Journal qf General Microbioiogj?(I gp),71, 35-52 Printed in Great Britain 35

Are Plastids Derived from Prokaryotic Micro-organisms? Action of on Chloroplasts of Euglena gracilis

By L. EBRINGER Department of Microbiology, Komensky University, Bratislava, Czechoslovakia

(Accepted for publication I January 1972)

SUMMARY Of 144 antibiotics examined with respect to their action on Euglena chloroplasts, 46 caused irreversible loss of plastids and most inhibited chlorophyll synthesis. These substances included structurally related compounds as well as degradation products of antibiotics. Antibiotics exhibiting bleaching activity were of two general types judged by their mechanisms of action in other systems : I. Inhibitors of DNA synthesis - anthramycin, edeine, porfiromycin, some mitomycins, myxin, nalidixic acid and its derivatives, novobiocin, primycin, rubi- flavin, sarkomycin and streptonigrin; 2. Inhibitors of protein synthesis - 29 antibiotics which carry a common mole- cular denominator in their structure (an aminohexose) and three antibiotics which lack aminosugar moieties : viomycin, and pactamycin. Only these two types of antibiotics permanently eliminated chloroplasts ; anti- biotics classified as possessing other mechanisms of action were not effective. All these bleaching antibiotics inhibited replication of plastids in concentrations having no effect on normal Euglena division. A diluting-out of pathological plastids is the explanation of this ‘bleaching phenomenon’.

INTRODUCTION was the first found to induce the permanent loss of plastids in Euglena gracilis (Provasoli, Hutner & Schatz, I 948 ; Jirovec, 1949). For a long time strepto- mycin was considered as the only antibiotic bringing about this ‘bleaching effect’ in Euglena. However, in 1961 we observed that exerted the same effect as streptomycin (Ebringer, I 96 I). Subsequently other antibiotics have been discovered which induce per- manent loss of chloroplasts in Euglena (Ebringer, 1962a, b, c, 1964, 1966, 1970, 1971; Ebringer, JurASek & Kada, 1967; Ebringer, Krkoska, MaEor, JurASek & Kada, 1967; Ebringer, Mego, JurASek & Kada, 1969; Zahalsky, Hutner, Keane & Burger, 1962; McCalla, 1962, 1965; Celmer & Ebringer, 1967; Lyman, 1967; McCalla & Baerg, 1969). Growing evidence supports the hypothesis of an exogenous origin for chloroplasts : (i) the presence of specific chloroplast DNA (Brawermann & Eisenstadt, 1964; Edelman, Schiff & Epstein, 1965); (ii) the presence in chloroplasts of 70s ribosomes which otherwise are found only in mitochondria and in prokaryotic micro-organisms (Boardman, Francki & Wildman, 1965; Kiintzel & Noll, 1967). These are different in many respects from 80 s ribosomes which are found in the cytoplasm of Euglena and all other plants and animals; (iii) the basically similar submicroscopical architecture of chloroplasts, mitochondria, bacteria and blue-green algae (Ris & Plaut, 1962). Euglena offers a convenient tool for studying the mechanisms of action of drugs or for antibiotic screening to find non-toxic drugs which attack the DNA of sensitive organisms 36 L. EBRINGER (Ebringer, 197I). The high sensitivity of chloroplasts and prokaryotic organisms towards antibacterial drugs suggests that in these two systems there is a common specific sensitive target (or targets) which is responsible for the damage or death of plastids and bacteria.

