state at about 50 min before the new steady-state rate of cell division is reached. The steady-state rates of nuclear division and length extension are reached at approximately the same time. The ratio of average cell length to numbers of nuclei per cell in exponential cultures is constant over a fourfold range of growth rates. These observations are consistent with: (i) surface growth zones which operate at a constant rate of length extension under any one growth condition, but which operate at an absolute rate proportional to the growth rate of the culture, (ii) a doubling in number of growth zones at nuclear segregation, and (iii) a requirement for deoxyribonucleic acid replication for the doubling in a number of sites.     

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.     

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.     

Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli.

The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.     

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.     

Regulation of RNA synthesis in Neurospora crassa : An analysis of a shift-up

A shift-up transition of growth from acetate to glucose is analyzed in Neurospora crassa. The rates of DNA and of protein accumulations remain at the preshift values for about 2 h, afterwards they increase to the rate characteristic of the new medium. The rate of RNA accumulation increases markedly 30 min after glucose addition initially at a rate greater than that of the new exponential growth which is achieved later on. An increase of the level of ribosomal proteins accompanies the increase of the rRNA content of the shifting cells, and 2–2.5 h after the shift the ribosomal level has reached the value characteristic of the new steady state of growth. The rate of rRNA methylation, which is strictly proportional to rRNA synthesis, remains almost unchanged in the 30 min following the shift; thereafter it increases to values greater than the final rate. It is interesting that the rate of rRNA synthesis is enhanced above the value typical of the new steady state as long as the ribosome level in the cells is below that characteristic of the new steady state, as if a compensatory mechanism were active.     

Temperature-Sensitive Simian Virus 40-Transformed Cells: Phenomena Accompanying Transition from the Transformed to the “Normal” State

Temperature-sensitive simian virus (SV 40)-transformed 3T3 cells (tsSV3T3), which express the transformed phenotype when growing at 32 C but not at 39 C, were used to study changes in growth behavior during shift-up or shift-down experiments. In cultures of tsSV3T3 cells which had reached or were beyond monolayer density at 32 C, DNA synthesis reached very low levels within 24 to 48 h after shift-up. When cells which had been allowed to grow to high densities at 32 C were shifted to 39 C, not only cell growth stopped, but within two to three days the cultures shed a large number of cells into the medium. These cells were nonviable, and shedding stopped only when the number of cells attached had been reduced to that characteristic of the saturation density at 39 C. The remaining attached cells were viable and after the shift to 32 C were again able to grow from the monolayer to high cell densities. This behavior has been compared with that of normal 3T3 and wild-type SV3T3 cells under different conditions. We have also isolated new tsSV3T3 lines, using cells which had been infected with non-mutagenized wild-type SV40. This further demonstrates that the temperature sensitivity of these lines is due to a cellular rather than a viral mutation.     

Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium

In previous studies, we had shown that the buoyant density ofEscherichia coli is determined by the osmolarity of the growth medium by varying the osmolarity of the medium with NaCl or sucrose. However, the buoyant density of the cells always exceeded that of the growth medium. Here we determined the effect of medium with a buoyant density greater than the expected buoyant density of cells by adding Nycodenz to Luria broth. Percoll gradients of cells were analyzed by laser light scattering. The buoyant density for 125- and 375-mOsM-grown cells was 0.002 g/ml and 0.003 g/ml more, respectively, for cells grown in the presence of Nycodenz than those grown without Nycodenz, while the buoyant density of 250-mOsM-grown cells was 0.005 g/ml less for cells grown in the presence of Nycodenz than those grown without Nycodenz. Cells grown in 500-mOsM medium with or without Nycodenz had the same buoyant density. the buoyant density of cultures grown in defined medium was the same as those grown in rich medium, with only the medium osmolarity correlating to buoyant density. We conclude from these experiments that neither buoyant density nor chemical make-up of the medium determines the buoyant density of cells grown in that medium. Only the medium osmolarity determines cell buoyant density, suggesting thatE. coli has no mechanisms to sense buoyant density.     

The regulation of RNA synthesis in yeast II: Amino acids shift-up experiments

Summary A study has been made of the effects of a casamino acids shift-up on a prototrophic strain of yeast growing under conditions of ammonium repression. The shift-up produced an increase in growth rate some 120 min after the addition of amino acids to the medium. This growth rate increase was slightly preceded by an increase in the rate of accumulation of DNA. In contrast, the rate of accumulation of protein increased immediately and that of RNA 15–20 min after the shift. RNA was initially accumulated at a rate greater than that required to sustain the new steady state. This was shown to be due to an increase in the rate of synthesis of the rRNA species derived from the 35S precursor. The rate of synthesis of 5S rRNA and of tRNA increased much later and to a lesser extent than that of the 35S derived species. The implications of these results for general theories of the regulation of RNA synthesis are discussed.Paper I in this series is Oliver and McLaughlin (1977)     

The relationships among RNA synthesis,RNA polymerase synthesis and guanosine tetraphosphate levels in Escherichia coli during nutritional shift-up

The relationships among the rate of RNA synthesis, RNA polymerase synthesis and activity, and guanosine tetraphosphate levels were investigated following nutritional shift-up in Escherichia coli. RNA synthesis continues at the preshift rate for 1.5 min after which an increase is observed that reaches a new steady-state rate at between 2 and 2.5 min. RNA polymerase activity measured in crude extracts increases immediately and by 10 min has increased 50%. RNA polymerase synthesis as measured by the synthesis of the β and β′ subunits lags for 2.5 min and then increases 75% by 10 min. Guanosine tetraphosphate levels decrease 50% by 3 min to levels characteristic of steady-state post-shift-up cells. The significance of these data to the regulation of RNA synthesis during shift-up is discussed.     

