EFFECTS OF CHLORAMPHENICOL ON GROWTH, SIZE DISTRIBUTION, CHLOROPHYLL SYNTHESIS AND ULTRASTRUCTURE OF EUGLENA GRACILIS

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1 J. Cell Sci. 4, (1969) 627 Printed in Great Britain EFFECTS OF CHLORAMPHENICOL ON GROWTH, SIZE DISTRIBUTION, CHLOROPHYLL SYNTHESIS AND ULTRASTRUCTURE OF EUGLENA GRACILIS Y. BENSHAUL AND Y. MARKUS laboratory for Electron Microscopy, TelAviv University, TelAviv, Israel SUMMARY Multiplication of Euglena cells treated by 0510 mg/ml chloramphenicol was not disturbed for the first 36 h and inhibition appeared only at later stages. The mean cell volume of treated dividing cells was decreased, although the initial rise in cell volume, which normally occurred during the first 12 h of incubation, was not prevented. The antibiotic also lowered the chlorophyll content of green dividing cells. In darkgrown cells transferred to light, inhibition of chlorophyll synthesis was immediate but not complete, and was followed by a decreased rate of plastid elongation and thylakoid formation. Our findings suggest that chloramphenicol does not cause the loss of existing pigment and that impaired chlorophyll synthesis is a secondary effect of inhibition of protein synthesis. The results also indicate that the greening process is more sensitive than cell division to the antibiotic. INTRODUCTION Chloramphenicol (CM) inhibits protein synthesis both in vivo and in vitro; according to most investigators the inhibition affects amino acid transfer from S RNA to the site of peptide bond formation (see review by Gale, 1963). Bacteria are inhibited by concentrations of 110/tg/ml (Brock, 1961). Fungi, higher plants and animal systems are not affected (Von Ehrenstein & Lipmann, 1961; Rendi & Ochoa, 1962). In bacteria incubated in the presence of CM, the rate of RNA synthesis increases and 15 s 'CM particles' are observed. These are considered to be precursors of ribosomes which are unable to aggregate due to inhibition of protein synthesis. Another possibility is that these particles are mrna which was prevented from binding to ribosomes by the CM (Gale, 1963). The selective effect of CM on different organisms is explained by the hypothesis that the interaction between ribosomes and mrna is affected. Accordingly bacteria are affected since 70s bacterial ribosomes bind the template RNA more weakly than the 80s ribosomes of plants and animal systems (Eisenstadt, 1967), or the CM is strongly attached to the 70s particles (Linnane & Stewart, 1967). Weisberger, Wolfe & Armentrout (1964) and Kucan & Lipmann (1964) maintain that only a new template RNA binding itself to the ribosomes is sensitive to CM, whereas informative RNA which is already bound is not inhibited.

