Journal qf General Microbioiogj? ( I g p ) , 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. E B R I N G E R Department of Microbiology, Komensky University, Bratislava, Czechoslovakia

(Accepted for publication I January 1972)

S U M M A R Y

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’.

I N T R O D U C T I O N

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 7 0 s 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. E B R I N G E R

(Ebringer, 197 I). 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

No.

I 2 3 4 5 6 7 8 9

I0

I1

12

13 14 I5 16 17 I8 19 20

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

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

Antibiotic

Anthram ycin Edein Mitomycin A Mitomycin B Mitomycin C N-Methyl mitomycin Porfiromycin Myxin Nalidixic acid 1-ethyl-7-methyl-

I : 8-naphtyridone-4-one+ carboxylic acid

+one-3-carboxylate)

naphtyridone-4-one-3-carboxylate)

acid

Ethyl(7-methyl-I : S-naphtyridone-

Ethyl(1-ethyl-7-methyl-I : 8-

5-Nitrofurfuril ester of nalidixic

Novobiocin Phleomycin Primycin RubiRavin Sarcomycin Streptonigrin Carzinophillin Actinomycin D Actinomycin C Nogalamycin Nogalarol Nogalarene 7-U-methylnogalarol Cinerubin B Daunomycin Mi thramycin Echinomycin Cordycepin Tubercidin Formycin Cytosin arabinoside Decoyinine Psicofuranine

Killing concn

(pglml)

80 5

I 0 60 30 5 0

I20 200

2 000

700

700

20

800 0. I

20 220

15000 I20

I 0

15 300 200 200 200 200 200 500

5 0 1.5

200 1000 200 I000 I000

I000

Bleached cells on the 9th

bleaching Colour of day after concn (pglml) cultures on plating pro-

causing the the 7th day duced by highest % of after addition ‘least concn’

bleached cells of antibiotics (%)

Least

60 4

NB 5 0

NB 40 80 I 0

500

W PG

PG

PG PG W W

-

-

87 27

40

55 72 I00

I00

-

-

500 NB 19 30

I 0 000 I00 NB NB NB NB NB NB NB NB NB NB NB NB NB NB NB NB NB

10 white colonies after 10 subcultur- ing gave the

following no. of bleached subcultures

9 5

8

9 9

I 0

I 0

- -

I0

5

-

I 0 - 6

9 9

I 0

- - - - - - - - - - - - - - - - -

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.

R E S U L T S

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. E B R I N G E R

Table 2. The action of inhibitors of protein synthesis and some antibiotics structurallr related to these inhibitors on Euglena chloroplasts

Bleached cells on the 9th day after

plating pro- duced by

‘least concn’ ( %) I00

I00 I00 100

I00

48 I a0 100

I00

79

74

1.5

-

-

-

10 white colonies after 10 subcultur- ing gave the

following no. of bleached subcultures

I 0 I 0 I 0

I 0 10 8

I 0 I 0 I 0

5

7

5

-

- -

Least bleaching

concn (yglml) causing the

highest %of bleached cells

I 0 I 0

I00 I00 I00

I00 200

50

NB 5

NB I

NB NB NB

700 1500 NB NB NB 200 NB 150

NB NB NB NB NB NB NB NB NB NB NB NB 500 100

700 700 700

2000

3000

I 0

0.5

800

4000

600 600 500 500 500 NB NB NB NB NB NB NB NB NB NB NB I 500 300

Colour of cultures on the 7th day

after addition of antibiotics

W W W W W PG W PG W PG

PG

PG

-

-

-

Killing concn

(pglml) 2000 I000

2000 2000 2000 4000 I000 I000 I000

15

15 400

I000

2 I

100

I00

I I 500 2000 1000

50 150 500

1000

200 300 1000

500 500 500

3 I00

100

500 500 500 I00

2000

700 5000 2000

2000 2000 5000 SO00 I200 900 700 700

2000 500

500 2000 2000 2000

1000

500 25 50 25 25

4000 3500

NO.

