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An extranuclear gene contributes to the regulation of cell division of the unicellular green alga Chlamydomonas reinhardtii
Plant Science, 59 (1989) 8 7 - 94
87
Elsevier Scientific Publishers Ireland Ltd.
AN E X T R A N U C L E A R GENE CONTRIBUTES TO T H E REGULATION OF CELL DIVISION OF T H E U N I C E L L U L A R GREEN ALGA CHLAMYDOMONAS R E I N H A R D T I I
JURGEN VOIGT and PETRA MUNZNER
Institut f~r AUgemeine Botanik und Botanischer Garten der Universitdt Hamburg, Ohnhorstst~zsse 18, D-~O00 Hamburg 52 (F.R.G.) (Received March 24th, 1988) (Revision received June 30th, 1988) (Accepted July 20th, 1988) Cell divisions of the unicellular green alga Chlamydomonas reinha~ltii are prevented by antibiotics, which specifically inhibit biosynthesis of RNA or protein in the organelles. Using synchronized, heterotrophically or mixotrophieaUy growing cultures, we have shown that this inhibition of cell division is not merely caused by effects on energy metabolism: When cell growth was stopped by terminating aeration and/or illumination during the third quarter of the growth period, transition to the division phase was not affected. Addition of chloramphenicol, tetracycline or rifampicin during the same time period, however, prevented cell divisions. DNA polymerase a could not be detected in such cell cycle-arrested cultures, whereas an aphidicolin-resistant DNA polymerase activity was found to be induced. The same effects were caused by the topoisomerase inhibitor nalidixic acid, which was recently found to affect gune expression in Chlamydomonas chloroplasts. Therefore, we conclude that an extranuelear gene contributes to the regulation of cell division in Chlamydomonas.
Key words: cell division; Chlamydomonas reinhardti~" DNA polymerases; extranuclear genes; nalidixic acid; rifampicin
Introduction Like other unicellular green algae, the phyto flagellate Chlamydomonas reinhardtii is able to grow both under photoautotrophic, mixotrophic and heterotrophic growth conditions [1-3]. Photoautotrophically growing cultures of unicellular green algae can be synchronized by alternating periods of light and darkness [4--6]. In the case of C. reinhardtii, synchronization by light-dark cycling was also achieved in the presence of acetate [7,8], which is metabolized by this phytofiagellate. When grown under optimal conditions, Chlamydomonas cells and other unicellular algae are able to multiply their cell mass prior to initiation of cell division [4,9--12]. During the following division phase, which normally occurs during the dark period, up to 32 zoospores are formed from a single mother cell by successive cell divisions. Photosynthesizing cells of Chlorella [13,14] and Chlamydomonas [11,15,16] attain a commit-
ment to divide before starting replication of nuclear DNA and thereafter they will complete cell divisions without requirement for growth. Cell divisions of C. reinhardtii were found to be prevented by antibiotics, which specifically inhibit biosynthesis of RNA or proteins in the organelles, when these antibiotics were added during early stages of cell growth [15,17]. When added during late stages of the growth period, these antibiotics no longer inhibited the transition to the division phase [15,17]. Sager and coworkers [18] have reported that addition of these drugs to photoautotrophically growing cultures of C. reinhardtii prevented replication of nuclear DNA, but did not affect replication of chloroplast and mitochondrial DNA. Obviously, there is a remarkable coincidence between the time, when the cell cycle switches from sensitivity to resistance against antibiotics, which inhibit gene expression in the organelies. Therefore, we have investigated whether an extranuclear gene contributes to the regulation
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
88
of cell division or whether the described effects are merely due to an inhibition of cell growth. Materials and methods The cell wall-deficient mutant (7. reinhardtii CW-15 [19] was grown at 21°C in high-salt
.
.
,
medium [20] supplemented with 2 g 1-1 sodium acetate and synchronized by light.lark cycling without aeration [8]. The cultures were gently mixed by stirring bars. Transfer to heterotrophic growth conditions was performed 24 h after beginning of the preceding growth period by bubbling the cultures with
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Tins ( h ) Fig. L Effect of antibiotics on the cell cycle of heterotrophically growing C. reinh~rdtii cells. The cell wall-deficient mutant CW-15 was grown in high-salt medium supplemented with sodium acetate (2 g 1-1) and synchronized by light-dark cycling without aeration. At zero time, e.g. 24 h after beginning of the preceding light period, the cultures were bubbled with filtered air and further incubated in the dark. (A) DNA synthesis by isolated nuclei. (B) Cell number ( • • ) and percentage of pseudosporangia (O. . . . . . . O). (C,D) Effect of antibiotics on cell number (closed symbols) and percentage of pseudosporangia (open symbols). Chloramphenieol (V,V: 600 mg 1-1),cycloheximide ( 4~, ~ : 10 mg 1-1),rifampicin ( I , D : 300 mg 1-1)or tetracycline (jk,A: 200 mg 1-1)were added to 50-ml aliquots taken 9 h (C) and 15 h after beginning of the growth period (D). All the cultures were incubated in the dark, and aeration was stopped 15 h after beginning of the growth period. Addition of antibiotics is marked by arrows.