METHODS Euglena gracilis strain z was grown in a proteose-peptone-tryptone medium (Mego, 1964). Stock cultures were grown in test tubes containing 10 ml of the medium, a 4-day culture at the end of logarithmic growth serving as inoculum. Usually the inoculum con- tained I 0 000 organisms/ml. Methods of cultivation, of determination of chlorophyll, of counting irreversibly bleached organisms, and of counting the chloroplasts per organism were as published by Ebringer, Neniec, Santovh & Foltinova (1970). In this paper we introduce a new expression, the ‘bleaching index’. The numerator in the bleaching index is a ratio of the killing concentration to the least bleaching concentration (in ,ug/ml) which causes the highest yo of permanently bleached cells. The denominator represents the difference between the killing concentration and the bleaching concentra- tion. I express to the following my deepest gratitude for gifts of antibiotics: Dr F. Arcamone, Farmitalia, Milan (daunomycin) ; Dr A. Aszales, The Squibb Institute for Medical Research, New Brunswick (rubiflavin) ; Dr V. Betina, Bratislava (citrinin, cyanein) ;Dr Zofia Borowska, New York (edeine); Dr W. D. Celmer, Chas. & Co. (carbomycin, , oleando- mycin and its derivatives, erythromycin, anisomycin, streptidin, cladinose, erythralosamine, , triacetyldesosamine) ; Dr L. Delcambe, International Centre for Information on Antibiotics, LiCge (phleomycin, actinomycin D, cinerubin B, bluensomycin, , geodin, erdin, primycin, anthramycin, stendomycin); Dr R. Donovick, The Squibb Institute for Medical Research, New Brunswick (methymycin); Dr J. Greenberg, Palo Alto Medical Research Foundation (carzinophillin) ; Dr L. Hanka, The Upjohn Company, Kalamazoo (amicetin and its derivatives, , ) ; Dr R. Hochster, Cell Biology Institute, Canadian Department of Agriculture, Ottawa (myxin) ; Dr J. Hoogerheide, Mycofarm, Delft (pimaricin); Dr K. Kagiwada, Kaken Chemical Co. Ltd, Tokyo (dihydro- deoxystreptomycin); Dr G. Lemonofides, The Winthrop Products Co., Surbiton, Surrey (nalidixic acid and its derivatives); Dr 0. Gonqalves de Lima, Tnstituto de Antibioticos, Recife, Brazil (lapachol); Dr H. E. Machamer, Parke-Davis Co., Detroit (viridogrisein, streptimidone); Dr T. J. McBride, Chas. Pfizer & Co. (streptonigrin, mithramycin); Dr J. L. Mego, Biology Department, University of Alabama (gougerotin); Dr J. Nakaya, Kyowa Hakko Kogyo Co. Ltd, Tokyo (mitomycins A, B, C, N-methylmitomycin); Dr N. Otake, The University of Tokyo (blasticidin S); Dr V. Prelog, Zurich (angolamycin, lanka- mycin, picromycin, rifamycin B); Dr F. M. Rottman, Michigan State University (cordy- cepin) ; Dr 2. kehBEek, Czechoslovak Academy of Sciences, Prague (antimycin A) ; Dr J. C. Sylvester, Abbott Laboratories, Chicago (hydroxystreptomycin, dihydroxystreptomycin, ); Dr H. Thrum, Jena (streptothricin) ; Dr D. Vazquez, Madrid (chloram- phenicols, streptogramin) ; Dr F. P. Willey, The Upjohn Co., Kalamazoo (tubercidin, nogalamycin and its derivatives, streptovitacin A, cycloheximide, cytosin arabinoside, decoyinine, pactamycin, pactamycate, porfiromycin) ; Dr W. K. Woo, Parke-Davis Co., Detroit (chalcomycin); Dr A. Zugaza, Antibioticos, S.A. Madrid ( and its derivatives, ). Antibiotics not listed above were purchased from com- mercial sources. Chloroplasts of Euglena gracilis 37

Table I. The action of inhibitors of nucleic acids and of synthesis of purine and pyrimidine nucleotides on Euglena chloroplasts

Bleached cells 10 white Least on the 9th colonies after bleaching Colour of day after 10 subcultur- concn (pglml) cultures on plating pro- ing gave the Killing causing the the 7th day duced by following no. concn highest % of after addition ‘least concn’ of bleached No. Antibiotic (pglml) bleached cells of antibiotics (%) subcultures I Anthram ycin 80 60 W 87 9 2 Edein 5 4 PG 27 5 3 Mitomycin A I0 NB - - - 4 Mitomycin B 60 50 PG 40 8 5 Mitomycin C 30 NB - - - 6 N-Methyl mitomycin 50 40 PG 55 9 7 Porfiromycin I20 80 PG 72 9 8 Myxin 200 I0 W I00 I0 9 Nalidixic acid 1-ethyl-7-methyl- 2 000 500 W I00 I0 I :8-naphtyridone-4-one+ carboxylic acid I0 Ethyl(7-methyl-I :S-naphtyridone- 700 I0 +one-3-carboxylate) I1 Ethyl(1-ethyl-7-methyl-I:8- 700 5 naphtyridone-4-one-3-carboxylate) 12 5-Nitrofurfuril ester of nalidixic 20 - acid 13 Novobiocin 800 500 I0 14 Phleomycin 0.I NB - I5 Primycin 20 19 6 16 RubiRavin 220 30 I0 17 Sarcomycin 15000 I0000 9 I8 Streptonigrin I20 I00 9 19 Carzinophillin I0 NB - 20 Actinomycin D 15 NB - 21 Actinomycin C 300 NB - 22 Nogalamycin 200 NB - 23 Nogalarol 200 NB - - 24 Nogalarene 200 NB 25 7-U-methylnogalarol 200 NB - 26 Cinerubin B 200 NB - - 27 Daunomycin 500 NB NB - 28 Mi thramycin 50 - 29 Echinomycin 1.5 NB 30 Cordycepin 200 NB - 31 Tubercidin 1000 NB - Formycin 200 NB - 32 - 33 Cytosin arabinoside I000 NB Decoyinine I000 NB - 34 - 35 Psicofuranine I000 NB

The following abbreviations will be used: NB, no bleaching activity; G, green colonies or cultures; W, white colonies or cultures; PG, pale green colonies or cultures.

RESULTS Table I shows that among inhibitors of nucleic acid synthesis only those antibiotics which attack DNA synthesis exhibited a bleaching effect. Among the 19 antibiotics tested (Table I, Compound no. I to 19) 14 exerted bleaching activity. Those exhibiting an especially favourable bleaching index were myxin, rubiflavin and nalidixic acid. Derivatives of nalidixic acid (Compound no. 10 to 12) had either no bleaching activity or lower activity than the darent substance. Phleomycin, mitomycins A and C and carzinophillin lacked bleaching effect. These compounds, especially phleomycin, had a high toxicity against Euglena. No antibiotic which inhibited RNA synthesis or purine and pyrimidine nucleotides synthesis (Table I, Compound no. 20 to 35) showed permanent bleaching activity, even in nearly lethal concentrations. Table 2 concerns inhibitors of protein synthesis. Most of this group consists of structurally 38 L. EBRINGER