I 2

3 4 5 6 7 8 9 I 0 I 1 I2

13 14 15 16 I7 18

19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 5 1 52 53 54 55 56 57 58 59 60 61 62 63 64

Antibiotic

Streptomycin Hydroxystreptomycin Dihydrostreptomycin Dihydrohy droxystreptomycin Dihydrodesoxystreptomycin Streptomycin osim Bluensomycin Kanamycin Spectinomycin Gentamycin Acetylgentamycin Paromomycin Neomycin Hygromycin B Kasugamycin Kasugamicinic acid Kasuganobiosamin Streptothricin Viomycin Amicetin Puromycin Blasticidin S Gougerotin Streptogramin Viridogrisein Pactamycin Pactamycate D(- )threo-chloramphenicol D(- lerythro-chloramphenicol L(- )erythro-chloramphenicol L(+ )three-chloramphenicol Cycloherimid Streptovitacin Anisomycin Tetracycline Chlortetracycline Ox ytetracycline Sparsomycin Angoiamycin Carbomycin Erythromycin Kitasamycin base Kitasamycin tartrate Acetylki tasamycin Oleandom ycin Triacet yloleandomycin Spiramycin Spiramycin I11 Neospiramycin 111 Forocidin 111 Tylosin Meth ymycin Picromycin Chalcomycin Lankamycin Rifamycin B Rifampicin Cyanein Nystatin Amphotericin B Pimaricin Filipin Lincom ycin Clindamycin

- PG PG PG -

35 I00

48

7 I 0

9

- W

PG - -

78 9 -

- I

1 0 I 0

1 0 I 0 I 0

I 0

9 I 0

9 8 8 7

I 0

I

W w W W W W W W PG PG PG PG W

I00 I00 I00

87 83 90 94

100 49 41 25 18 I00

Chlorophsts of Euglena grucilis

Table 3. Antibiotics and related compounds with different mechanism of action but with no permanent bleacliing activity

39

No. I

2

3 4 5 6 7 8 9

I 0 I 1 12

I 3

14 15 1 6 17 18 19 20 21 22

23

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Antibiotic Antiinycin A Oligomycin Rutamycin Lysozynie Penicillin Ampicillin Cephalosporin Baci tracin Cycloserin Granicidin Vankomycin Ris tocetin Trypacidin Geodin Erdin Griseofiilvin Citrinin S tendomycin Azalomy cin Monorden Cytochalazin A Azaserin Hadacidin Lapachol Cladinose M ycarose Erythralosamine Pentaacety 1-N-methyl-L-glucosaniine Desosamine HC1 Triacetyldesosamine HCl Streptidin sulphate Methylstreptobiosamid tetraacetate 6-Amino-~-glucose HCI 2-Desoxy-streptamin 2 HBr Methyl-6-amino-2-~-glucopyranoside HCI Oleandrin Lana tosid Lanacordal Strophantin K Digi toxin Cyt osamine Triacet ylcytosamine Cytymidin Kojic acid Streptimidone

Killing concn in ,~ig/ml

i00

50

4 000

100

1 0 000

1 0 000

5 000

2 000

20

200 2 000

3 000

2 000

2 000

2 000

1000

700 100

10

I 00

150 400

600 200

2 000

I 000 2 000

I 500 2 000 2 000

2 000

1 000

2 000 2 000

2 000 2 000

I o m I 000 I 000

10

2 500 3 000

4 000

2 000

1 0

related substances containing the aminohexose moiety as well as some unrelated antibiotics. Therefore it is not surprising that of 18 aminoglycoside antibiotics belonging to the ' strepto- mycin-like' family, I 3 exerted bleaching activity. In this group were antibiotics with the most favourable bleaching index: streptomycins, 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 macrolide antibiotics (Compound no.

