Synchronization of Division in Vitamin B12-Starved Euglena gracilis

SYNOPSIS Vitamin B₁₂ deficiency arrests cell division in Euglena gracilis. B₁₂ starvation for short periods made it possible to induce synchronous growth by addition of the vitamin. Culture conditions were established to optimize replenishment synchrony. The DNA content of E. gracilis in steady state culture and vitamin B₁₂ deficiency culture was measured by flow cytofluorometry and was consistent with colorimetric determinations. The cell volume and DNA distributions of E. gracilis in synchronous culture were analyzed and the sequential changes during the division cycle were computed. Synchronous culture permits more definitive studies of shifts in cell volume and DNA distributions, in which the biochemical events required for cell division are presumably synchronized.     

DNA synthesis in relation to a varying vitamin B12 content in Propionibacterium shermanii cells

The content of DNA in Propionibacterium cells is nearly twice as low at vitamin B12 deficiency comparing with normal cells. The rate of labeled adenine incorporation into the DNA depends on vitamin B12 content in the cells. The addition of adenosylcobalamin and thymine to the medium makes DNA content rise in the cells of the B12-deficient culture. The addition of thymine to a suspension of B12-deficient cells accelerates the incorporation of labelled adenine into DNA. The authors discuss the phenomenon of bacterial "unbalanced division" and the control of replication by changing the level of DNA precursors.     

Growth and cell volume of Euglena gracilis in different media.

It is necessary to propagate Euglena gracilis cells for several days after transfer from one medium to another to establish the steady state of balanced growth. Steady-state growth was established in minimal and in complex medium. Specific growth rates and cell volume distributions were computed for each culture medium. Mean cell volume of E. gracilis is not uniquely correlated with the specific growth rate.     

Growth and cell volume of Euglena gracilis in different media.

It is necessary to propagate Euglena gracilis cells for several days after transfer from one medium to another to establish the steady state of balanced growth. Steady-state growth was established in minimal and in complex medium. Specific growth rates and cell volume distributions were computed for each culture medium. Mean cell volume of E. gracilis is not uniquely correlated with the specific growth rate.     

Inhibition of an early event in the cell division cycle of Escherichia coli by FL1060, an amidinopenicillanic acid.

Analysis of exponential and synchronous cultures of Escherichia coli B/r after the addition of FL1060 indicates a block point for division by this agent some 15 to 20 min before the end of the preceding cell division cycle, a time corresponding to the beginning of the C period of the cell division cycle. Morphological examination of FL1060-treated synchronous cultures of E. coli /r was consistent with inhibition by FL1060 of a very early event in the cell division cycle. This event appears to be essential for normal cell surface elongation in a rod configuration. Temporary treatment of synchronous cultures of E. coli B/r with FL1060 resulted in division delay, the extent of which was a function of the duration of exposure to FL1060. However, even after relatively long times of FL1060 treatment the delayed divisions were still synchronous. Although FL1060 had no direct effect on deoxyribonucleic acid (DNA) synthesis, the synchronous delayed division occuring after temporary treatment with FL1060 were accompanied by a delay in the attainment of resistance of cell division to inhibitors of DNA, ribonucleic acid, and protein synthesis. These results suggest aht an FL1060-sensitive event initiates at the beginning of the C period of the cell division cycle of E. coli and is responsible for normal cell elongation. This cell elongation pathway procedes independently of DNA synthesis, but there is an interaction between this pathway and termination of a round of DNA replication in which a normal rod configuration is necessary to allow a signal for cell division to be generated upon completion of DNA replication.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : II. Recovery from Unbalanced Growth Induced by Vitamin B12 Deficiency

When vitamin B₁₂ is added to B₁₂-deficient cultures of Euglena gracilis, the cells undergo two relatively synchronous cell divisions within a shorter than usual period of time, apparently as a result of a transitory shortening of the cell cycle. The first cell division pulse, occurring 4.5 h after addition of B₁₂, is preceded by the completion of DNA duplication, but appears to involve no net synthesis of RNA or protein. Before the second round of cell division at about 11 h, a significant amount of DNA synthesis is observed. This time it is accompanied by a minor increase in the RNA and protein content of the culture. The cellular contents of RNA and protein were observed to decrease steadily after the resumption of cell division in B₁₂-depleted cultures receiving the vitamin. Ultimately all three macromolecules returned to their nondeficient, plateau stage levels; by this time, cell division had ceased.     

