Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent or very low in cell extracts prepared from heat-treated E. coli cells. It is concluded that during intraperiplasmic growth B. bacteriovorus degrades some 20 to 40% of the ribonucleotides derived from the initial E. coli RNA into the base and ribose-1-phosphate moieties. The ribose-1-phosphate is further metabolized by B. bacteriovorus both for energy production and for biosynthesis, of non-nucleic acid cell material. In addition, the data indicate that during intraperiplasmic growth B. bacteriovorus can metabolize ribose only if this compound is available to it as the ribonucleoside monophosphate.     

Effects of methotrexate on intraperiplasmic and axenic growth of Bdellovibrio bacteriovorus.

The intraperiplasmic growth rate and cell yield of wild-type Bdellovibrio bacteriovorus 109J, growing on Escherichia coli of normal composition as the substrate, were not markedly inhibited by 10-3 M methotrexate (4-amino-N10-methylpteroylglutamic acid). In contrast, the growth rate and cell yield of the mutant 109Ja, growing axenically in 0.5% yeast extract +0.15% peptone, were strongly inhibited by 10-4 and 10-3 M methotrexate. Thymine, thymidine, and thymidine-5'-monophosphate, in increasing order of effectiveness, partially or completely reversed the inhibition. E. coli depleted of tetrahydrofolate and having an abnormally high protein/deoxyribonucleic acid (DNA) ratio was obtained by growing it in the presence of methotrexate. B. bacteriovourus grew at a normal rate on these depleted E. coli cells but with somewhat reduced cell yield. Mexthotrexate (10-3 M) inhibited intraperiplasmic growth of bdellovibrio on the depleted E. coli somewhat more than it inhibited growth on normal E. coli, but the effects were small compared with inhibition of axenic growth of the mutant. Total bdellovibrio DNA after growth on the depleted E. coli in the presence or absence of methotrexate exceeded the initial quanity of E. coli DNA present. Thymidine-5'-monophosphate (10-3 M) largely reversed the inhibition and increased the amount of net synthesis of DNA. The data are consistent with the prediction that intraperiplasmic growth of B. bacteriovorus should be insensitive to all metabolic inhibitors that act by specifically preventing synthesis of essential monomers. The data also indicate that B. bacteriovorus possesses thymidylate synthetase, thymidine phosphorylase, and thymidine kinase, and has the potential to carry out de novo DNA synthesis from non-DNA precursors during intraperiplasmic growth. The results also suggest that methionyl tRNAfMet is not required for initiation of protein synthesis by B. bacteriovorus.     

Growth Cycle of Predacious Bdellovibrios in a Host-Free Extract System and Some Properties of the Host Extract

Host-free growth and reproduction of a host-dependent strain of Bdellovibrio bacteriovorus incubated with an extract from host cells were studied. The morphological changes occurring in the cells were correlated with deoxyribonucleic acid (DNA) synthesis as measured by labeled nucleotide or orthophosphate incorporation. The host-free developmental cycle of Bdellovibrio is similar to that of the two-membered system; the early loss of flagella, the elongation into filaments, and multiple fission into flagellated progeny are typical for both host-free and intraperiplasmic development of bdellovibrios. Filament length and time of division appear to depend on the concentration of the host extract. Host extract was found to be heat stable and DNase stable, and Pronase sensitive and RNase sensitive. Addition of ribonucleic acid to the extract medium at various times during the Bdellovibrio growth cycle demonstrated that host extract is required continuously during the cycle for growth. The observations reported give a unified picture of Bdellovibrio development and allow for the suggestion that wild-type bdellovibrios depend upon the presence of some host factor for induction of DNA synthesis, whereas depletion of host factor triggers division. The ecological implications of such host dependence are discussed.     

A comparison of the survival of intraperiplasmic and attack phase bdellovibrios with reduced oxygen

The ability of intraperiplasmic and attack phase bdellovibrios to survive and/or grow under anoxic and microaerobic conditions was examined. Both halotolerant and nonhalotolerant bdellovibrio strains were examined. In all instances, the bdellovibrio strains were unable to grow under anoxic conditions, but were able to survive for periods of time in both the extracellular and intraperiplasmic forms. However, the intraperiplasmic organisms were observed to survive longer. Increased temperature hastened the loss of viability of both forms of the predatory bacteria in oxic and anoxic environments. Under microaerobic conditions, halotolerant bdellovibrios were observed to grow, although at a slightly reduced rate than in atmospheric oxygen, while two nonhalotolerant isolates survived but did not grow. The ability of attack phase bdellovibrios to survive in an anoxic environment for up to nine days and their growth or survival under microaerobic conditions greatly expands the possible ecological niches in which the predators may be active members of the microbial community. Correspondence to: A.J. Schoeffield.     