2 628 Y. BenShaul and Y. Markus CM inhibits greening in darkgrown Euglena cultures exposed to light (Pogo & Pogo, 1965; Aaronson, Ellenbogen, Yellen &Hutner, 1967; Linnane & Stewart, 1967). Since Euglena cytoplasmic ribosomes are similar to those of mammals and chloroplast ribosomes to those of bacteria, it was suggested that in Euglena CM inhibits protein synthesis in chloroplasts and to a lesser extent protein synthesis in the cytoplasm. In the light of the hypothesis that chloroplasts are selfreplicating entities (Schiff & Epstein, 1965) it was of special interest to examine the effect of CM on the transformation of proplastids into chloroplasts. When mrna synthesis in proplastids is induced by exposure to light, the messenger formed in the organelle attaches itself to ribosomes constantly present in the proplastids and serves as a template for the synthesis of plastid structural proteins and enzymes (Gnanam & Kahn, 1967). Thus, CM could serve as a tool in the study of the correlation between protein synthesis and the formation of plastid structure. Only scarce data are available on the effect of CM on the greening of dividing cells and the fine structure of Euglena. The aim of this work was to examine changes, particularly ultrastructural, in Euglena cells growing in a medium containing CM, and to correlate these changes with other physiological parameters such as cell division and chlorophyll synthesis. MATERIALS AND METHODS Euglena gracilis var. bacillaris was cultured in Hutner's ph 33 growing medium (dividing cells) (Greenblatt & Schiff, 1959) or in resting medium composed of mannitol, MgCl 2 and KH 2 PO 4 (nondividing cells). The cultures were illuminated by optimal light of 100 ftc (Stern, Epstein & Schiff, 1964) from fluorescent bulbs, with continuous shaking at 2426 C. Darkgrown cells were obtained by periodic transfers to fresh growing medium in the dark. Chloramphenicol was dissolved in the media by prolonged shaking. Counting of cells was carried out in a Model B Coulter Counter, using a ioo/t aperture, and size distribution graphs were obtained with an accessory plotter which was carefully calibrated with a microhaematocrit. Chlorophyll was extracted with 80% acetone, and optical densities measured and recorded in a Cary Model 14 recording spectrophotometer. Chlorophylls a + b were estimated according to Mackinney (1941), using the following extinction coefficients: for chlorophyll a, 1009 an d T 5"8 for the wavelengths 662 m/t and 647 m/x; for chlorophyll b, 62 and 620 for the same wavelengths. Euglena cells were prepared for electron microscopy in the following way. Pellets obtained by gentle centrifugation were fixed with 5% glutaraldehyde in oi M phosphate buffer at 4 C for 1 h and postfixed with 1 % osmium tetroxide in the same buffer for 3 h. After dehydration by 10min transfers through a graded ethanol series followed by propylene oxide, the pellets were embedded in Epon 812. Sections of A were prepared using glass knives on an LKB ultramicrotome and collected on Formvarcoated, carbonreinforced grids. The sections on grids were stained overnight with a saturated solution of uranyl acetate in 30% ethanol and

3 Effects of chloramphenicol on Euglena 629 poststained for 12 min with lead citrate according to Venable & Coggeshall (1965). The stained sections were examined in a Jeolco Jem7 electron microscope. RESULTS Cell division Populations of Euglena cells growing under continuous light or cells transferred from dark to light showed a typical Sshaped pattern of growth. 320r h x 160 I 120 U Hours Fig. i. The effect of chloramphenicol on division rate of lightgrown cells.'cells at stationary phase were transferred to fresh growing medium. Samples were taken after various periods for determination of cell number, size distribution and chlorophyll content., Control;, CM, 05 mg/ml;, CM, io mg/ml. Table 1 A. Stimulation of divisions in Euglena cells treated with CM for 36 h (in % of stimulation as compared to controls). (In greening cells the antibiotic was applied for 12 h in the dark and for an additional 24 h after exposure to light) Lightgrown green Darkgrown cells exposed to light (greening cells) cells, * ^ + 05 mg/ml CM +02 mg/ml CM +03 mg/ml CM +05 mg/ml CM S9'3 n7 5'5 60 Table 1B. The increase (%) in the mean cell volume of dividing lightgrown and darkgrown Euglena cells treated for 12 h with CM Lightgrown cells Darkgrown cells Control + 05 mg/ml CM Control + 05 mg/ml CM +20 mg/ml CM

4 630 Y. BenShaul and Y. Markus When grown in a medium containing oi mg/ml CM, the growth rate was similar to the control. When higher concentrations of CM, 05 and io mg/ml, were applied, the rate of cell division during the first 36 h increased (Table 1 A, Fig. 1). Only later was inhibition of cell division observed. In darkgrown cells transferred to light, the early increase in cell division was small; in green cells, however, the increase was pronounced and was significantly observed in four independent experiments (Table 1 A). In CM concentrations of 24 mg/ml the inhibition of cell division was complete and viability of cells stopped after 48 h. Cells kept in resting medium did not divide and their viability was not affected by the antibiotic material. Size distribution In order to study the effect of the antibiotic material on the volume of the cells, their size distributions were recorded at different periods after the application of the CM (Fig. 2). The calculations of mean cell volumes were made according to Brecher et al. (1962) Relative cell size Fig. 2. Coulter counter plot depicting relative frequency distribution of cell sizes. Details as in Fig. 1. A, after 12 h in the fresh medium, B, after 72 h in the fresh medium. C, after 72 h in medium containing 05 mg/ml CM. It was found that in nondividing cells the mean cell volume did not change in either control or treated cells. In dividing cells, both treated and untreated, however, a marked increase in cell volume was observed after the first 12 h of incubation