Changes in buoyant density and cell size of Escherichia coli in response to osmotic shocks.

The buoyant density of Escherichia coli was shown to be related to the osmolarity of the growth medium. This was true whether the osmolarity was adjusted with either NaCl or sucrose. When cells were grown at one osmolarity and shocked to another osmolarity, their buoyant density adjusted to nearly suit the new osmolarity. When cells were subjected to hyperosmotic shock, they became denser than expected. When cells were subjected to hypoosmotic shock they occasionally undershot the new projected density, but the undershoot was not as dramatic as the overshoot seen with hyperosmotic shocks. Shrinkage and swelling of the cells in response to osmotic shocks could account for the change in their buoyant density. The changes in cell size after osmotic shocks were measured by two independent methods. The first method measured cell size with a Coulter Counter, and the second method measured cell size by stereologic analysis of Nomarski light micrographs. Both methods gave qualitatively similar results and showed the cells to be flexible. The maximum swelling recorded was 23% of the original cell volume, while the maximum shrinkage observed was 33%.     

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.     

Effects of sudden temperature shifts on pure cultures of four strains of freshwater bacteria

Three psychrotrophic and one mesophilic strains were isolated from winter water samples of different freshwater biotopes and identified asCytophaga johnsonae (C-21),Cytophaga sp. (M-17),Pseudomonas fluorescens (KD), andEnterobacter cloacae (BS-2). Temperature shift-up experiments with emphasis on low temperatures were carried out with aerated pure batch cultures in glucose mineral medium. The effects of sudden temperature increases on growth rates and substrate conversion were investigated. All three psychrotrophic strains in the temperature increase experiments at low temperatures showed differing reactions within the linear zone of the Arrhenius plot. TheC. johnsonae (C-21) shift-up cultures adjusted the growth rate immediately to the rate of the temperature adapted cultures, whereasCytophaga sp. (M-17) shift-up cultures showed a lower andP. fluorescens (KD) a higher growth rate. The mesophilicE. cloacae (BS-2), likeC. johnsonae (C-21), adjusted immediately to the new growth rate. Substrate conversion increased in all experiments immediately after the shift-up. The extracellular substrate conversion byP. fluorescens (KD) of glucose to gluconate and 2-ketogluconate was particularly affected by the sudden temperature increase.     

Changes in nuclear non-histone protein composition during normal differentiation and carcinogenesis of intestinal epithelial cells

Nuclei from colonic epithelial cells were isolated and fractionated by centrifugation in discontinuous sucrose density gradients. Nuclei differing in buoyant density differ in size, non-histone protein to DNA ratio, and capacity for DNA synthesis in vivo. They do not differ in histone content or in proportions of the major histone classes. The distribution of cell nuclei after density gradient centrifugation corresponds functionally to their histological localization in the colonic mucosa, as judged by the nuclear capacity for DNA synthesis in both normal and tumor tissues. The nuclei of colonic epithelial cells contain a heterogeneous set of non-histone proteins which change in total amount and in relative proportions during normal differentiation. The complement of nuclear proteins differs in normal intestinal epithelial cells and in colon tumors induced by the carcinogen, 1,2-dimethylhydrazine. There is a striking increase in the nuclear content of two major protein classes (of mol. wt ca 44 000 and 62 000) during carcinogenesis. The accumulation of these proteins in the nuclei of carcinogen-treated cells follows early, selective increases in their rates of synthesis.     

Involvement of FtsZ protein in shift-up-induced division delay in Escherichia coli.

A nutritional shift-up from glucose minimal medium to LB broth was previously shown to cause a division delay of about 20 min in synchronized cultures of Escherichia coli, and a similar delay was observed after a nutritional pulse (a shift-up followed rapidly by a return to glucose minimal medium). Using synchronized cultures, we show here that the pulse-induced division delay does not require protein synthesis during the period in LB broth, suggesting that a nonprotein signal is generated by the shift-up and transmitted to the cell division machinery. The cell division protein FtsZ, target of the SOS-associated division inhibitor SfiA (or SulA), seems to be involved in the postshift division delay. Mutants in which the FtsZ-SfiA interaction is reduced, either sfiA (loss of SfiA) or ftsZ(SfiB) (modification of FtsZ), have a 50- to 60-min division delay after a shift-up. Furthermore, after a nutritional pulse, the ftsZ(SfiB) mutant had only a 10- to 16-min delay. These results suggest that the FtsZ protein is the target element of the cell division machinery to which the shift-up signal is transmitted.