L. EBRINGER

l r

I, A C E A C E A C E A C E A C E A C E

B D F B D F B D F R D F B D F B D F B D F A C E

3 4 5

Time of cultivation (daya ) 6 chlorophyll

I A C E

B D F chlorophyll

, A C E A C E ,

B D F B D F A C E

B D F 4 C E A C E A C E

B D F B D F B D F 7 4 5 Time of cultivation (days)

6

I A C E A C E A C E A C E A C E A C E A C E

B D F B D F B D F B D F B D F B D F B D F 3 1 3 4 _s 6 ch lo ro ph y 1 1

Time of cultivation (days)

Fig. I a, b and c

Chloroplasts of Euglena gracilis

L A C E A C E A C E A C E A C E A C ' E A C E

B D F B D F B D F B D F B D F B D F B D F 1 1 I 3 4 5 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 IOO yo 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 IOO yo bleached cells. Among antibiotics at the lowest concentration (10 pglml), 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. E B R I N G E R

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

Antibiotic 500

Streptomycin 478 Dihydros trep tomycin 460 Kanam ycin 426 Spec t inomycin 397 Viomycin 395 Bluensomycin 399 Control = 525

200 I00

480 525 490 520 539 540 399 425 423 427 433 456

7

10

542 500 540 432 520

498

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.

No. I

2

3

4

5

6

7

8

9

I 1

I 1

12

I3

14

15

16

I 7

I 8

19

AT! tibio t ic Sarkomycin

Primycin

Mitomycin C

Porfiromycin

Phleomycin

Myxin

Novobiocin

Ru biflavin

Nalidixic acid

Ethyl(7-methyl-I : S-naphtyridone-4-one-3-

Ethyl( I -ethyl-7-methyl- I : 8-naph tyridone-

Decoy inine

Cytosin arabinoside

Tubercidin

Nogalamycin

Daunomycin

Actinomycin D

Mithramycin

Streptomycin

carboxylic acid)

4-one-3-carboxylate)

Concn (Puglml)

I00

I0

I0 I

I0 I

I 0 I

I 0’1

I00 I 0

I00 I 0

I00 I 0

I00 I 0

I00 I0

I00 I 0

I00 I 0

I00 I 0

I00 I 0

I00 I0

I00 I0

I0 I

I00 I 0

I00

I0

Chlorophyll

123 109

I07 223 92

215 I I4 71 79 7 4

( %I

I02

I I 2 101

37 46 52

113

104 I I 8 I 28 I 14 107

23 37

103

64 93

138 I49 105 127 64 76

6 13

I02

111

Bleached colonies after plating ( %I

0 0

0 0

0

0

0

0

0

0

100

100

0 0

100

41 47 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0

0

0

100

94

Chloroplasts of Euglena gracilis

Table 5 (cont).

43

No. 20

21

27

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

4'

42

43

44

45

Antibiotic Dihydrostreptomycin

Kanamycin

Spec t inomycin

Neomycin

BI uensomycin

Ace t ylgen tamycin

Hygromycin B

Paromomycin

Kasugamycin

Viomycin

Ace t y 1 ki tasamycin

Kitasamycin tartrate

Oleandomycin

Triacet yloleandomycin

Erythromycin

Carbomy cin

Ty losin

Picromycin

Lankamycin

Chalcomycin

Rifampicin

Lincomycin

Clindamycin

Cy anein

D( - )threo-chloramphenicol

L( + )threo-chloramphenicol

Concn (PuSh1) I00 I0

I00

I 0

I00 I 0

I00 I0

I00 I0

I00

I0

I 0'1

I 0 I

I

0'1

I00

I0

I00 I0

I00

I0

I00

I0

I00 I0

I00 I 0

I00 I 0

I00 I 0

I00

I0

I00 I 0

I00 10

I00

I0

I00 I 0

I00

I 0

I00

I0

I00 I0

I00 I 0

Chlorophyll (96)

48 45 105

5 30

142 130 14

107

57 95 29

132

47 95 88 92 15

105

6 19

30 23 37 19 37 6

46

1 0

I 2

2

27 8

37 19 42 30 39 26 46 98 91 75 91 9

28 I I 2 I02

102

99 98

I 08

Bleached colonies after plating (%)

96 82 I00

0

I00

37 0

0

97 17

0 0

0

0

7 0

0

0

I00

19 0

0

5 0

0

0

0

0

35

31 0

0

0

0

0

0

0 0

0

0

0 0

0

0

95 10

0

0

0

0

0 0

44 L. E B R I N G E R

No.