Table 2. The action of inhibitors of protein synthesis and some antibiotics structurallr related to these inhibitors on Euglena chloroplasts Bleached cells 10 white Least on the 9th colonies after bleaching Colour of day after 10subcultur- concn (yglml) cultures on plating pro- ing gave the Killing causing the the 7th day duced by following no. concn highest %of after addition ‘least concn’ of bleached NO. Antibiotic (pglml) bleached cells of antibiotics ( %) subcultures

I Streptomycin 2000 I0 W I00 I0 2 Hydroxystreptomycin I000 I0 W I00 I0 3 Dihydrostreptomycin 2000 I00 W I00 I0 4 Dihydrohy droxystreptomycin 2000 I00 W 100 I0 5 Dihydrodesoxystreptomycin 2000 I00 W I00 10 6 Streptomycin osim 4000 3000 PG 48 8 7 Bluensomycin I000 I00 W I a0 I0 8 Kanamycin I000 200 PG 100 I0 9 Spectinomycin I000 50 W I00 I0 I0 Gentamycin 15 I0 PG 79 5 I1 Acetylgentamycin I000 NB - - - I2 15 5 PG 74 7 - 13 400 NB - - 14 Hygromycin B 2 I PG 1.5 5 15 Kasugamycin I NB - - - 16 Kasugamicinic acid 100 NB I7 Kasuganobiosamin I00 NB - 18 Streptothricin I 0.5 PG 35 7 19 Viomycin I 500 700 PG I00 I0 20 Amicetin 2000 1500 PG 48 9 21 1000 NB - 22 Blasticidin S 50 NB 23 Gougerotin 150 NB - 24 Streptogramin 500 200 W 25 Viridogrisein 1000 NB - - 26 Pactamycin 200 150 PG 78 9 27 Pactamycate 300 NB - 28 D(- )threo- 1000 NB 29 D(- lerythro-chloramphenicol 500 NB 30 L(- )erythro-chloramphenicol 500 NB 31 L(+ )three-chloramphenicol 500 NB 32 Cycloherimid 3 NB 33 Streptovitacin I00 NB 34 Anisomycin 100 NB 35 500 NB 36 500 NB 37 500 NB -

38 Sparsomycin I00 NB I I 39 Angoiamycin 2000 500 W I00 10 40 Carbomycin 700 100 w I00 I0 41 Erythromycin 5000 800 W I00 10 42 Kitasamycin base 2000 700 W 87 I0 43 Kitasamycin tartrate 2000 700 W 83 I0 44 Acetylki tasamycin 2000 700 W 90 I0 45 Oleandom ycin 5000 4000 W 94 9 46 Triacet yloleandomycin SO00 2000 W 100 I0 47 I200 600 PG 49 9 48 Spiramycin I11 900 600 PG 41 8 49 Neospiramycin 111 700 500 PG 25 8 50 Forocidin 111 700 500 PG 18 7 51 Tylosin 2000 500 W I00 I0 52 Meth ymycin 500 NB 53 Picromycin 500 NB 54 Chalcomycin 2000 NB 55 Lankamycin 2000 NB 56 Rifamycin B 2000 NB 57 Rifampicin 1000 NB 58 Cyanein 500 NB 59 Nystatin 25 NB 60 Amphotericin B 50 NB 61 Pimaricin 25 NB 62 Filipin 25 NB 63 Lincom ycin 4000 I 500 64 Clindamycin 3500 300 Chlorophsts of Euglena grucilis 39 Table 3. Antibiotics and related compounds with different mechanism of action but with no permanent bleacliing activity

Killing concn No. Antibiotic in ,~ig/ml

I Antiinycin A i00 2 Oligomycin 50 3 Rutamycin 100 4 Lysozynie 4 000 5 Penicillin 10000 6 Ampicillin 10000 7 Cephalosporin 5 000 8 Baci tracin 2 000 9 Cycloserin 20 I0 Granicidin 200 I1 Vankomycin 2 000 12 Ris tocetin 3 000 I3 Trypacidin 2 000 14 Geodin 2 000 15 Erdin 2 000 16 Griseofiilvin 1000 17 Citrinin 700 18 Stendomycin 100 19 Azalomycin 10 20 Monorden I 00 21 Cytochalazin A 150 22 Azaserin 400 23 Hadacidin 200 24 Lapachol 600 25 Cladinose 2 000 26 Mycarose I 000 27 Erythralosamine 2 000 28 Pentaacety1-N-methyl-L-glucosaniine I 500 29 Desosamine HC1 2 000 30 Triacetyldesosamine HCl 2 000 31 Streptidin sulphate 2 000 32 Methylstreptobiosamid tetraacetate 1 000 33 6-Amino-~-glucoseHCI 2 000 34 2-Desoxy-streptamin 2 HBr 2 000 35 Methyl-6-amino-2-~-glucopyranosideHCI 2 000 36 Oleandrin 2 000 37 Lana tosid I om 38 Lanacordal I 000 39 Strophantin K I 000 40 Digitoxin 10 41 Cyt osamine 2 500 42 Triacetylcytosamine 3 000 43 Cytymidin 2 000 44 Kojic acid 4 000 45 Streptimidone 10 related substances containing the aminohexose moiety as well as some unrelated antibiotics. Therefore it is not surprising that of 18 antibiotics belonging to the ' strepto- mycin-like' family, I 3 exerted bleaching activity. In this group were antibiotics with the most favourable bleaching index: , spectinomycin, bluensomycin and kanamycin. Table 2 includes four antibiotics structurally different from antibiotics no. I to 18. These were viomycin, pactamycin, streptogramin and amicetin. Other inhibitors of protein synthesis (Compound no. 27 to 38) did not have bleaching activity. In Table 2 there is a structurally uniform group of antibiotics (Compound no. L. EBRINGER

lr

ACE ACE ACE ACE ACE ACE ACE BDF BDF BDF RDF BDF BDF BDF I, 3 4 5 6 chlorophyll Time of cultivation (daya)