5 Effects of chloramphenicol on Euglena 631 (Table 1 B, Fig. 3). This period coincided with a lag phase in which no divisions were observed. Later, a slow decrease in the mean cell volume was observed which was probably due to the increasing division rate. When the growth curves reached their plateaus, the mean cell volumes were also in a steady state (Fig. 3). It seems that the treated cells differed significantly from the untreated only by a consistently lower mean volume after the first 12 h (Fig. 3). A possible explanation would be that the treated cells were prevented from growing in volume once division started. Identical results were obtained both for green cells and for darkgrown cells exposed to light. & x 3 0 "^25 0> o "5 15 S Hours 120 Fig. 3. Effect of CM on the mean cell volumes of lightgrown, cells. Details as in Fig. 1., Control;, CM, oi mg/ml;, CM, 10 mg/ml. In an additional experiment, green cells maintained in resting medium were treated with io mg/ml CM for 44 h and then transferred to growing medium containing the same concentration of CM. The prolonged preincubation of the cells in the resting medium with CM did not prevent the 'jump' in the cell volume in growing medium. The increase in mean cell volume was similar to that recorded for cells which underwent the same procedure but without the antibiotic. The recorded increase in mean cell volume was 62% for untreated cells and 66 % for cells treated with io mg/ml CM. This experiment indicates that the lack of effect of CM on the increase in volume which takes place during the first 12 h in growing medium is not due to slow permeability into the cells. The sizedistribution curves (Fig. 2 B, c) are not symmetrical; each of them is composed of two symmetrical curves, one representing most of the cells which were small, the other representing a few cells in the state of division or cells just divided but still connected to each other. The latter cells contained double volumes and were therefore recorded in the 'high' volume 'windows'. A symmetrical curve was obtained only after 12 h in CM (Fig. 2 A). This was the

6 632 Y. BenShaul and Y. Markus only synchronic phase in which, as mentioned above, increase in volume with no cell division was found. Chlorophyll content In green cells maintained in resting medium no change in chlorophyll content was found in either control or treated cells. In untreated dividing cells, the amount of total chlorophyll per cell decreased during the first 36 h (Fig. 4). After 36 h an increase in chlorophyll content was observed which reached a plateau after about 72 h. During that period the rate of divisions was decreasing (Fig. 1). In cells treated with OIIO mg/ml CM the decrease in chlorophyll content during the first 36 h was more pronounced and with 0510 mg/ml no recover)' of the pigment synthesis was observed (Fig. 4). 10 c 9 8 ^> n o 6 > a. 2 3 U Hours 72 Fig. 4. Effect of CM on chlorophyll content of lightgrown cells. Details as in Fig. 1., Control;, CM, oi mg/ml;, CM, io mg/ml. In greening experiments, darkgrown cells were preincubated for 12 h with the antibiotic and then exposed to light. This was done to ensure penetration of CM into the cells. It was found that in both dividing and nondividing cells CM concentrations of OIIO mg/ml inhibited chlorophyll synthesis. The degree of inhibition was proportional to the concentration of CM. In oi mg/ml the inhibition was observed only in the first few days, whereas in concentrations of 0510 mg/ml CM the inhibition was more pronounced and greening was very slow. In all experiments the curve depicting chlorophyll synthesis of control cells was exponential and that of treated cells linear (Fig. 5). The inhibition of chlorophyll synthesis was never complete, even in cells treated with high concentrations of CM (Table 2). The inhibition of chlorophyll synthesis by 05 mg/ml CM was immediate and was obsen'ed already after illumination for 4 h (Fig. 5).