Ribonucleotide reductase activity in vitamin B12-deficient Euglena gracilis.

The ribonucleotide reductase activities in vitamin B12-sufficient and -deficient cells of Euglena gracilis were measured. We found that the cells progress into vitamin B12 deficiency the enzyme activity increases, reaching a maximum value of 20-fold in advanced deficiency. No signigicant differences in the activities were found to result as a consequence of different growth conditions. We propose that the increased activity in vitamin B12-deficient cells is due to an increase in enzyme protein.     

Studies on Bacterial Cell Multiplication

Chemical composition of the filamentous cells of Lactobacillus delbrueckii produced by vitamin B₁₂ deficient culture was studied. Protein and RNA contents per unit cell volume of the filamentous cells were nearly equal to those of normal cells, but the DNA content was much reduced. A cytoplast of the filamentous cell possessed about twice as large volume as that of the normal cell. A cytoplast of either filamentous or normal cell seems to contain the same amount of DNA. DNA level and membrane formation necessary for cell division remained as future problems.     

Production of antioxidant vitamins, beta-carotene, vitamin C, and vitamin E, by two-step culture of Euglena gracilis Z

Euglena gracilis Z is one of the few microorganisms which simultaneously produces antioxidant vitamins such as beta-carotene and vitamins C and E. Photoheterotrophically cultured E. gracilis Z produced larger levels of biomass but with a lower content of antioxidant vitamins than photoautotrophically grown cultures. For efficient production of these vitamins, a two-step culture was performed. Cells were grown photoheterotrophically and then transferred to photoautotrophic conditions. When E. gracilis Z cells were grown in fed-batch culture under photoheterotrophic conditions, their density reached 19 g/L after 145 h. Subsequent transfer of these cells to photoautotrophic conditions increased vitamin content, enhancing the total vitamin yields, which were 71.0 mg/L of beta-carotene, 30.1 mg/L of vitamin E, and 86.5 mg/L of vitamin C. (c) 1997 John Wiley & Sons, Inc.     

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.     

Synchronous Growth and Plastid Replication in the Naturally Wall-less Alga Olisthodiscus luteus

Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B₁₂ to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B₁₂, plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.     

Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations.

A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.     

Influence of penicillin and nalidixic acid on growth and cell division of Escherichia coli K-12

Ultrastructure of E. coli K-12 cells and the synthesis of DNA in bacteria treated with low concentration of nalidixic acid and penicillin was investigated. In E. coli both drugs caused inhibition of cell division in period D of the life cycle although nalidixic acid inhibits division at an earlier stage of septum formation. The ability of cells to form filaments in the presence of nalidixic acid depends on their age, i.e. time at which cells are taken from synchronous culture.     

Folic acid fortification: why not vitamin B12 also?

Folic acid fortification of cereal grains was introduced in many countries to prevent neural tube defect occurrence. The metabolism of folic acid and vitamin B12 intersect during the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine catalyzed by B12-dependent methioine synthase. Regeneration of tetrahydrofolate via this reaction makes it available for synthesis of nucleotide precursors. Thus either folate or vitamin B12 deficiency can result in impaired cell division and anemia. Exposure to extra folic acid through fortification may be detrimental to those with vitamin B12 deficiency. Among participants of National Health And Nutrition Examination Survey with low vitamin B12 status, high serum folate (>59 nmol/L) was associated with higher prevalence of anemia and cognitive impairment when compared with normal serum folate. We also observed an increase in the plasma concentrations of total homocysteine and methylmalonic acid (MMA), two functional indicators of vitamin B12 status, with increase in plasma folate under low vitamin B12 status. These data strongly imply that high plasma folate is associated with the exacerbation of both the biochemical and clinical status of vitamin B12 deficiency. Hence any food fortification policy that includes folic acid should also include vitamin B12.     

Sequential changes in cell volume distribution during vitamin B12 starvation of Euglena gracilis.

During vitamin B12 starvation of Euglena, a new peak appears in the cell volume distribution. Some cells are inhibited at a unique point in the cell cycle between the initiation of DNA synthesis and nuclear division. The mechanism of inhibition of other cells differs.     