I ACE ACE ,4CE ACE ACE ACE ACE BDF BDF BDF BDF BDF BDF BDF 7 4 5 6 chlorophyll Time of cultivation (days) ,

ACE ACE ACE ACE ACE ACE ACE BDF BDF BDFI BDF BDF BDF BDF 1 3 3 4 _s 6 ch lo ro ph y 11 Time of cultivation (days) Fig. I a, b and c Chloroplasts of Euglena gracilis

ACE ACE ACE ACE ACE AC'E ACE BDF BDF BDF BDF BDF BDF BDF 1 1 I 3 4 L5 6 chlolophyll Time of cultit ation (days) Fig. ~d Fig. I. Proportions of bleached colonies obtained after growth of Euglena gracilis in various con- centrations of antibiotics for varying lengths of time and the chlorophyll content on the 6th day of cultivation. (a) 500 ,ug/ml, (b) zoo ,ug/mI, (c) roo ,ug/ml, (d) 10,ug/ml. After the indicated period of cultivation with the antibiotics, the organisms were washed and plated on antibiotic-free media. Bleached and green colonies were counted after 9 days. A, Streptomycin; B, dihydrostreptomycin; C,kanamycin ; D, spectinomycin; E, viomycin ; F, bluensomycin. 39 to 51), which contain both aminohexose and neutral hexose moieties and which exerted bleaching activity. Some of these had a very favourable bleaching index: angolamycin, carbomycin, erythromycin, kitasamycins and tylosin. Methymycin and picromycin (Com- pound no. 52 to 53), which contain only the aminohexose moiety, and chalcomycin and lankamycin (Compound no. 54 to 55), which are neutral macrolide antibiotics bearing only neutral hexoses, did not permanently bleach euglenas. Other neutral macrolide-like anti- biotics (Compound no. 56 to 58) as well as polyene antibiotics (Compound no. 59 to 62) showed no bleaching activity. On the other hand, lincomycin and chdamycin (Compound no. 63 to 64), which lack the macrocyclic lactone ring but have a rare aminosugar in their molecules, were highly effective bleaching antibiotics. Antibiotics which are believed to attack targets other than DNA or protein synthesis include many diverse chemical structures and properties and did not bleach Euglena (Table 3). Here were included also some sugar components mostly obtained from parent antibiotics as well as cardiotonic glycosides or some components isolated from them. All these com- pounds were tested up to the killing concentrations and showed no bleaching activity. Fig. I shows that bleaching activity of the various antibiotics depended on the concentra- tions used as well as on the duration of its contact with the cells. The most active were streptomycin and spectinomycin, which in concentrations of 500,200 and IOO ,ug/ml induced IOO yo permanently bleached colonies after 3 days of contact with the Euglena cells. Dihydro- streptomycin and bluensomycin at 500 ,.ug/ml induced IOOyo bleached colonies after 4 days and at 2oo,ug/ml did the same after 5 days, while kanamycin showed an effect only after 5 days of contact with the multiplying culture and then only in the highest concentration tested (500,ug/ml). The weakest bleaching activity was shown by viomycin, which in the highest concentrations tested (500 ,ug/ml) did not produce IOOyo bleached cells. Among antibiotics at the lowest concentration (10pglml), only streptomycin induced 100 yo bleached colonies after 5 days of its presence in the multiplying culture. Fig. I shows that cultures contained quite a large amount of chlorophyll at the end of cultivation (6th day), although 42 L. EBRINGER Table 4. Efects of some antibiotics on growth of Euglena gracilis The numbers represent thousands of organismslml medium after 6 days at 24 “C. ,uglml 7 Antibiotic 500 200 I00 10 Streptomycin 478 480 525 542 Dihydrostrep tomycin 460 490 520 500 Kanam ycin 426 539 540 540 Spect inomycin 397 399 425 432 Viomycin 395 423 427 520 Bluensomycin 399 433 456 498 Control = 525

Table 5. The inhibition of chlorophyll synthesis and formation of chloroplasts by antibiotics when added to the dark-adapted cells Aftecaddition of the drugs the temporarily bleached cells were transferred to the normzl light conditions. Bleached Concn Chlorophyll colonies after No. AT!tibio t ic (Puglml) ( %I plating ( %I I Sarkomycin I00 123 0 I0 109 0 2 Primycin I0 I02 0 I I07 0 3 Mitomycin C I0 223 0 I 92 0 4 Porfiromycin I0 215 0 I I I4 0 5 Phleomycin I 71 0 0’1 79 0 6 Myxin I00 7 100 I0 4 100 7 Novobiocin I00 I I2 0 I0 101 0 8 Ru biflavin I00 37 100 I0 46 41 9 Nalidixic acid I00 52 47 I0 113 0 I1 Ethyl(7-methyl-I :S-naphtyridone-4-one-3- I00 104 0 carboxylic acid) I0 I I8 0 I1 Ethyl( I -ethyl-7-methyl-I :8-naph tyridone- I00 I 28 0 4-one-3-carboxylate) I0 I 14 0 12 Decoyinine I00 107 0 I0 I02 0 I3 Cytosin arabinoside I00 23 0 I0 37 0 14 Tubercidin I00 111 0 I0 103 0 15 Nogalamycin I00 64 0 I0 93 0 16 Daunomycin I00 138 0 I0 I49 0 I7 Actinomycin D I0 105 0 I 127 0 I8 Mithramycin I00 64 0 I0 76 0 19 Streptomycin I00 6 100 I0 13 94 Chloroplasts of Euglena gracilis 43