7 doj Electron microscopy Effects of chloramphenicol on Euglena 633 No structural changes were observed in green cells maintained in resting medium for 85 h in the presence of 05 mg/ml CM. However, in growing green cells maintained for 60 h in medium containing io mg/ml the following structural changes were observed. The number of lamellae per plastid was much lower (Fig. 9) than that found in untreated cells (Fig. 8). The number of thylakoids per lamella was variable and the thylakoids were swollen. In many cells the mitochondria were abnormal = 10 V * ^ j >' A / / A / T / ". ^ /, 08 3" t 07 " u ~ 0 _ / s^ / ^r A >' / / / / i / ^^^^^ Houn Fig. 5. The effect of 05 mg/ml CM on chlorophyll synthesis in darkgrown cells exposed to light. Darkgrown cells at stationary phase were transferred to fresh growing medium (G.M.) or resting medium (R.M.). After 12 h of preincubation in the dark the cells were exposed to light (time o). Samples were taken for determination of chlorophyll content and for electron microscopy., G.M., control;, R.M., control;, G.M. + CM;, R.M. + CM. Table 2. Inhibition of chlorophyll synthesis (% as compared with controls) in darkgrown cells exposed for 24 h to light in the presence of CM + zo mg/ml CM + 40 mg/ml CM Dividing cells Nondividing cells Coll Sci. 4

8 634 Y. BenShaul and Y. Markus (Fig. 9). In the cytoplasm electrondense round bodies which seem to be of lipid nature were often seen (Fig. 10). In order to study the development of plastids in cells treated with 05 mg/ml CM, samples of darkgrown cells exposed to light were taken at different developmental stages for cell counts, pigment extraction and electron microscopy. In the presence of CM the rate of plastid elongation and thylakoid formation was markedly reduced 9r a. O o Hours 48 Fig. 6. The effect of CM on chloroplast elongation. Details as in Fig. 5., G.M. control;, R.M., control;, G.M. + CM;, R.M. + CM fio B 8 1 < Hours 48 Fig. 7. The effect of CM on thylakoid formation. Details as in Fig. 5., G.M. control;, R.M., control;, G.M. + CM;, R.M. + CM.

9 Effects of chbramphenicol on Euglena 635 (Figs. 6, 7). The average number of thylakoids per lamella was lower than in untreated cells in all developmental stages, indicating that the antibiotic decreased the rate of thylakoid fusion into lamellae. The inhibition effects of CM were similar in both dividing and nondividing cells with one exception; in nondividing cells the inhibition of plastid elongation was demonstrated only during the first 24 h (Fig. 6). Statistically the differences in structure between treated and untreated cells were significant with at least 99% confidence. The only insignificant change was the one after 4 h. The proplastids of darkgrown cells were about 1 /i in diameter, and contained ribosomes (Fig. 11). After 4 h in light few thylakoids were seen in the plastids (Fig. 12). A marked elongation of chloroplasts and the organization of thylakoids in lamellae were observed after 12 h in the light (Fig. 13). In treated cells after 12 h of illumination the plastids were not developed and contained only a very small number of unfused thylakoids. In these plastids, particularly those of treated resting cells (Figs. 14, 15), the stroma contained several aggregates of amorphous granular material. These dense granules seemed to be smaller than the ribosomes observed in plastids of untreated cells. After 24 h in the light, plastids of treated cells (Fig. 17) were less developed than those of untreated cells (Fig. 16). Normal chloroplasts after 48 h were very long and contained a large number of lamellae (Fig. 18). In treated cells the chloroplasts were smaller, and contained fewer, mostly unfused, thylakoids. Sometimes the chloroplasts of treated cells were abnormally large and contained unorganized lamellae (Fig. 19). DISCUSSION The rate of cell division in lightgrown Euglena was significantly higher during the first 36 h in the presence of CM (Table 1). A possible explanation could be that in CM the mean cell volumes are smaller. This decrease in volume might be a result of inhibition of protein synthesis. Normally cells reach a certain critical volume before they start to divide. Treated cells probably divide before that critical volume. It is interesting to note in this connexion that in resting cells which do not divide, CM does not affect the volume. CM perhaps enables the division of small cells to occur, and thus promotes division. The inhibition of cell divisions appears late in treated cells and might be a result of exhaustion of protein 'pools'. In treated darkgrown cells exposed to light no promotion of cell division was detected. In this case a major part of the protein 'pool' is probably used for greening and therefore the decrease in cell volume is not accompanied by promotion of cell division. Chloramphenicol inhibits chlorophyll synthesis in cells transferred from dark to light. In untreated green dividing cells the initial decrease in chlorophyll content per cell (Fig. 4) can be explained by the inability of the cells to form the pigment at a rate comparable to the rate of cell division. This is in contrast to the situation in treated dividing cells in which a dilution of existing pigment among the progeny was probably accompanied by an inhibition of new pigment synthesis. 402