46

47

48

49

50

51

52

53

54

55

56

57

Antibiotic

L( - )erythro-chloramphenicol

D( - )erythro-chloramphenicol

Pactamycin

Pactamycate

Anisom ycin

Cycloheximide

0-Me t hy 1 t hreonine

Streptogramin

Tetracyclin

Puromycin

Sparsomycin

Ci trinin

Table 5 (cont.)

Concn (iuglml)

LOO I 0

I00 I 0

I00 I 0

I00 I 0

I00 I0

I

0'1

100 I 0

I00 I 0

I00 10

I 00 I 0

I00

I0

I00 I 0

Chlorophyll ( %) 104

98 103

27 56 34 57 13 35 31 92 39 94 9

161

25 98 24 57 3 0

I 16

105

I 0 1

111

Bleached colonies after plating (%I

0 0

0

0

8 0

0 0

0 0

0

0

0

0

42 0

0

0

0 0

0

0

0

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 0.1 pglml). 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

10 9 -

7 8 7

2 5 -

5 3 - c ' 7 -

1 -

5 fj

5 4 -

- -

4

#

45

- L- * - 100 ) - 100 10 - 0- - -n,(12< 1 - 100 . \ - 90

D.

9 - - ' O ( I O 0 1 - 90 x - 8 0 - f 8 - b x > 6 - x-x* - 80 --

:\x,x-x - 70 =d 5 7 - \\ \ \ - 70 =- - '\, \ - 60 5, 5 6 - '\ \ - 60 =,

'0 ,po 1 - 50 rc, - 40 2

"0. !\ -

\ --- '- --. b. - b--- \

0%. \ - 50 % 7 j -

'\ \., ( I00 ) - 40 g g- 4 - Q. - 20 Li c ' 7 -

'-.. - - q l l N )

- 30 3 z- > - '\ 0 - 30 3 - 7

20

.O..' , - --. '

',, , i I O O )

. \ 9. x --. '

I I B 'Qid - 10 . I0 1 - ..,\I 100 1-

\Jo I 0" ' ' ' ' I h o 1 I I

" 0 1 2 3 4 5 6 7 8 No. of cell divisions

100 90

& 40

20 I0

- 6 30

1 9 3 4 5 6 7 No. of cell divisions

( I 00 :I)

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, 10 pg/ml of streptomycin; x --- x , 500 ,ug/ml of streptomycin; O---O, 500 pg/ml of dihydrostreptomycin.

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 aminoglycosides (Table 5, Compound no. 19 to 22, 24, 29) as well as basic macrolides (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

10

L. E B R I N G E R

I

0 1 2 3 4 5 6 7 8 Xo of ccll dn ivons

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.

D I S C U S S I O N

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 19 inhibitors 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 17 r111151.'

47

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

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

B Carbomycin Actinomycin D NB Erythromycin Nogalamycin NB Chalcomycin NB Cinerubin NB Lankamycin NB Chromomvcin NB Methymycin NB Daunomycin NB Pikromycin NB Echinomycin NB Spiramycins Mithramycin NB Oleandomycin Olivomycin NB S treptogramins Rifamycin NB Viridogrisein NB Rifampicin NB Lincomycins

Sparsomycin NB

Edeine Hygromycin B F

Angolamycin Cytosin arabinoside NB

4 h l 1 C f I

L. E B R I N G E R

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 3 9 , 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

48

49 Chloroplasts of Euglena gracilis 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 tetracyclines, 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. E B R I N G E R 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.