89
D N A synthesis DNA synthesis was measured as recently described [8]: the reaction mixture (20 ~1) contained 55 mM Tris--HCI (pH 8.0), 150 mM KCI, 2.25 mM MgCI~, 0.002% (w/v) spermidine, 125 mM sorbitol, 0.520/0 (w/v) Ficoll, 12.5% (v/v) glycerol, 2.5 mM dithiothreitol, 2.50/0 (v/v) dimethylsulfoxide, 1 mM dATP, 1 mM dGTP, 1 mM TTP, 1 ~Ci [a-82p]dCTP (spec. radioact., 10 Ci mmo1-1) and 5"108 nuclei. After addition of nuclei, the reaction mixture was incubated at 25°C for 1 h. After incubation, 5 / d stop mix, containing 50/0 (w/v) sodium dodecyl sulfate and 50 mM EDTA were added and the reaction mixtures plated onto Whatman 3MM filters. The filters were washed once with 10o/0 (w/v) trichloroacetic acid (TCA), twice with 5% (w/v) TCA and twice with methanol, dried and measured for radioactivity after addition of 5 ml Lipoluma (Baker). Blank values (reaction mixtures without incubation) were subtracted.
filtered air. Cell concentrations were determined by hemocytometer counting. Although the mutant strain CW-15 is cell wall deficient, the daughter cells stick together for several hours after cytokinesis. Each of these 'pseudosporangia' was counted as one cell. Isolation of nuclei Cells were collected by centrifugation, washed and resuspended to a final concentration of 5" 107 cells m1-1 in homogenization buffer containing 375 mM sorbitol, 40 mM Tris--HCI (pH 7.5), 20 mM KCI, 5 mM MgCls, 1 mM MnC12, 2 mM EDTA and 14 mM 2-mercaptoethanol. Cells were lysed by addition of 10°/0 (v/v) NP40 to a final concentration of 0.50/0. Crude nuclei were collected by centrifugation at 1000 × g for 5 rain, washed with homogenization buffer and resuspended to a final concentration of 1"109 nuclei m1-1 in storage buffer containing 2.5% {w/v) Ficoll, 0.5 M sorbitol, 20 mM T r i s - H C l (pH 7.5), 0.0080/0 (w/v) spermidine, 1 mM dithiothreitol, 5 mM MgCI 2 and 500/0 (v/v) glycerol. The isolated nuclei were stored at - 70 °C.
Results and discussion Acetate-adapted cells synchronized by light-
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Fig. 2. Effectof varied growth periods on the cell cycle.Cultures of C. reinhardti{ strain CW-15growingin the presence of sodium acetate were synchronizedby light-dark cyclingas in Fig. 1. At zero time, e.g. 24 h after beginningof the preceding light period,the cultures were bubbled with filtered air and incubatedin the dark (A,B)or illuminatedwith 9,0000 Ix (CJ3). Cell growth was stopped by terminating aeration (A,B)or illumination(C,D)9 h or 15 h after beginningof the growth period (indicated by arrows). Open symbols:9 h growth period; closed symbols:15 h growth period. Cell number and percentage of 'pseudosporangia'were determinedas in Fig. 1.
90
dark cycling [8] continue to divide synchronously for one cell cycle period when transferred to heterotrophic growth conditions (Fig. 1B). Cell divisions were found to be prevented when eycloheximide, chloramphenicol, tetracycline or
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rifampiein were added 9 - 1 0 h after beginning of aeration (Fig. 1C). When these antibiotics were added 15 h after beginning of heterotrophic growth, e.g. at the beginning of the Sphase (Fig. 1A), cell divisions were no longer
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Fig. 3. Effect of nalidixic acid, novobiocin and rifampicin on the cell cycle. Cultures of C. reinhardtii strain CW-15 growing in the presence of sodium acetate were synchronized by light-dark cycling as in Fig. 1. A t zero time, e.g. 24 h after beginning of the preceding light period, the cultures were either bubbled with filtered a/r and incubated in the dark (A--D) or illuminated with 20 000 Ix (E-- H). Nalidixic acid (A. . . . . -A, • . . . . . . • : 60 mg l-l), novobiocin ( ~ O, • • : 150 mg I-I) or rifampicin (I"I. . . . . . [:], • . . . . . . • : 500 mg I-I) were added 9 h (A,B) and 10.5 h after beginning of heterotrophie growth (C,D), respectively. In case of mixotrophieally growing cultures, the antibiotics were added 7 h (E,F) and I0 h after beginning of the final growth period (G,H), respectively. (O O, • • ) Untreated control cultures.