Table 5 (cont). Bleached Concn Chlorophyll colonies after No. Antibiotic (PuSh1) (96) plating (%) 20 Dihydrostreptomycin I00 10 96 I0 48 82 21 Kanamycin I00 45 I00 I0 105 0 27 Spect inomycin I00 5 I00 I0 30 37 23 Neomycin I00 142 0 I0 130 0 24 BI uensomycin I00 14 97 I0 107 17 25 Ace t ylgen tamycin I00 57 0 I0 95 0 26 Hygromycin B I 29 0 0'1 132 0 27 Paromomycin I0 47 7 I 95 0 28 Kasugamycin I 88 0 0'1 92 0 29 Viomycin I00 15 I00 I0 105 19 30 Acet y 1ki tasamycin I00 6 0 I0 19 0 31 Kitasamycin tartrate I00 I2 5 I0 30 0 32 I00 23 0 I0 37 0 33 Triacetyloleandomycin I00 19 0 I0 37 0 34 Erythromycin I00 6 35 I0 46 0 35 Carbomycin I00 2 31 I0 27 0 36 Ty losin I00 8 0 I0 37 0 37 Picromycin I00 19 0 I0 42 0 38 Lankamycin I00 30 0 I0 39 0 39 Chalcomycin I00 26 0 10 46 0 40 Rifampicin I00 98 0 I0 91 0 4' Lincomycin I00 75 0 I0 91 0 42 Clindamycin I00 9 95 I0 28 10 43 Cy anein I00 I I2 0 I0 I02 0 44 D( - )threo-chloramphenicol I00 102 0 I0 99 0 45 L( + )threo-chloramphenicol I00 98 0 I0 I 08 0 44 L. EBRINGER Table 5 (cont.) Bleached Concn Chlorophyll colonies after No. Antibiotic (iuglml) ( %) plating (%I 0 46 L( - )erythro-chloramphenicol LOO 104 I0 I01 0 0 47 D( - )erythro-chloramphenicol I00 98 I0 103 0 8 48 Pactamycin I00 27 I0 56 0 49 Pactamycate I00 34 0 I0 57 0 0 50 Anisom ycin I00 13 I0 35 0 0 51 Cycloheximide I 31 0'1 92 0 0 52 0-Met hy 1t hreonine 100 39 I0 94 0 53 Streptogramin I00 9 42 I0 161 0 54 Tetracyclin I00 25 0 10 98 0 55 Puromycin I 00 24 0 I0 57 0 56 Sparsomycin I00 30 0 I0 I 16 0 57 Ci trinin I00 105 0 I0 111 0 after plating the colonies were commonly totally bleached. The highest content of chloro- phyll on the 6th day of cultivation was found in the presence of kanamycin and viomycin. In cultures with kanamycin and viomycin at 500pg/ml, more than 60% of the original chlorophyll was found in spite of their completely bleached colonies after plating. None of the antibiotics tested showed a significant inhibiting effect on the growth of euglenas at the concentrations used (Table 4). Some weak inhibition was observed with spectinomycin. Nevertheless, this antibiotic exhibited strong bleaching activity. Table 5 shows the action of 57 antibiotics on chlorophyll synthesis and on induction of bleached colonies when temporarily dark-bleached cells were transferred into continuous light at the moment of addition of the antibiotics. In this type of experiment we used two antibiotic concentrations, usually IOO and ropg/ml, but if the antibiotic was toxic, two lower concentrations were used (10 and I pg/ml or I and 0.1pglml). When comparing the results in Table 5 with those in Tables I and 2 we can generally say that antibiotics which bleached euglenas under the usual conditions also induced mutants without plastids when euglenas were transferred from dark-adapted growth into the light. Among 57 antibiotics tested in this manner 37 inhibited chlorophyll synthesis and 16 produced permanent loss of plastids. I wish to emphasize here that when using higher concentrations the frequency of bleaching increased proportionally. Among inhibitors of DNA synthesis, again the strongest bleachers were myxin, rubiflavin and nalidixic acid. Under these conditions nalidixic acid induced 47 % mutants in a concentration of IOO ,ug/ml, while under usual conditions, as in Tables I and 2, the same concentrations did not induce bleached mutants. Mitomycin C and porfiromycin at 10,ug/ml stimulated chlorophyll synthesis (Table 5, Compound no. 3 to 4). Chloroplasts of Euglena gracilis 45 100 90

-& 40 6 30 20 (I 00 :I) I0

"012345678 1934567 No. of cell divisions No. of cell divisions Fig. 2 Fig. 3 Fig. 2. Average number of plastids per flagellate during growth of EugZena gracilis after treatment with streptomycin and dihydrostreptomycin. A-A, Control; 0-17, 10 pg/ml of strepto- mycin; x - - - x , 500 ,ug/ml of streptomycin; O---O, 500 ,ug/ml of dihydrostreptomycin. Fig. 3. Chlorophyll content and "/, of bleached colonies during growth of Euglena gracilis after treatment with streptomycin and dihydrostreptomycin. After the indicated period of cultivation with the antibiotics, the organisms were washed, plated on antibiotic-free media, and % of bleached and green colonies counted after 9 days (numbers in parentheses). In another sample the chlorophyll content was determined. 0-0, 10pg/ml of streptomycin; x --- x , 500 ,ug/ml of streptomycin; O---O,500 pg/ml of dihydrostreptomycin.