R E F E R E N C E S

ANDERSON, L. A. & SMILLIE, R . M . (1966). Binding of chloramphenicol by ribosomes from chloroplasts Biochemical and Biophysical Research Communications 23,535-539,

BOARDMAN, N. K., FRANCKI, R. I . B. & WILDMAN, S . G. (1965). Protein synthesis by cell-free extracts from tobacco leaves. 2. Association of activity with chloroplast ribosomes. Biochemistry 5, 872-878.

BRAWERMAN, G. & EISENSTADT, J. M. (1964). Deoxyribonucleic acid from the chloroplasts of Euglena gracilis. Biochimica et biophysica acta 91, 477-485.

CELMER, W. D. & EBRINGER, L. (1967). Effects of certain O-acyl derivatives of oleandomycin on chloroplasts of Euglena gracilis. Journal of Protozoology 14, 263-267.

COHEN, S. S. (1947). Streptomycin and desoxyribonuclease in the study of variations in the propsrties of a bacterial virus. Journal of Biological Chemistry 168, 51 1-526.

COHEN, S. S. & LICHTENSTEIN, J. (1960). The isolation of deoxyribonucleic acid from bacterial extracts by precipitation with streptomycin. Journal of Biological Chemistry 235, PC55-PC56.

EBRINGER, L. (1961). Die Erythromycinwirkung auf die Flagellaten Euglena gracilis Klebs. Naturwissen- schaften 48,606-607.

EBRINGER, L. ( I 962 a). Erythromycin- and streptomycin-like antibiotics as bleaching factors for Euglena gracilis. Naturwissenschaften 14, 3 34-3 3 5 .

EBRINGER, L. (1962 b). Erythromycin-induced bleaching of Euglena gracilis. Journal of Protozoology 9,

373-374s EBRINGER, L, (1962~). Side-effect of kanamycin on a green protista. Journal of Antibiotics, Tokyo A 15,

113-114. EBRINGER, L. (1964). Bleaching of euglenas by antibiotics - a specific form of antagonism in Actinomycetes

Folia microbiologica, Praha 9, 249-255. EBRINGER, L. (1966). Antibiotics and apochlorosis. I. Macrolide antibiotics - their common mclecular

structure responsible for bleaching of Euglena gracilis. Folia microbiologica, Praha 11, 379-386. EBRINGER, L. (1970). The action of nalidixic acid on Euglena p!astids. Journal of General Microbiology 61,

EBRINGFR, L. (1971). The action of inhibitors of nucleic acids synthesis on Euglena. Experientia 27, 586-587. 14 1 -I 44.

Chloroplasts of Euglena gracilis 51 EBRINGER, L., JURASEK, A. & KADA, R. (1967). Antibiotics and apochlorosis. 111. The effects of 5-nitrofurans

on chloroplast system of Euglena gracilis. Folia microbiologica, Praha 12, 151-156. EBRINGER, L., KRKOSKA, P., MAEOR, M., JURASEK, A. & KADA, R. (1967). Furan derivatives - their common

molecular denominator responsible for bleaching of Euglena gracilis. Avchiv .fur Mikrobiologie 57, 61-67.

EBRINGER, L., MEGO, J. L. & JURASEK, A. (1969). Mitomycins and the bleaching of Euglenagracilis. Archil: fiir iMikrohiologie 64, 229-234.

EBRINGER, L., MEGO, J. L., JURASEK, A. & KADA, R. (1969). The action of streptomycins on the chloroplast system of Euglena gracilis. Journal of General Microbiology 59,203-209.

EBRINGER, L., NEMEC, P., SANTOVA, H. & FOLT~NOVA, P. (1970). Changes of the plastid system of Euglena gracilis induced with streptomycin and dihydrostreptomycin. Archiv fur Mikrobiologie 73, 268-280.

EDELMAN, M., SCHIFF, J. A. & EPSTEIN, H. T. (1965). Studies of chloroplast development in Euglena. XII. Two types of satellite DNA. Journal of Molecular Biology 11, 769-774.

