Journal qf General Microbioiogj?(I gp),71, 35-52 Printed in Great Britain 35 Are Plastids Derived from Prokaryotic Micro-organisms? Action of Antibiotics 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, streptogramin 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 Streptomycin was the first antibiotic 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 erythromycin 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. Pfizer & Co. (carbomycin, tylosin, oleando- mycin and its derivatives, erythromycin, anisomycin, streptidin, cladinose, erythralosamine, desosamine, triacetyldesosamine) ; Dr L. Delcambe, International Centre for Information on Antibiotics, LiCge (phleomycin, actinomycin D, cinerubin B, bluensomycin, hygromycin B, 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, lincomycin, clindamycin) ; 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, spectinomycin); 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 (kitasamycin and its derivatives, dihydrostreptomycin). 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