10 636 Y. BenShaul and Y. Markus In green resting cells and in green cells, both resting and dividing which were returned to the dark, chlorophyll content was similar. It seems, therefore, that when the cells are kept under conditions in which no chlorophyll synthesis occurs, the antibiotic does not cause loss of existing pigment. As reported, green dividing cells in the presence of CM have less chlorophyll and fewer thylakoids per chloroplast than controls. This inhibition of both chlorophyll and membranes, which was also found in greening treated cells, confirms the view of Pogo & Pogo (1965) that impaired chlorophyll synthesis is a secondary effect of the inhibition of enzymes and structural proteins, parts of which are needed for new chlorophyll synthesis. As mentioned, in the presence of CM no inhibition of division was observed during the first 36 h, whereas inhibition of greening was immediate. It seems that the greening process is more sensitive to CM than cell division, indicating that protein synthesis is a major and immediate need for greening. These findings are in accordance with works showing that plastid ribosomes are more sensitive to CM than cytoplasmic ribosomes (Aaronson et al. 1967; Eisenstadt, 1967; Linnane & Stewart, 1967). Indeed, it is possible that the aggregates of grainy, amorphous material observed in plastids of treated greening cells are identical or similar to the 'CM particles' described by Gale (1963). In treated greening cells both elongation of plastids and formation of thylakoids are inhibited. The curves depicting kinetics (Figs. 6, 7) show that in untreated resting cells the length of the plastids is tripled between 4 and 12 h after exposure to light; after this period the elongation is slower until it reaches a plateau. This 'jump' in length was attributed by BenShaul, Schiff & Epstein (1965) to a linear fusion of three proplastids into one chloroplast. In treated resting cells this 'jump' in elongation was found between 24 and 28 h. It can be speculated that CM caused a delay in proplastid fusion. It remains to be checked whether for up to 24 h in treated cells the number of proplastids is equal to the number found in darkgrown cells before fusion of proplastids. In treated growing cells no 'jump' in plastid length was observed (Fig. 6). This is probably due to a dilution factor resulting from cell divisions. The dilution seems to affect also the rate of thylakoid formation (Fig. 7). Whereas in resting cells both with or without CM the rate of thylakoid formation is exponential, in dividing cells both treated and untreated, an absolute decrease in the number of thylakoids between 1224 h was observed. In that time interval the rate of divisions is the highest. Since chloroplasts seem to divide lengthwise (Schiff & Epstein, 1965), the number of thylakoids is diluted, whereas the length is not affected. REFERENCES AARONSON, S., ELLENBOGEN, B. B., YELLEN, L. K. & HUTNER, S. H. (1967). In vivo differentiation of Euglena cytoplasmic and chloroplast protein synthesis with chloramphenicol and DLethionine. Biochem. biophys. Res. Commiin. 27, BENSHAUL, Y., SCHIFF, J. A. & EPSTEIN, H. T. (1964). Studies of chloroplast development in Euglena. VII. Fine structure of the developing plastid. PL Physiol., Lancaster, 39,