The role of vitamin B12 in the metabolism of Euglena gracilis var. bacillaris

1. The concentrations of RNA, DNA and protein are decreased in cells of Euglena gracilis var. bacillaris grown on suboptimum concentrations of vitamin B(12). 2. The addition of vitamin B(12) to deficient cells stimulates the incorporation of [(14)C]formate into the above cell components as well as into thymine of DNA and serine and methionine of protein. 3. In a cell-free system from vitamin B(12)-deficient cells, the incorporation of labelled formate into thymidylate is decreased to a greater extent with uridine than with deoxyuridine as the substrate. 4. The addition of unlabelled glutamate dilutes the radioactivity incorporated into thymine from labelled formate. 5. These results are interpreted to mean that, in DNA synthesis, vitamin B(12) has a greater role in the reduction of ribotides to deoxyribotides than in the reduction of formate to thymine methyl and that the vitamin B(12)-dependent conversion of glutamate into beta-methylaspartate also contributes to thymine synthesis.     

Localization of vitamin B12 binding in Euglena gracilis

Different fractionation procedures were used to determine the location of vitamin B12 binding sites in Euglena gracilis. Using uptake measurements, cell fractionation, and light and electron microscopy, the cuticle of the cell was found to be the fraction containing the majority of B12 binding sites. The apparent distribution of vitamin binding sites differed according to the cell lysis method used. The cuticle fraction was responsible for the binding of 80% of the vitamin taken up by the cell during both the rapid and the slow phase of uptake. These results suggest that vitamin B12 binding is regulated, in part, at the cuticle level, and support our previous conclusion that the secondary phase of uptake represents the synthesis of new receptor sites and not the unloading of vitamin inside the cell.     

X-Ray and Ultraviolet Sensitivity of Synchronously Dividing Escherichia coli

A paper pile filtration technique was used to obtain synchronously dividing populations of E. coli strains B and B/r from cultures in the exponential growth phase. Three generations of highly phased cell division were obtained by rapid pressure filtration which selected approximately 1 per cent of the exponentially growing culture. The sensitivity of E. coli strain B to x-ray and UV inactivation as a function of the cell division cycle was determined on synchronous populations. E. coli strain B showed a sharp decrease in sensitivity to inactivation by both radiations in the middle of the division cycle, and a further decrease near the end of the cycle. The sensitivity of E. coli strain B/r to x-irradiation was also investigated. Only the mid-cycle decrease in sensitivity was found during the division cycle of this strain. It was concluded that the repetition of the observed sensitivity patterns in both strains through the first three cycles after synchronization indicates that the same basic sensitivity patterns are probably also present in the individual cells of an exponential phase culture.     

Evolution of ornithine decarboxylase activity during the cell cycle of Euglena gracilis Z in synchronous culture. Influence of vitamin B-12.

Ornithine decarboxylase activity in Euglena gracilis Z was studied during the normal cell cycle and in vitamin B-12 deficiency. The cells were synchronized by means of alternating periods of light and dark. During the normal cell cycle, ornithine decarboxylase activity was very weak in the dark period, while three peaks of activity were recognized in the light period. The first peak, in the G1 phase, occurred when luminous stimulation started; the second preceded the S phase and the third was found in G2. In B-12-deficient cells, ornithine decarboxylase activity was greatly decreased and only the first peak remained. Elimination of the deficiency by addition of vitamin B-12 to the medium induced a very fast and significant increase in ornithine decarboxylase activity.     

A relationship between adenosine 3'-5'-cyclic monophosphate levels and deoxyribonucleic acid synthesis in Euglena

Using the binding protein method we found that cAMP levels in normal, exponentially growing Euglena stay constant on per cell and protein basis. The level rises slightly when cells enter the stationary stage. Cells growing in low vitamin B12 medium show the same pattern during predeficiency growth. Upon becoming vitamin B12 deficient, the cAMP level decreases. Replenishment of these cells with the vitamin causes an immediate drop, followed by a sharp rise in cAMP. This is followed by resumption of DNA synthesis. The cAMP level drops and rises again when DNA duplication is completed and during the G2 period. The level of the cAMP drops again followed by resumption of cell division. the data suggest a relation exists between cAMP level, resumption and completion of DNA synthesis, and cell division.