91
affected by chloramphenicol, tetracycline or rifampicin, but were still sensitive to cycloheximide (Fig. 1D). During the final stages of cell growth, e.g. between 9 and 15 h after beginning of the growth period, considerable amounts of RNA and protein are synthesized by C. reinhardtii cells [8,16,21]. These findings indicate that inhibition of cell divisions by chloramphenicol, tetracycline or rifampicin (Fig. 1C) could be due to effects on energy metabolism and cell growth. Therefore, we have investigated, whether or not cell divisions were prevented when cell growth was stopped 9 h after beginning of the growth period. Using 1-1 cultures or 4-1 cultures which were incubated in the presence of acetate in 2-1 and 5-1 Erlenmeyer flasks, respectively, and gently mixed by stirring bars, cell growth was only observed, when the cultures were either illuminated or bubbled with air, as revealed by measurements of RNA and protein (data not shown). Agitation of the cultures by stirring bars was found to be insufficient to promote heterotrophic growth. Therefore, cell growth could be easily interrupted. As shown in Fig. 2, cell divisions were not prevented, when heterotrophic or mixotrophic growth was stopped 9 h after beginning of the growth period by terminating aeration and/or illumination, whereas addition of antibiotics at the same time period caused a complete inhibition of cell divisions (Fig. 1C). However, the increase of cell density was found to be reduced, when cell growth was interrupted after 9 h (Fig. 2B,D). Furthermore, in the mixotrophically growing cultures illuminated for 15 h, transition to cell division was observed considerably earlier than in the cultures illuminated for 9 h only (Fig. 2C). As 'pseudosporangia' [8] were already observed prior to the end of the light phase in case of cultures illuminated for 15 h, transition to the division phase was initiated by maximal cell mass [8,12] rather than by timer control [16,21--24] in these cultures. The experiments described above clearly show that expression of extranuclear genes
during late stages of the growth period is a prerequisite for cell division, although cell growth is no longer required at this cell cycle stage. Therefore, we conclude that there is at least one extranuclear gene which is involved in the regulation of cell division and which has to be expressed during late stages of the growth period. To exactly define the cell cycle stage, where this extranuclear gene is expressed, we have added rifampicin at different times after beginning of the growth period (Fig. 3), because it has been reported that rifampicin is an inhibitor of prokaryotic and organellar RNA polymerases [25--30]. It has, however, to be mentioned that in vitro assays with transcriptionally active extracts from chloroplasts of higher plants did not reveal any sensitivity to rifampicin for chloroplast RNA polymerase [31]. Since it has been recently shown that the topoisomerase II inhib-
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16 18 24 Time (h ) Fig. 4. Cell-cycle dependent incorporation of deoxyribonueleoside triphosphates by isolated nuclei and its modulation by in vivo application of antibiotics. Antibiotics were added 9 h after beginning of the growth period (indicated by arrow) to synchronized, heterotrophically growing Chlamydomonas cells as in Figs. I and 3. Crude nuclei were prepared and analyzed for DNA polymerase activity in the absence of aphidicolin as in Fig. 1. Cam ( V - - . - V ) , chloramphenicol; Tet (A • • • A), tetracycline; Nal (A . . . . . . . A), nalidixic acid; Novo ( 0 • ), novobiocin; Rif (11. . . . . . . U), rifampicin; ( • • ), control.
92
itors novobiocin and nalidixic acid [32] differentially affect the expression of chloroplast genes in Chlamydomonas [33], both drugs were also tested in these experiments. The compounds were added when cell growth was stopped by terminating aeration or illumination. In case of heterotrophically growing cultures (Figs. 3A-3D), cell divisions were found to be completely inhibited, when rifampicin or nalidixic acid were added 9 h after beginning of aeration. When added 1.5 h later, e.g. 10.5 h after beginning of the growth period, both antibiotics no longer prevented cell division, but caused a considerable delay of cell divisions (Figs. 3C and 3D). Novobiocin, however, did not affect cell division. Essentially the same effects were observed in case of mixotrophically growing cultures (Figs. 3E--3H). However, in order to completely inhibit the transition to the division phase, rifampicin or nalidixic acid had to be added about 3 h earlier than in case of heterotrophically growing cultures. To figure out whether the observed inhibition of cell divisions is mediated by effects on replication of nuclear DNA as deduced from the data of Blamir et al. [18], we have measured the incorporation of deoxyribonucleoside triphosphates by