11 Effects of chloramphenicol on Euglena 637 BRECHER, G., JAKOBEK, E. F., SCHNEIDERMAN, M. A., WILLIAMS, G. Z. & SCHMIDT, P. J. (1962). Size distribution of erythrocytes. Ann. N.Y. Acad. Set. 99, BROCK, T. D. (1961). Chloramphenicol. Bad. Rev. 25, EISENSTADT, J. M. (1967). Protein synthesis in chloroplasts and chloroplast ribosomes. In Biochemistry of Chloroplasts, vol. 2 (ed. T. W. Goodwin), pp New York: Academic Press. GALE, E. F. (1963). Mechanisms of antibiotic action. Pharmac. Rev. 15, GNANAM, A. & KAHN, J. S. (1967). Biochemical studies on the induction of chloroplast development in Euglena gracilis. III. Ribosome metabolism associated with chloroplast development. Biochivi. biophys. Acta 142, GREENBLATT, C. L. & SCHIFF, J. A. (1959). A pheophytinlike pigment in darkadapted Euglena gracilis. J. Protozool. 6, KUCAN, Z. & LIPMANN, F. (1964). Differences in chloramphenicol sensitivity of cellfree amino acid polymerization systems. J. biol. Chem. 239, LINNANE, A. W. & STEWART, P. R. (1967). The inhibition of chlorophyll formation in Euglena by antibiotics which inhibit bacterial and mitochondrial protein synthesis. Biochem. biophys. Res. Comniun. 27, MACKINNEY, G. (1941). Absorption of light by chlorophyll solutions. J. biol. Chem. 140, Poco, B. G. T. & Poco, A. O. (1965). Inhibition by chloramphenicol of chlorophyll and protein synthesis and growth in Euglena gracilis. J. Protozool. 12, RENDI, R. & OCHOA, S. (1962). Effect of chloramphenicol on protein synthesis in cellfree preparations of Escherichia coli.j. biol. Chem. 237, SCHIFF, J. A. & EPSTEIN, H. T. (1965). The continuity of the chloroplast in Euglena. In Reproduction: Molecular, Subcellular, and Cellular, pp New York: Academic Press. STERN, A. I., EPSTEIN, H. T. & SCHIFF, J. A. (1964). Studies of chloroplast development in Euglena. VI. Light intensity as a controlling factor in development. PI. Physiol., Lancaster 39, VENABLE, J. H. & COCGESHALL, R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, VON EHRENSTEIN, G. & LIPMANN, F. (1961). Experiments on hemoglobin biosynthesis. Proc. natn. Acad. Sci. U.S.A. 47, WEISBEHGER, A. S., WOLFE, S. & ARMENTROUT, S. (1964). Inhibition of protein synthesis in mammalian cellfree systems by chloramphenicol. J. exp. Med. 120, (Received 21 August 1968 Revised 18 November 1968)

12 6 3 8 Y. BenSlmul and Y. Markus ABBREVIATIONS ON PLATES e endosome 8 golgi apparatus I lamella Id lipid droplets m mitochondria n nucleus ne nuclear envelope P plastid pe Pr py r t u plastid envelope paramylon pyrenoid ribosomes thylakoid unknown body Fig. 8. Section through a lightgrown cell after 60 h in growing medium (G.M.) x Fig. 9. Section through a lightgrown cell after 60 h in growing medium containing io mg/ml CM. Note abnormal mitochondrion, x

13 Effects of chloramplienicol on Euglena 639 ne m

14 640 Y. BenShaul and Y. Markus Fig. 10. Section through a lightgrown cell after 60 h in G.M. containing io mg/ml CM. Note the lipid droplets, x Fig. 11. Proplastids in section through a darkgrown cell after 12 h of preincubation in the dark, x Fig. 12. Section through a darkgrown cell in resting medium (R.M.) after 4 h in light. Details as in Fig. 5. x Fig. 13. Section through a darkgrown cell in G.M. after 12 h in light. Details as in Fig. 5. x

15 12 Effects of chloramphenicol on Euglena 641

16 642 Y. BenShaul and Y. Markus Fig. 14. Section through a darkgrown cell in R.M. containing CM after 4 h in light. Details as in Fig. 5. x Fig. 15. Section through a darkgrown cell in R.M. containing CM after 12 h in light. Details as in Fig. 5. x Fig. 16. Section through a darkgrown cell in R.M. after 24 h in light. Details as in Fig. 5. x Fig. 17. Section through a darkgrown cell in G.M. containing CM after 24 h in light. Details as in Fig. 5. x

17 Ejfects of chloramphenicol on Euglena 643

18 644 Y. BenShaul and Y. Marktis Fig. 18. Section through a darkgrown cell in G.M. after 48 h in light. Details as in Fig. 5. x Fig. 19. Section through a darkgrown cell in G.M. containing CM after 48 h in light. Details as in Fig. 5. x