10 - L- * - 100 ) 100 10 - 0- - -n,(12< 1 - 100 D. - . 9- 'O(IO0 1 - 90 9- - 90 \ - 78 x - 80- f 8-bx>6- x-x* - 80 -- 7 :\x,x-x - 70 =d 5 7- \\ \ \ - 70 =- 5 fj - '\, \ - 60 5, 5 6- '\ \ - 60 =, - b--- \ - b. 2 5- 0%. \ - 50 % 7 j- --.'- '0,po 1 - 50 rc, --qllN) --- 5 4- '-.. - 40 g g- 4- Q. \ - 40 2 '\ ( ) 4 .O..' , -7 \., I00 5 3- - 30 3 z- >- '\ 0 - 30 3 # --..\' - c '7- 9. x - 20 Li c '7- "0. !\ - 20 --...,\I ' 100 1- ',, ,iIOO ) 1- \Jo I .I0 1- 'Qid - 10 0" ' ' ' ' Ih o1 I I IIB

Antibiotics inhibiting RNA synthesis (e.g. cytosine arabinoside, mithramycin, nogala- mycin, etc.) did not prevent the formation of plastids in spite of a remarkable inhibition of chlorophyll synthesis. Among inhibitors of protein synthesis the strongest inhibitors of chlorophyll synthesis as well as of the formation of chloroplasts were some streptomycin-like (Table 5, Compound no. 19 to 22, 24, 29) as well as basic (Compound no. 34 to 35) and clindamycin and streptogramin (Compound no. 42, 53). Here again viomycin and kanamycin were stronger bleachers under these conditions; at only IOO ,ug/ml they induced IOO yo bleached mutants. Many antibiotics in spite of a strong inhibition of chlorophyll synthesis did not induce permanent loss of plastids (Table 5, Compound no. 30, 32, 33, 36, 37 to 39, 50 to 52, 54 to L. EBRINGER 10

I 012345678 Xo of ccll dnivons Fig. 6. Average number of plastids per cell, chlorophyll content and yo of bleached colonies after treatment with 500 pg/ml of nalidixic acid. For key see Fig. 4.

56). When increasing concentrations of some of these antibiotics were used they produced totally unpigmented cells, but only temporarily. After transferring into a fresh, antibiotic- free medium, the cells produced chlorophyll and chloroplasts normally. Fig. z to 6 show that after the addition of a bleaching antibiotic in a multiplying culture, chloroplasts were gradually diluted out. Fig. z shows that streptomycin at 500 pg/ml quickly induced dilution-out of chloroplasts, while dihydrostreptomycin in the same concentration was slower. Even after four generation times I measured a slight increase in numbers of chloroplasts per cell. Under the conditions used in my laboratory and described in Methods, euglenas were able to multiple for only five to six generations. After this period the culture reached the maximal concentration of organisms and growth stopped. This is why I needed to dilute the organisms after five to six generations in order to allow the subsequent multipli- cation of cells. Thus dihydrostreptomycin at 500 pg/ml and streptomycin at Iopg/ml (Fig. 2, 3) diluted-out the chloroplasts and chlorophyll slowly. Nalidixic acid had a very similar action (Fig. 6). Rubiflavin as well as carbomycin caused a very rapid dilution of plastids (Fig. 4, 5). After the addition of antibiotics the content of chlorophyll also gradually decreased (Fig. 3 to 6), but this depended on the bleaching activity of the particular antibiotic. Dihydrostreptomycin at 500 pg/ml depigmented euglenas slowly (Fig. 3), while rubiflavin and carbomycin at IOO pg/ml (Fig. 4, 5) and streptomycin at 500 ,ug/ml (Fig. 3) depigmented euglenas rapidly.

DISCUSSION The results presented above indicate that bleaching activity is common among antibiotics. Most of the compounds tested were antibiotics but some which were not were included in the screening because of their structural relationship with the antibiotics. Out of 144 com- pounds tested, 46 showed bleaching activity. All of these have been shown to be inhibitors of DNA synthesis or protein synthesis in other systems. Not one antibiotic which inhibited wall synthesis in bacteria, or RNA synthesis, or purine and pyrimidine synthesis, or which caused changes in membrane permeability, or inhibited electron transport, showed bleaching effectiveness. Among 19inhibitors of DNA synthesis tested against Euglena were five with no bleaching activity. Their lack of bleaching potency can be explained on the basis of their high toxicity. For instance phleomycin has a high affinity also for nuclear DNA, and therefore the cells did not survive the attack of this drug. A similar observation was made in the mitomycin group of antibiotics (Ebringer, Mego & JurBSek, 1969). The most toxic derivatives of mito- mycin (mitomycin A and C) cannot be added to Euglena culture in sufficient concentrations Chloroplasts of Euglena gracilis 47 17 r111151.'