isolated nuclei. As shown in Fig. 4, DNA synthesis by isolated nuclei was found to be considerably increased when the cells were pretreated with chloramphenicol, tetracycline, nalidixic acid or rifampicin. These findings were quite unexpected, since Blamir et al. [18] have reported that replication of nuclear DNA is prevented by addition of antibiotics which inhibit the expression of extranuclear genes. Therefore, we have investigated whether or not the induced DNA polymerase activity is DNA polymerase a which is responsible for replication of nuclear DNA [34]. As shown in Table I, the induced DNA polymerase activity was found to be resistant against aphidicolin, a specific inhibitor of DNA polymerase a [34,35]. Aphidicolin-sensitive DNA polymerase a was detected in untreated and novobiocin-treated C. reinhardtii cells, but was never found in cellcycle arrested cells (Table I). Therefore, chlor-
Table I. In vivo effects of antibiotics on aphidicolin-resistent and total DNA polymerase activities of isolated nuclei. Antibiotics were added 9 h after beginning of the growth period to synchronized, heterotrophieally growing cultures of the ceU-wall deficient mutant CW-15 as in Figs. 1 and 3. Crude nuclei were prepared and analyzed for DNA polymerase activities as in Fig. 1. The DNA polymerase a inhibitor aphidicolin was added to the reaction mixtures from a 2.8 mmol 1-1 stock in dimethylsulfoxide to a final concentration of 70 ~anol 1-l. The same volume of dimethylsulfoxide was added to the reaction mixtures without aphidicolin. Antibiotic
Time period after beginning of cell growth (h)
Incorporation of [a-~P]dCTP (108 cpm/106 nuclei _ S.E.) - aphidicolin
+ aphidicolin
None
17 20
25.2 ± 2.2 11.8 ± 1.0
11.3 ± 1.6 6.4 ± 0.8
Novobiovin
17 20
26.4 ± 2.1 12.6 ± 0.9
10.2 ± 1.4 7.0 ± 0.6
Nalidixic acid
17 20
33.4 ± 3.5 34.8 ± 3.2
37.6 ± 4.1 34.4 ± 3.9
Rifampicin
17 20
34.2 ± 3.4 35.2 ± 3.7
39.0 ± 4.1 42.3 ± 4.7
Chloramphenicol
17 20
55.2 _ 6.4 67.6 ± 7.4
59.0 ± 7.2 72.8 ± 8.3
Tetracycline
17 20
49.6 ± 5.3 59.1 ± 6.4
55.8 ± 6.1 66.7 ± 7.4
amphenicol, tetracycline, rifampicin and nalidixic acid obviously inhibit transition to the S-phase. As clearly demonstrated by our findings described above, an extranuclear gene contributes to the regulation of cell division in C. reinhardtii. This gene is expressed during a very defined time period just prior to replication of nuclear DNA. Acknowledgement This work has been supported by a grant from the Deutsche Forschungsgemeinschaft (Vo 327/1).
93
References 1
H.C. Bold, The cultivation of algae. Bot. Rev., 8 (1942) 138. 2 R. Sager and S. Granick, Nutritional studies with Chlamydomonas reinhardtK Ann. New York Acad. Sci, 56 (1953) 831 -- 838. 3 M.R. Droop, Heterotrophy of carbon, in: W.D.P. Stewart (Ed.), Algal Physiology and Biochemistry, Blackwell Science Publishers, 1974, pp. 530-559. 4 U.G. Schl6sser, Enzymatiseh gesteuerte Freisetzung von Zoosporen bei Chlamydomonas reinhardtii Dangeard in Synehronkultur. Arch. Mikrobiol., 54 (1966) 129-- 159. 5 H. Tamiya, Synchronous cultures of algae. Annu. Rev. Plant Physiol., 17 (1966) 1 - 26. 6 A. Pirson and H. Lorenzen, Synchronized dividing algae. Annu. Rev. Plant Physiol., 17 (1966) 439-458. 7 D. Grant, D.C. Swinton and K.-S. Chiang, Differential patterns of mitochondrial, chloroplastie and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomwnas reinhardti~ Planta, 141 (1978) 259- 267. 8 J. Voigt and P. Mfinzner, The Chlamydomonas cell cycle is regulated by a Hght/dark-responsive cell-cycle switch. Planta, 172 (1987) 463-- 472. 9 E. Bernstein, Physiology of an obligate photoautotroph (Chlamydomonas moe~usii). I. Characteristics of synchronously and randomly reproducing cells and an hypothesis to explain their population curves. J. Protozool., 11 (1964) 56--74. I0 S. Mihara and E. Hase, Studies on the vegetative life cycle of Chlamydomonas reinhardtii Dangeard in synchronous culture. I. Some characteristics of the cell cycle. Plant Cell Physiol., 12 (1971) 225--236. 11 J.L. Spudich and R. Sager, Regulation of the Chlamydomonas cell cycle by light and dark. J. Cell Biol., 85 (1980) 136-- 145. 12 R.A. Craigie and T. Cavalier-Smith, Cell volume and the control of the Chlamydomonas cell cycle. J. Cell SCI., 54 (1982) 173-191. 13 H. Tamiya, T. Iwamura, K. Shibata, E. Hase and T. Nihei, Correlation between photosynthesis and lightindependent metabolism in the growth of Chlorell~ Biochim. Biophys. Acta, 12 (1953) 23--40. 14 F. Wanka, Untersuchungen zur Wirkung des Lichts auf die Zellteflung von ChloreUa pyrenoidos~ Arch. Mikrobiol., 34 (1959) 161 -- 188. 15 S.H. Howell, W.J. Blasebo and C.M. Drew, Inhibitor effects during the cell cycle in Chlamydomonas reinhardti~ determination of transition points in asynchronous cultures. J. Cell Biol., 67 (1975) 126-- 135. 16 L. Donnan and P.C.L. John, Cell cycle control by timer and sizer in Chlamydomonas. Nature, 304 (1983) 630-633. 17 S. Mihara and E. Hase, Studies on the vegetative life cycle of Chlamydomonas reinhardtii Dangeard in syn-