Incorporation of Substrate Cell Lipid A Components into the Lipopolysaccharide of Intraperiplasmically Grown Bdellovibrio bacteriovorus

The composition of Bdellovibrio bacteriovorus lipopolysaccharide (LPS) was determined for cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas putida. The LPS of axenically grown bdellovibrios contained glucose and fucosamine as the only detectable neutral sugar and amino sugar, and nonadecenoic acid (19:1) as the predominant fatty acid. Additional fatty acids, heptose, ketodeoxyoctoic acid, and phosphate were also detected. LPS from bdellovibrios grown intraperiplasmically contained components characteristic of both axenically grown bdellovibrios and the substrate cells. Substrate cell-derived LPS fatty acids made up the majority of the bdellovibrio LPS fatty acids and were present in about the same proportions as in the substrate cell LPS. Glucosamine derived from E. coli LPS amounted to about one-third of the hexosamine residues in intraperiplasmically grown bdellovibrio LPS. However, galactose, characteristic of the E. coli outer core and O antigen, was not detected in the bdellovibrio LPS, suggesting that only lipid A components of the substrate cell were incorporated. Substrate cell-derived and bdellovibrio-synthesized LPS materials were conserved in the B. bacteriovorus outer membrane for at least two cycles of intraperiplasmic growth. When bdellovibrios were grown on two different substrate cells successively, lipid A components were taken up from the second while the components incorporated from the lipid A of the first were conserved in the bdellovibrio LPS. The data show that substrate cell lipid A components were incorporated into B. bacteriovorus lipid A during intraperiplasmic growth with little or no change, and that these components, fatty acids and hexosamines, comprised a substantial portion of bdellovibrio lipid A.     

Prey-derived signals regulating duration of the developmental growth phase of Bdellovibrio bacteriovorus.

The filamentous elongation typical of growth-phase cells of the predatory bacterium Bdellovibrio bacteriovorus is mediated by regulatory signals that are derived from the prey cell itself. These signals regulate the differentiation of growth-phase cells into the attack phase and appear to be required for continued filamentous growth by prey-dependent wild-type bdellovibrios and their prey-independent mutant derivatives alike. Using a prey-independent bdellovibrio strain, we have developed an assay for the detection and quantification of the growth-extending signal activity present in extracts of prey cells. This prey-derived regulatory activity was shown to be independent of its nutritional contribution to the bdellovibrios and was found to occur in heat-stable, proteinlike compounds of a variety of native molecular weights within the soluble fraction of extracts from both gram-negative and gram-positive bacteria.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus on heat-treated Escherichia coli.

Heat treatment (55 degrees C for 40 min) of cell suspensions in buffer (ca. 3 x 10(9) cells per ml) of Escherichia coli ML35 caused a 4- to 4.5-log loss of cell viability. Similar results were found for several other E. coli strains that were examined. As a result of this heat treatment, 260-nm- and 280-nm-absorbing materials were released into the suspending buffer, along with about 10% of the total cellular radioactivity, when cells uniformly labeled with (14)C were used. In comparison with untreated cells, heat-treated E. coli ML35 cells showed (i) no significant changes in macromolecular composition other than ca. 22% less RNA content, (ii) an increased permeability to o-nitrophenyl-beta-d-galactopyranoside (a compound to which untreated cells are impermeable), (iii) almost complete loss of respiratory potential, and (iv) substantial losses of numerous glycolytic enzyme activities in cell extracts prepared from these cells. Intraperiplasmic development of Bdellovibrio bacteriovorus 109J with heat-treated E. coli ML35 as substrate cells appeared normal when observed microscopically, although bdellovibrio attachment and resultant bdelloplast formation were slightly retarded. No significant changes were observed in cell yields or in the ratios and contents of DNA, RNA, or protein between bdellovibrios harvested from untreated cells and those from heat-treated substrate cells after single-developmental-cycle growth on these cells. The average Y(ATP) values for intraperiplasmic growth on untreated and heat-treated substrate cells were 16.0 and 17.9, respectively. It is concluded that intraperiplasmic bdellovibrio growth on gently heat-treated E. coli substrate cells is very similar to growth on untreated substrate cells, even though the former substrate cells are nonviable and substantially impaired in many metabolic activities.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.     

Regulated breakdown of Escherichia coli deoxyribonucleic acid during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J.