G F Fig. 7. The distribution of antibiotics according to their site of action in other sensitive organisms. NB indicates no permanent bleaching activity. A C E Rubiflavin Streptomycins Puromycin NB Anthramycin Neomycin NB Blasticidin NB Mi tomycins Kanamycin Amicetin Sarkomycin Paromomycin Gougerotin NB Streptonigrin Gentamycin Edeine Hygromycin B F Primycin Viomycin Hadacidin NB Nalidixic acid Spectinomycin Azaserin NB Myxin Tetracycline NB Novobiocin Chlortetracycline NB G Phleomycin NB Oxytetracycline NB Cordycepin NB Carzinophillin NB Edeine Tubercidin NB Nitrosoguanidine Formycin NB Nitrofurans D Psicofuranin NB Ultraviolet light Chloramphenicols NB Decoyinine NB Angolamycin Cytosin arabinoside NB B Carbomycin Actinomycin D NB Erythromycin Nogalamycin NB Chalcomycin NB Cinerubin NB Lankamycin NB Chromomvcin NB Methymycin NB Daunomycin NB Pikromycin NB Echinomycin NB Mithramycin NB Oleandomycin Olivomycin NB Rifamycin NB Viridogrisein NB Rifampicin NB Lincomycins Sparsomycin NB

4 hl1C fI 48 L. EBRINGER to induce bleaching without killing the cells. This accounts also for the action of carzino- phillin. Among antibiotics inhibiting protein synthesis there were many with bleaching activity. Almost all members of the ‘ streptomycin-like ’ family exhibited strong bleaching activity with the exception of neomycin, acetylgentamycin, kasugamycin and its derivatives. In the group of macrolide antibiotics there was again a high frequency of bleachers. In this group the most effective bleaching agents were basic macrolides with two sugar com- ponents in their molecules, the one a neutral hexose, the other an aminohexose (Table 2, Compound no. 39 to 46). Spiramycin and spiramycin I11 contain three sugar components (I neutral and 2 aminosugars) and were weak bleaching agents, and their derivatives (products ofcheniical degradation) - neospiramycin and forocidin - were even weaker than the parent substances. On the other hand the neutral macrolide antibiotics, lankamycin and chalcomycin, which contain only the neutral sugars L-arcanose and L-lancavose (syn. chalcose and D-mycinose), did not permanently bleach euglenas. A similar situation was observed with the basic macrolide antibiotics picromycin and methymycin which contain aminosugar components bound to the macrocyclic lactone ring. In spite of this observation, forocidin - a degradation product of spiramycin containing only I aminosugar in the molecule (I aminohexose and I neutral hexose were chemically removed from the original spiramycin molecule) - still exerted some weaker bleaching activity. In this respect there is a complete correlation between bleaching ability of macrolides and their capacity to bind to the erythromycin A site on the 50 s ribosomal subunits. Only those macrolides which contain two hexose moieties (one of them an aminosugar) bind very efficiently to this site (Wilhelm, Oleinick & Corcoran, 1968). Macrolides lacking aminosugar moieties do not bind to it. These authors’ results indicate that lincomycins too must bind to the ‘macrolide site’ of 50 s ribosomal subunits. There is again a correlation because linco- mycins showed bleaching activity although they contain only one sugar moiety. Of course we do not know if this is the mode of action of macrolides on bacteria in vivo or even on Euglena because this organism represents a different system. The possibility that these anti- biotics bind firmly to DNA of bacteria and chloroplasts, thus causing the prevention of replication of chloroplast (or bacterial) DNA cannot be excluded. There are some indications that certain antibiotics bind more efficiently to DNA than to other polyanions (Zimmer, Triebel & Thrum, 1967; Cohen & Lichtenstein, 1960). Antibiotics belonging to the group of polyene macrolides which are known to change membrane permeability in various systems did not induce bleaching in spite of the presence of an aminosugar moiety (D-mycosamine) in their molecules. Antibiotics with no sugar components (rifamycin, rifampicin, cyanein, monorden, cytochalazin), structurally in some respects related to macrolide antibiotics, also did not bleach euglenas. Certain fragments of these bleaching antibiotics (mostly sugar components : Table 3, Compound no. 25 to 39, as well as some cardiac glycosides which contain some rarer sugars closely related to a hexose (e.g. oleandrose), are formed in nature only as cardiac glycosides and antibiotics (oleandrin and oleandomycin). These fragments and cardiac glycosides did not bleach euglenas. Only antibacterially active antibiotics exerted bleaching activity. After chemically or physically changing the molecules, thus removing their antibacterial activity, these drugs also lost their antiplastid activity. On the other hand, a slight chemical change of some molecules can significantly increase their antibacterial and antiplastid activity, as shown by comparing lincomycin with clindamycin, oleandomycin with triacetyloleando- mycin, mitomycin C with porfiromycin, and streptomycin with dihydrostreptomycin. It has Chloroplasts of Euglena gracilis 49 recently been shown that there is an exact correlation in antibacterial and antiplastid activity of these pairs (Celmer & Ebringer, 1967). There is evidence that all aminoglycosides of the streptomycin family have the Same mechanism of action (Gorini, 1967). Similarly we can expect one site of action for all the macrolide antibiotics. In the group of inhibitors of protein synthesis there are some structural exceptions. For example amicetin belongs to the group of aminoacyl antibiotics which