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chronous culture. II. Effects of chloramphenicol and eycloheximide on the length of cell cycle. Plant Cell Physiol., 12 (1971) 237-241. J. Blamir, V.R. Flechtner and R. Sager, Regulation of nuclear DNA replication by the chloroplast in Chlamydomonas. Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 2867 - - 2871. D.R. Davies and A. Plaskitt, Genetical and structural analysis of cell-wall formation in Chlamydomonas reinhardti~ Genet. Res., 17 (1971) 33-43. N. Sueoka, Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardti~ Proc. Natl. Acad Sci. U.S.A., 46 (1960) 83--89. M.J. Rollins, J.D.I. Harper and P.C.L. John, Synthesis of individual proteins, including tubulins and chloroplast membrane proteins, in synchronous cultures of the eukaryote Chlamydomonas reinhardti~ Elimination of periodic changes in protein synthesis and enzyme activity under constant environmental conditions. J. Gen. Microbiol., 129 (1983) 1899-- 1919. V.G. Bruce, The biological clock in Chlamydomonas reinhardti~ J. Protozool., 17 (1970) 334--340. S.C. Straley and V.G. Bruce, Stickiness to glass. Circadian changes in the cell surface of Chlamydomonas reinhardti£ Plant Physiol., 63 (1979) 1175-- 1181. P.C.L. John, Control of the cell division in Chlamydomonas. Microbiol. Sci., 1 (1984) 96-101. G. Hartmann, K.O. Honikel, F. Kniisel and J. Nfiesch, The specific inhibition of the DNA-directed RNA synthesis by rifamycin. Biochim. Biophys. Acta, 145 (1967) 843-- 844. S. Surzycki, Genetic functions of the chloroplast of Chlamydomonas reinhardti~ effect of rifampicin on chloroplast DNA~lependent RNA polymerase. Proc. Natl. Acad. Sei. U.S.A., 63 (1969) 1327 - 1334. M.N. Gadaleta, M. Greco and C. Saccone, The effect of rifampicin on mitochondrial RNA polymerase from rat liver. FEBS Lett, 10 (1970) 54--56. H. Kfintzel and K.P. Schiifer, Mitochondrial RNA polymerase from Neurospora crasso~ Nature (New Biol.), 231 (1971) 265-- 269. E. Br~tndle and K. Zetsche, Die Wirkung yon Rifampicin auf die RNA- und Proteinsynthese sowie die Morphogenese und den Chlorophyllgehalt kernhaltiger und kernloser Acetabularia-Zellen. Planta, 99 (1971) 46--55. J.J. Armstrong, S. Surzycki, B. Moll and R.P. Levine, Genetic transcription and translation specifying chloroplast components in Chlamydomonas reinhardti£ Biochemistry, 10 (1971) 6 9 2 - 701. W. Gruissem, B.M. Greenberg, G. Zurawski and R.B. Hallick, Chloroplast gene expression and promoter identification in chloroplast extracts, in: A. Weissbach and H. Weissbach (Eds.), Methods in Enzymology, Vol. 118, Academic Press, 1986, pp. 253--270. M. Gellert, DNA topoisomerases. Annu. Rev. Biochem., 50 (1981) 879-- 910.
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34
R.J. Thompson and G. Mosig, An ATP~lependent supercoiling topoisomerase of Chlamydomonas reinhardtii affects accumulation of specific chloroplast transcripts. Nucleic Acids Res., 13 (1985) 873--891. A. Weissbach, Eukaryotic DNA polymerases. Annu. Rev. Biochem., 46 (1977) 2 5 - 47.
35
F. Sala, B. Parisi, D. Burroni, A.R. Amileni, G. PedraliNoy and S. Spadari, Specific and reversible inhibition by aphidicolin of the a-like DNA polymerase of plant cells. FEBS Lett., 117 (1980) 93--98.
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Elsevier Scientific Publishers Ireland Ltd.
AN E X T R A N U C L E A R GENE CONTRIBUTES TO T H E REGULATION OF CELL DIVISION OF T H E U N I C E L L U L A R GREEN ALGA CHLAMYDOMONAS R E I N H A R D T I I
JURGEN VOIGT and PETRA MUNZNER
Institut f~r AUgemeine Botanik und Botanischer Garten der Universitdt Hamburg, Ohnhorstst~zsse 18, D-~O00 Hamburg 52 (F.R.G.) (Received March 24th, 1988) (Revision received June 30th, 1988) (Accepted July 20th, 1988) Cell divisions of the unicellular green alga Chlamydomonas reinha~ltii are prevented by antibiotics, which specifically inhibit biosynthesis of RNA or protein in the organelles. Using synchronized, heterotrophically or mixotrophieaUy growing cultures, we have shown that this inhibition of cell division is not merely caused by effects on energy metabolism: When cell growth was stopped by terminating aeration and/or illumination during the third quarter of the growth period, transition to the division phase was not affected. Addition of chloramphenicol, tetracycline or rifampicin during the same time period, however, prevented cell divisions. DNA polymerase a could not be detected in such cell cycle-arrested cultures, whereas an aphidicolin-resistant DNA polymerase activity was found to be induced. The same effects were caused by the topoisomerase inhibitor nalidixic acid, which was recently found to affect gune expression in Chlamydomonas chloroplasts. Therefore, we conclude that an extranuelear gene contributes to the regulation of cell division in Chlamydomonas.