During growth of Bdellovibrio bacteriovorus on [2-14C]deoxythymidine-labeled Escherichia coli, approximately 30% of the radioactivity was released to the culture fluid as nucleoside monophosphates and free bases; the remainder was incorporated by the bdellovibrio. By 60 min after bdellovibrio attack, when only 10% of the E. coli deoxyribonucleic acid (DNA) had been solubilized, the substrate cell DNA was degraded to 5 X 10(5)-dalton fragments retained within the bdelloplast. Kinetic studies showed these fragments were formed as the result of sequential accumulation of single- and then double-strand cuts. DNA fragments between 2 X 10(3) and 5 X 10(5) daltons were never observed. Chloramphenicol, added at various times after initiation of bdellovibrio intraperiplasmic growth on normal or on heated E. coli, which have inactivated deoxyribonucleases, inhibited further breakdown and solubilization of substrate cell DNA. Analysis of these intraperiplasmic culture deoxyribonuclease activities showed that bdellovibrio deoxyribonucleases are synthesized while E. coli nucleases are inactivated. It is concluded that continuous and sequential synthesis of bdellovibrio deoxyribonucleases of apparently differing specificities is necessary for complete breakdown and solubilization of substrate cell DNA, and that substrate cell deoxyribonucleases are not involved in any significant way in the degradation process.     

Unbalanced growth as a normal feature of development of Bdellovibrio bacteriovorus

In this study we have investigated the rates and spatial patterns of chromosome replication and cell elongation during the growth phase of wild-type and facultatively prey-independent mutant strains of Bdellovibrio bacteriovorus. For the facultatively prey-independent mutants, the total DNA content of synchronously growing cultures was found to increase exponentially, as the multiple chromosomes within each filamentous cell replicated simultaneously. Cell mass, measured as total cellular protein, also increased exponentially during this period, apparently by means of multiple elongation sites along the filament wall. The relative rates of DNA and protein synthesis were unbalanced during growth, however, with the cellular concentration of DNA increasing slightly faster than that of protein. The original cellular DNA: protein ratio was restored in the progeny cells by continued protein synthesis during the septation period that follows the termination of DNA replication. Because of technical problems, these experiments could not be conducted on the wild-type cells, but similar results are assumed. This unusual pattern of unbalanced growth may represent an adaptation by bdellovibrios to maximize their progeny yield from the determinate amount of substrate available within a given prey cell.     

Translocation of an outer membrane protein into prey cytoplasmic membranes by bdellovibrios.

Within minutes of Bdellovibrio bacteriovorus attack on prey cells, such as Escherichia coli, the cytoplasmic membrane of the prey is altered. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified invaded prey cell (bdelloplast) membranes revealed the appearance of a noncytoplasmic membrane protein. This protein is not observed in preparations of noninvaded E. coli membranes and migrates in a manner similar to that of E. coli OmpF. Isoelectric focusing and two-dimensional gel electrophoresis of bdelloplast cytoplasmic membrane preparations also revealed the presence of a protein with electrophoretic properties similar to those of OmpF and the major Bdellovibrio outer membrane proteins. The protein appears in cytoplasmic membrane preparations within minutes of attack and persists throughout most of the intraperiplasmic developmental cycle. The appearance of this protein is consistent with our hypothesis that bdellovibrios translocate a pore protein into the bdelloplast cytoplasmic membrane to kill their prey and to gain access to the cytoplasmic contents for growth.     

Reversible alterations in the mitotic cycle of chick embryo cells in various states of growth regulation

Chick embryo cells which have been kept overnight at pH 6.8 in the absence of serum multiply very slowly. Only a small fraction of cells is in the S period at any given time, and the rate of uptake of 2-deoxy-D-glucose is very low. Upon raising the pH to 7.4 and adding serum (“turn-on”) the uptake of 2-deoxy-D-glucose increases immediately; the rate of DNA synthesis increases after a lag of about 4 hours, and represents an increase in the fraction of cells synthesizing DNA. The uptake of 2-deoxy-D-glucose is rapidly returned to its original low rate at any time by again lowering the pH and removing serum (“turn-off”). The synthesis of DNA in the culture remains constant or continues to rise at a markedly reduced rate following the same treatment. Lowering pH or removing serum independently of each other is less efficient at inhibiting the increase in DNA synthesis than the combined treatment but each accomplishes a similar result. Cultures which have been “turnedoff” during the early stages of the rapid increase in DNA synthesis, resume their prior rate of increase immediately if “turned-on” again within 2.5 hours. If the cultures have been “turned-off” for 5.5 hours before restoring the “turn-on,” there is a 2 hour delay before they resume an increased rate of DNA synthesis. The results indicate that chick embryo cells do not become committed to the initiation of DNA synthesis until shortly before, or at the time of the onset of the S period. Up to 96% of the cells in post-confluent cultures growing in conventional medium become labeled upon continuous, prolonged exposure to ³H-thymidine. Seventy-eight percent of the cells in serum-deprived cultures growing at a very low rate become labeled. These and other considerations suggest that the inhibition of cell multiplication by high population density or serum deprivation is caused by a lengthening of the time cells remain in the prereplicative G₁ period rather than by shifting cells into a qualitatively distinct G₀ period. There may, however, be a period common to all cells regardless of growth rate, in which cells are not progressing toward the S period. The length of this variable period would then determine the growth rate of a population of cells.     