include puromycin, blasticidin S and gougerotin. Puromycin, blasticidin S and gougerotin do not bleach euglenas but amicetin does. One possible explanation of this diversity in action is that amicetin contains an aminohexose (amosamine), a stereoisomer of mycaminose which is a sugar component of carbomycin, kitasamycin, spiramycins and tylosin. Chloramphenicol and its stereoisomers, which are known to inhibit protein synthesis in organisms containing 70s ribosomes, as well as in chloroplasts (Pogo & Pogo, 1965; Anderson & Smille, 1966), do not irreversibly eliminate plastids from euglenas. Similarly inactive are the , anisomycin, cycloheximide, sparsomycin and other inhibitors of protein synthesis (D-L-ethionine, etc.). These observations suggest that not only DNA inhibitors, but perhaps streptomycin and other aminoglycosides also, may bleach euglenas by inhibiting DNA replication or by some other mechanism influencing DNA in chloroplasts. There is some support for this hypothesis in other areas. Although since its discovery strepto- mycin has been one of the most extensively studied antibiotics it still appears to be one of the most obscure in its lethal action. There is also evidence that streptomycin has an affinity for DNA (Cohen, 1947; Stern, Barnet & Cohen, 1968). Marjai, Kiss & Ivhnovics (1970) suggest that a direct action of streptomycin on chromosomal replication might be involved in the genesis of some mutants of Bacillus subtilis. Obe (1970) found streptomycin very effective in inducing achromatic lesions in human chromosomes in vitro. Sager (1962) and Sager & Tsube (1962) demonstrated, too, that streptomycin is a mutagen for ‘non-chromo- SOma1’ genes. The absence of the bleaching activity in the group of antibiotics inhibiting RNA synthesis can be explained on the basis of the existence of cistrons for coding of RNA of chloroplast-ribosomes, not only on the chloroplast DNA but also on nuclear DNA (Scott, 1969). Thus there are two potential sites for synthesis of ribosomal RNA of chloroplasts. If the site in the chloroplast is inhibited by an inhibitor with binding affinity for chloroplast DNA (e.g. nogalamycin) RNA synthesis can be directed by the cistrons located on nuclear DNA. This explains why inhibitors of RNA synthesis do not permanently bleach euglenas. The absence of bleaching activity with chloramphenicols, tetracyclines and some inhibitors of protein synthesis can be explained similarly. DNA from spinach nuclei, from chloroplasts, and from animal mitochondria definitely stimulates protein synthesis in the Escherichiu coli cell-free system. On the other hand nuclear DNA from rat liver gives no significant stimula- tion of protein synthesis. This must be due not only to the presence of a bacterium-like protein-synthesizing apparatus in chloroplasts and mitochondria but also to the presence of genetic information concerning plastids directed by nuclear DNA of plants but not animals (Rabussay, Herzlich, Schweiger & Zillig, 1969). Perhaps some antibiotics which are believed to kill bacteria by inhibiting protein synthesis exert a different mechanism of action in chloroplasts. Kirk (1968) suggests that chlorophyll synthesis is dependent on protein synthesis and, like Schiff & Epstein (1965)~thinks the cause of bleaching is the inhibition of protein syn- thesis in chloroplasts. If this is true all inhibitors with primary effect on protein synthesis (chloramphenicols, tetracyclines) should be bleaching agents. These antibiotics specifically inhibit protein synthesis in chloroplasts only, in spite of which they do not cause permanent loss of chloroplasts. On the other hand, antibiotics which inhibit protein synthesis on the L. EBRINGER 50 80 s ribosomal apparatus (e.g. cycloheximide) were expected not to inhibit protein synthesis in chloroplasts but they do (Kirk & Allen, 1965; Kirk, 1968). This also indicates co-operative interaction between chloroplast and surrounding cytoplasm and nucleus. All the aminoglycosides which induce misreading contain a deoxystreptamine or strept- amine residue (streptomycins, kanamycin, neomycin, paromomycin, hygromycin B, gentamycin). Those aminoglycosides which do not contain such a residue do not induce misreading (spectinomycin and kasugamycin). Isolated a-deoxystreptamine causes mis- reading (Tanaka, Masukawa & Umezawa, 1967) but does not bleach euglenas. These results are incompatible with the hypothesis that the misreading of chloroplast RNA is responsible for permanent loss of plastids. At the present time we do not know if there is a dichotomy in the mechanism of action of antibiotics against bacteria and plastids. We are actively attempting to resolve this problem. However, the action of chloramphenicol, which is a specific inhibitor of protein synthesis in chloroplasts, strongly suggests that more than inhibition of protein synthesis is involved in bleaching. It seenis more reasonable to believe that substances which attack chloroplast DNA are more likely to induce changes resulting in irreversible chloroplast loss than are substances which attack any other site. Fig. 7, which shows a distribution of bleaching anti- biotics according to their site of action in other systems, partially supports this hypothesis. The sensitivity of chloroplasts towards antibiotics and plastid loss from seemingly normal euglenas also supports the hypothesis of the exogenous origin of chloroplasts.

The author gratefully acknowledges the assistance of Mrs Gabriela Smutna-Baker and Miss Marika RuEkova.

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