Key words: cell division; Chlamydomonas reinhardti~" DNA polymerases; extranuclear genes; nalidixic acid; rifampicin
Introduction Like other unicellular green algae, the phyto flagellate Chlamydomonas reinhardtii is able to grow both under photoautotrophic, mixotrophic and heterotrophic growth conditions [1-3]. Photoautotrophically growing cultures of unicellular green algae can be synchronized by alternating periods of light and darkness [4--6]. In the case of C. reinhardtii, synchronization by light-dark cycling was also achieved in the presence of acetate [7,8], which is metabolized by this phytofiagellate. When grown under optimal conditions, Chlamydomonas cells and other unicellular algae are able to multiply their cell mass prior to initiation of cell division [4,9--12]. During the following division phase, which normally occurs during the dark period, up to 32 zoospores are formed from a single mother cell by successive cell divisions. Photosynthesizing cells of Chlorella [13,14] and Chlamydomonas [11,15,16] attain a commit-
ment to divide before starting replication of nuclear DNA and thereafter they will complete cell divisions without requirement for growth. Cell divisions of C. reinhardtii were found to be prevented by antibiotics, which specifically inhibit biosynthesis of RNA or proteins in the organelles, when these antibiotics were added during early stages of cell growth [15,17]. When added during late stages of the growth period, these antibiotics no longer inhibited the transition to the division phase [15,17]. Sager and coworkers [18] have reported that addition of these drugs to photoautotrophically growing cultures of C. reinhardtii prevented replication of nuclear DNA, but did not affect replication of chloroplast and mitochondrial DNA. Obviously, there is a remarkable coincidence between the time, when the cell cycle switches from sensitivity to resistance against antibiotics, which inhibit gene expression in the organelies. Therefore, we have investigated whether an extranuclear gene contributes to the regulation
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
88
of cell division or whether the described effects are merely due to an inhibition of cell growth. Materials and methods The cell wall-deficient mutant (7. reinhardtii CW-15 [19] was grown at 21°C in high-salt
.
.
,
medium [20] supplemented with 2 g 1-1 sodium acetate and synchronized by light.lark cycling without aeration [8]. The cultures were gently mixed by stirring bars. Transfer to heterotrophic growth conditions was performed 24 h after beginning of the preceding growth period by bubbling the cultures with
.
-40
i.
~
..
Is
1-
"I°
9
Ig
15
18
II
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Tins ( h ) Fig. L Effect of antibiotics on the cell cycle of heterotrophically growing C. reinh~rdtii cells. The cell wall-deficient mutant CW-15 was grown in high-salt medium supplemented with sodium acetate (2 g 1-1) and synchronized by light-dark cycling without aeration. At zero time, e.g. 24 h after beginning of the preceding light period, the cultures were bubbled with filtered air and further incubated in the dark. (A) DNA synthesis by isolated nuclei. (B) Cell number ( • • ) and percentage of pseudosporangia (O. . . . . . . O). (C,D) Effect of antibiotics on cell number (closed symbols) and percentage of pseudosporangia (open symbols). Chloramphenieol (V,V: 600 mg 1-1),cycloheximide ( 4~, ~ : 10 mg 1-1),rifampicin ( I , D : 300 mg 1-1)or tetracycline (jk,A: 200 mg 1-1)were added to 50-ml aliquots taken 9 h (C) and 15 h after beginning of the growth period (D). All the cultures were incubated in the dark, and aeration was stopped 15 h after beginning of the growth period. Addition of antibiotics is marked by arrows.
89
D N A synthesis DNA synthesis was measured as recently described [8]: the reaction mixture (20 ~1) contained 55 mM Tris--HCI (pH 8.0), 150 mM KCI, 2.25 mM MgCI~, 0.002% (w/v) spermidine, 125 mM sorbitol, 0.520/0 (w/v) Ficoll, 12.5% (v/v) glycerol, 2.5 mM dithiothreitol, 2.50/0 (v/v) dimethylsulfoxide, 1 mM dATP, 1 mM dGTP, 1 mM TTP, 1 ~Ci [a-82p]dCTP (spec. radioact., 10 Ci mmo1-1) and 5"108 nuclei. After addition of nuclei, the reaction mixture was incubated at 25°C for 1 h. After incubation, 5 / d stop mix, containing 50/0 (w/v) sodium dodecyl sulfate and 50 mM EDTA were added and the reaction mixtures plated onto Whatman 3MM filters. The filters were washed once with 10o/0 (w/v) trichloroacetic acid (TCA), twice with 5% (w/v) TCA and twice with methanol, dried and measured for radioactivity after addition of 5 ml Lipoluma (Baker). Blank values (reaction mixtures without incubation) were subtracted.
filtered air. Cell concentrations were determined by hemocytometer counting. Although the mutant strain CW-15 is cell wall deficient, the daughter cells stick together for several hours after cytokinesis. Each of these 'pseudosporangia' was counted as one cell. Isolation of nuclei Cells were collected by centrifugation, washed and resuspended to a final concentration of 5" 107 cells m1-1 in homogenization buffer containing 375 mM sorbitol, 40 mM Tris--HCI (pH 7.5), 20 mM KCI, 5 mM MgCls, 1 mM MnC12, 2 mM EDTA and 14 mM 2-mercaptoethanol. Cells were lysed by addition of 10°/0 (v/v) NP40 to a final concentration of 0.50/0. Crude nuclei were collected by centrifugation at 1000 × g for 5 rain, washed with homogenization buffer and resuspended to a final concentration of 1"109 nuclei m1-1 in storage buffer containing 2.5% {w/v) Ficoll, 0.5 M sorbitol, 20 mM T r i s - H C l (pH 7.5), 0.0080/0 (w/v) spermidine, 1 mM dithiothreitol, 5 mM MgCI 2 and 500/0 (v/v) glycerol. The isolated nuclei were stored at - 70 °C.