Control of cell division in the yeast Saccharomyces cerevisiae cultured at different growth rates

Saccharomyces cerevisiae has been grown with different generation times by alterations in media richness and by altering the flow rate of the limiting nutrient, glucose in a chemostat. Within the generation time range 2.89-approx. 8.0 h the time from the initiation of DNA synthesis to cell division was independent of generation time and was approx. 2 h. Thus the cell cycle of yeast can be divided into an expandable phase from cell division to the initiation of DNA synthesis, the length of which is dependent on growth rate and a constant phase from the initiation of DNA synthesis to cell division which takes a constant time independent of generation time. In cells growing with generation times longer than 8.6 h this constant phase expands somewhat in time. These results are reminiscent of the observation that in the bacterium Escherichia coliB/R the time from initiation of DNA synthesis to cell division is constant except at very slow growth rates.     

Evidence for a Relationship Between Equine Abortion (Herpes) Virus Deoxyribonucleic Acid Synthesis and the S Phase of the KB Cell Mitotic Cycle

Autoradiographic analyses of deoxyribonucleic acid (DNA) synthesis in randomly growing KB cell cultures infected with equine abortion virus (EAV) suggested that viral DNA synthesis was initiated only at times that coincided with the entry of noninfected control cells into the S phase of the cell cycle. Synchronized cultures of KB cells were infected at different stages of the cell cycle, and rates of synthesis of cellular and viral DNA were measured. When cells were infected at different times within the S phase, viral DNA synthesis was initiated 2 to 3 hr after infection. However, when cells in G1 and G2 were infected, the initiation of viral DNA synthesis was delayed and occurred only at times corresponding to the S phase. The times when viral DNA synthesis began were independent of the time of infection and differed by as much as 5 hr, depending on the stage of the cell cycle at which cells were infected. Viral one-step growth curves were also related to the S phase in a manner which indicated a relationship between the initiation of viral DNA synthesis and the S phase. These data support the concept that initiation of EAV DNA synthesis is dependent upon some cellular function(s) which is related to the S phase of the cell cycle.     

Glycolytic and tricarboxylic acid cycle enzyme activities during intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli.

Selected enzyme activities were measured in extracts of the total cell pellets obtained at various times during aerobic intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on anaerobically grown Escherichia coli substrate cells. Initially, the glycolytic enzyme activities were associated with the input of E. coli and the tricarboxylic acid cycle enzyme activities with the input of bdellovibrios. During the first 90 min of Bdellovibrio development, the glycolytic activities declined about 25 to 60%, whereas the tricarboxylic acid cycle activities increased about 10%. Between 110 and 180 min, the glycolytic activities decreased to trace levels and tricarboxylic acid cycle activities increased about 50 to 90%. Both bdellovibrio cell extracts and the cell-free growth menstruum (obtained after bdellovibrio growth on E. coli) caused the inactivation of glycolytic enzymes in E. coli extracts.     

Regulation of DNA synthesis in bacteria: Analysis of the Bates/Kleckner licensing/initiation-mass model for cell cycle control

Bates and Kleckner have recently proposed that bacterial cell division is a licensing agent for a subsequent initiation of DNA replication. They also propose that initiation mass for DNA replication is not constant. These two proposals do not take into account older data showing that initiation of DNA replication can occur prior to the division event. This critical analysis is derived from measurements of DNA replication during the division cycle in cells growing at different, and more rapid, growth rates. Furthermore, mutants impaired in division can initiate DNA synthesis. The data presented by Bates and Kleckner do not support the proposal that initiation mass is variable, and the proposed pattern of DNA replication during the division cycle of the K12 cells analysed is not consistent with prior data on the pattern of DNA replication during the division cycle.     

A conjugation procedure for Bdellovibrio bacteriovorus and its use to identify DNA sequences that enhance the plaque-forming ability of a spontaneous host-independent mutant.