Results and discussion Acetate-adapted cells synchronized by light-
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Fig. 2. Effectof varied growth periods on the cell cycle.Cultures of C. reinhardti{ strain CW-15growingin the presence of sodium acetate were synchronizedby light-dark cyclingas in Fig. 1. At zero time, e.g. 24 h after beginningof the preceding light period,the cultures were bubbled with filtered air and incubatedin the dark (A,B)or illuminatedwith 9,0000 Ix (CJ3). Cell growth was stopped by terminating aeration (A,B)or illumination(C,D)9 h or 15 h after beginningof the growth period (indicated by arrows). Open symbols:9 h growth period; closed symbols:15 h growth period. Cell number and percentage of 'pseudosporangia'were determinedas in Fig. 1.
90
dark cycling [8] continue to divide synchronously for one cell cycle period when transferred to heterotrophic growth conditions (Fig. 1B). Cell divisions were found to be prevented when eycloheximide, chloramphenicol, tetracycline or
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rifampiein were added 9 - 1 0 h after beginning of aeration (Fig. 1C). When these antibiotics were added 15 h after beginning of heterotrophic growth, e.g. at the beginning of the Sphase (Fig. 1A), cell divisions were no longer
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Fig. 3. Effect of nalidixic acid, novobiocin and rifampicin on the cell cycle. Cultures of C. reinhardtii strain CW-15 growing in the presence of sodium acetate were synchronized by light-dark cycling as in Fig. 1. A t zero time, e.g. 24 h after beginning of the preceding light period, the cultures were either bubbled with filtered a/r and incubated in the dark (A--D) or illuminated with 20 000 Ix (E-- H). Nalidixic acid (A. . . . . -A, • . . . . . . • : 60 mg l-l), novobiocin ( ~ O, • • : 150 mg I-I) or rifampicin (I"I. . . . . . [:], • . . . . . . • : 500 mg I-I) were added 9 h (A,B) and 10.5 h after beginning of heterotrophie growth (C,D), respectively. In case of mixotrophieally growing cultures, the antibiotics were added 7 h (E,F) and I0 h after beginning of the final growth period (G,H), respectively. (O O, • • ) Untreated control cultures.
91
affected by chloramphenicol, tetracycline or rifampicin, but were still sensitive to cycloheximide (Fig. 1D). During the final stages of cell growth, e.g. between 9 and 15 h after beginning of the growth period, considerable amounts of RNA and protein are synthesized by C. reinhardtii cells [8,16,21]. These findings indicate that inhibition of cell divisions by chloramphenicol, tetracycline or rifampicin (Fig. 1C) could be due to effects on energy metabolism and cell growth. Therefore, we have investigated, whether or not cell divisions were prevented when cell growth was stopped 9 h after beginning of the growth period. Using 1-1 cultures or 4-1 cultures which were incubated in the presence of acetate in 2-1 and 5-1 Erlenmeyer flasks, respectively, and gently mixed by stirring bars, cell growth was only observed, when the cultures were either illuminated or bubbled with air, as revealed by measurements of RNA and protein (data not shown). Agitation of the cultures by stirring bars was found to be insufficient to promote heterotrophic growth. Therefore, cell growth could be easily interrupted. As shown in Fig. 2, cell divisions were not prevented, when heterotrophic or mixotrophic growth was stopped 9 h after beginning of the growth period by terminating aeration and/or illumination, whereas addition of antibiotics at the same time period caused a complete inhibition of cell divisions (Fig. 1C). However, the increase of cell density was found to be reduced, when cell growth was interrupted after 9 h (Fig. 2B,D). Furthermore, in the mixotrophically growing cultures illuminated for 15 h, transition to cell division was observed considerably earlier than in the cultures illuminated for 9 h only (Fig. 2C). As 'pseudosporangia' [8] were already observed prior to the end of the light phase in case of cultures illuminated for 15 h, transition to the division phase was initiated by maximal cell mass [8,12] rather than by timer control [16,21--24] in these cultures. The experiments described above clearly show that expression of extranuclear genes
during late stages of the growth period is a prerequisite for cell division, although cell growth is no longer required at this cell cycle stage. Therefore, we conclude that there is at least one extranuclear gene which is involved in the regulation of cell division and which has to be expressed during late stages of the growth period. To exactly define the cell cycle stage, where this extranuclear gene is expressed, we have added rifampicin at different times after beginning of the growth period (Fig. 3), because it has been reported that rifampicin is an inhibitor of prokaryotic and organellar RNA polymerases [25--30]. It has, however, to be mentioned that in vitro assays with transcriptionally active extracts from chloroplasts of higher plants did not reveal any sensitivity to rifampicin for chloroplast RNA polymerase [31]. Since it has been recently shown that the topoisomerase II inhib-
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16 18 24 Time (h ) Fig. 4. Cell-cycle dependent incorporation of deoxyribonueleoside triphosphates by isolated nuclei and its modulation by in vivo application of antibiotics. Antibiotics were added 9 h after beginning of the growth period (indicated by arrow) to synchronized, heterotrophically growing Chlamydomonas cells as in Figs. I and 3. Crude nuclei were prepared and analyzed for DNA polymerase activity in the absence of aphidicolin as in Fig. 1. Cam ( V - - . - V ) , chloramphenicol; Tet (A • • • A), tetracycline; Nal (A . . . . . . . A), nalidixic acid; Novo ( 0 • ), novobiocin; Rif (11. . . . . . . U), rifampicin; ( • • ), control.
92
itors novobiocin and nalidixic acid [32] differentially affect the expression of chloroplast genes in Chlamydomonas [33], both drugs were also tested in these experiments. The compounds were added when cell growth was stopped by terminating aeration or illumination. In case of heterotrophically growing cultures (Figs. 3A-3D), cell divisions were found to be completely inhibited, when rifampicin or nalidixic acid were added 9 h after beginning of aeration. When added 1.5 h later, e.g. 10.5 h after beginning of the growth period, both antibiotics no longer prevented cell division, but caused a considerable delay of cell divisions (Figs. 3C and 3D). Novobiocin, however, did not affect cell division. Essentially the same effects were observed in case of mixotrophically growing cultures (Figs. 3E--3H). However, in order to completely inhibit the transition to the division phase, rifampicin or nalidixic acid had to be added about 3 h earlier than in case of heterotrophically growing cultures. To figure out whether the observed inhibition of cell divisions is mediated by effects on replication of nuclear DNA as deduced from the data of Blamir et al. [18], we have measured the incorporation of deoxyribonucleoside triphosphates by isolated nuclei. As shown in Fig. 4, DNA synthesis by isolated nuclei was found to be considerably increased when the cells were pretreated with chloramphenicol, tetracycline, nalidixic acid or rifampicin. These findings were quite unexpected, since Blamir et al. [18] have reported that replication of nuclear DNA is prevented by addition of antibiotics which inhibit the expression of extranuclear genes. Therefore, we have investigated whether or not the induced DNA polymerase activity is DNA polymerase a which is responsible for replication of nuclear DNA [34]. As shown in Table I, the induced DNA polymerase activity was found to be resistant against aphidicolin, a specific inhibitor of DNA polymerase a [34,35]. Aphidicolin-sensitive DNA polymerase a was detected in untreated and novobiocin-treated C. reinhardtii cells, but was never found in cellcycle arrested cells (Table I). Therefore, chlor-
Table I. In vivo effects of antibiotics on aphidicolin-resistent and total DNA polymerase activities of isolated nuclei. Antibiotics were added 9 h after beginning of the growth period to synchronized, heterotrophieally growing cultures of the ceU-wall deficient mutant CW-15 as in Figs. 1 and 3. Crude nuclei were prepared and analyzed for DNA polymerase activities as in Fig. 1. The DNA polymerase a inhibitor aphidicolin was added to the reaction mixtures from a 2.8 mmol 1-1 stock in dimethylsulfoxide to a final concentration of 70 ~anol 1-l. The same volume of dimethylsulfoxide was added to the reaction mixtures without aphidicolin. Antibiotic
Time period after beginning of cell growth (h)
Incorporation of [a-~P]dCTP (108 cpm/106 nuclei _ S.E.) - aphidicolin
+ aphidicolin
None
17 20
25.2 ± 2.2 11.8 ± 1.0
11.3 ± 1.6 6.4 ± 0.8
Novobiovin
17 20
26.4 ± 2.1 12.6 ± 0.9
10.2 ± 1.4 7.0 ± 0.6
Nalidixic acid
17 20
33.4 ± 3.5 34.8 ± 3.2
37.6 ± 4.1 34.4 ± 3.9
Rifampicin
17 20
34.2 ± 3.4 35.2 ± 3.7
39.0 ± 4.1 42.3 ± 4.7
Chloramphenicol
17 20
55.2 _ 6.4 67.6 ± 7.4
59.0 ± 7.2 72.8 ± 8.3
Tetracycline
17 20
49.6 ± 5.3 59.1 ± 6.4
55.8 ± 6.1 66.7 ± 7.4
amphenicol, tetracycline, rifampicin and nalidixic acid obviously inhibit transition to the S-phase. As clearly demonstrated by our findings described above, an extranuclear gene contributes to the regulation of cell division in C. reinhardtii. This gene is expressed during a very defined time period just prior to replication of nuclear DNA. Acknowledgement This work has been supported by a grant from the Deutsche Forschungsgemeinschaft (Vo 327/1).
93
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F. Sala, B. Parisi, D. Burroni, A.R. Amileni, G. PedraliNoy and S. Spadari, Specific and reversible inhibition by aphidicolin of the a-like DNA polymerase of plant cells. FEBS Lett., 117 (1980) 93--98.