Wild-type bdellovibrios are obligate intraperiplasmic parasites of other gram-negative bacteria. However, spontaneous mutants that can be cultured in the absence of host cells occur at a frequency of 10(-6) to 10(-7). Such host-independent (H-I) mutants generally display diminished intraperiplasmic-growth capabilities and form plaques that are smaller and more turbid than those formed by wild-type strains on lawns of host cells. An analysis of the gene(s) responsible for the H-I phenotype should provide significant insight into the nature of Bdellovibrio host dependence. Toward this end, a conjugation procedure to transfer both IncQ and IncP vectors from Escherichia coli to Bdellovibrio bacteriovorus was developed. It was found that IncQ-type plasmids were capable of autonomous replication in B. bacteriovorus, while IncP derivatives were not. However, IncP plasmids could be maintained in B. bacteriovorus via homologous recombination through cloned B. bacteriovorus DNA sequences. It was also found that genomic libraries of wild-type B. bacteriovorus 109J DNA constructed in the IncP cosmid pVK100 were stably maintained in E. coli; those constructed in the IncQ cosmid pBM33 were unstable. Finally, we used the conjugation procedure and the B. bacteriovorus libraries to identify a 5.6-kb BamHI fragment of wild-type B. bacteriovorus DNA that significantly enhanced the plaque-forming ability of an H-I mutant, B. bacteriovorus BB5.     

The timing of initiation of macronuclear DNA synthesis is set during the preceding cell cycle in Paramecium tetraurelia. Analysis of the effects of abrupt changes in nutrient level

In many eukaryotic organisms, initiation of DNA synthesis is associated with a major control point within the cell cycle and reflects the commitment of the cell to the DNA replication-division portion of the cell cycle. In Paramecium, the timing of DNA synthesis initiation is established prior to fission during the preceding cell cycle. DNA synthesis normally starts at 0.25 in the cell cycle. When dividing cells are subjected to abrupt nutrient shift-up by transfer from a chemostat culture to medium with excess food, or shift-down from a well-fed culture to exhausted medium. DNA synthesis initiation in the post-shift cell cycle occurs at 0.25 of the parental cell cycle and not at either 0.25 in the post-shift cell cycle or at 0.25 in the equilibrium cell cycle produced under the post-shift conditions. The long delay prior to initiation of DNA synthesis following nutritional shift-up is not a consequence of continued slow growth because the rate of protein synthesis increases rapidly to the normal level after shift-up. Analysis of the relation between increase in cell mass and initiation of DNA synthesis following nutritional shifts indicates that increase in cell mass, per se, is neither a necessary nor a sufficient condition for initiation of DNA synthesis, in spite of the strong association between accumulation of cell mass and initiation of DNA synthesis in cells growing under steady-state conditions.     

Periodic synthesis of phospholipids during the Caulobacter crescentus cell cycle.

Net phospholipid synthesis is discontinuous during the Caulobacter crescentus cell cycle with synthesis restricted to two discrete periods. The first period of net phospholipid synthesis begins in the swarmer cell shortly after cell division and ends at about the time when DNA replication initiates. The second period of phospholipid synthesis begins at a time when DNA replication is about two-thirds complete and ends at about the same time that DNA replication terminates. Thus, considerable DNA replication, growth, and differentiation (stalk growth) occur in the absence of net phospholipid synthesis. In fact, when net phospholipid synthesis was inhibited by the antibiotic cerulenin through the entire cell cycle, both the initiation and the elongation phases of DNA synthesis occurred normally. An analysis of the kinetics of incorporation of radioactive phosphate into macromolecules showed that the periodicity of phospholipid synthesis could not have been detected by pulse-labeling techniques, and only an analysis of cells prelabeled to equilibrium allowed detection of the periodicity. Equilibrium-labeled cells also allowed determination of the absolute amount of phosphorus-containing macromolecules in newborn swarmer cells. These cells contain about as much DNA as one Escherichia coli chromosome and about four times as much RNA as DNA. The amount of phosphorus in phospholipids is about one-seventh of that in DNA, or about 3% of the total macromolecular phosphorus.     

Effect of tunicamycin on cell cycle progression in budding yeast

Tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharides, prevents bud formation and growth in Saccharomyces cerevisiae cells that are either growing exponentially or recovering from different cell cycle arrests at start. Analysis of tunicamycin-treated cells by flow microfluorometry clearly shows that cells have a postsynthetic DNA content, but there is no evidence of an increase in binucleate cells. Therefore tunicamycin affects bud emergence and initiation of DNA synthesis, two events correlated under physiological conditions, in different ways. A bulk glycoprotein synthesis is shown to be required for bud emergence and localized chitin deposition, probably to sustain directional secretory vesicle transport, which allows polar growth of the bud. No evidence for a glycoprotein requirement for entrance into the S phase is obtained from the present experiments.