GORINI, L. (1967). Induction of code ambiguity by aminoglycoside antibiotics. Federation Proceedings 26,5-8. J~ROVEC, 0. (1949). Der Einfluss des Streptomycin und Patulins auf einige Protozoen. Experientia 5, 74-79. KIRK, J. T. 0. (1968). Studies on the dependence of chlorophyll synthesis on protein synthesis in Euglena

gracilis, together with a nomogram for determination of chlorophyll concentration. Planta 78, 200-207.

KIRK, J. T. 0. & ALLEN, R. L. (1965). Dependence of chloroplast pigment synthesis on protein synthesis: effect of actidione. Biochemical and Biophysical Resedrch Communications 2, 523-530.

KUNTZEL, H. & NOLL, H. (1967). Mitochondria1 and cytoplasmic polysomes from Neurospora crtrssa. Nature, London 215, 1340-1345.

LYMAN, H. (1967). Specific inhibition of chloroplast replication in Euglena gracilis by nalidixic acid. Journal of Cell Biology 35, 726730.

MCCALLA, D. R. (1962). Chloroplast of Euglena gracilis affected by furadantin. Science, New York 137, 225-226.

MCCALLA, D. R. (1965). Effect of nitrofurans on the chloroplast system of Euglena gracilis. Journal of

MCCALLA, D. R. & BAERG, W. (1969). Action of myxin on the chloroplast system of Euglenagracilis. Journal of Protozoology 16,425-428.

MARJAI, E., KISS, I. & IVANOVICS, G. (1970). Auxotrophic mutation associated with low streptomycin resistance and slow growth in Bacillus subtilis. Acta microbiologica Academiae scientiarum hungaricae 17, 133-145-

MEGO, J. L. (1964). The effect of hadacidin on chloroplast development in non-dividing Euglena cells. Biochiinica et biophysicd acta 79, 22 1-225.

OBE, G. (1970). Die Wirkung von Streptomycin und Dihydrostreptomycin auf menschliche Chromosomen in vitro. Molecular and General Genetics 107,361-365.

POGO, B. G. T. & POGO, A. 0. (1965). Inhibition by chloramphenicol of chlorophyll and protein synthesis and growth in Euglena gracilis. Journal of Protozoology 12, 96-100.

PROVASOLI, L., HUTNER, S. H. & SCHATZ, A. ( I 948). Streptomycin-induced chlorophyll-less races of Euglena. Proceedings of the Society for Experimental Biology and Medicine 69, 279-282.

RABUSSAY, D., HERZLICH, P., SCHWEIGER, M. & ZILLIG, W. (1969). More evidence for bacteria-like protein synthesizing apparatus in chloroplasts and mitochondria. FEBS Letters 4.55-59.

RIS, H. & PLAUT, W. (1962). Ultrastructure of DNA-containing areas in the chloroplast of Chlamydomonas. Journal of Cell Biology 13,383-391.

SAGER, R. (1962). Streptomycin as a mutagen for nonchromosomal genes. Proceedings of the National Academy of Sciences of the United States of America 48, 2018-2026.

SAGER, R. & TSUBE, Y. (1962). Mutagenic effects of streptomycin in Chlamydomonas. Archivfiir Mikro- biologie 4, 159-175.

SCHIFF, J. A. & EPSTEIN, H. T. (1965). The continuity of the chloroplast in Euglena. In Reproduction: Mole- cular, Subcellular and Cellular, pp. 131-187. Edited by M. Locke, New York: Academic Press.

SCOTT, N. S. (1969). The direction of chloroplast ribosomal RNA synthesis by DNA. (Abstract.) Annual Meeting of the Australian Biochemical Society, Adelaide.

STERN, J. I..., BARNET, H. D, & COHEN, S. S. (1966). The lethality of streptomycin and the stimulation of RNA synthesis in the absence of protein synthesis. Journal of Molecular Biology 17, 188-217.

TANAKA, N., MASUKAWA, H. & UMEZAWA, H. (1967). Structural basis of kanamycin for miscoding activity. Biochemical and Biophysical Research Communication 26, 544-549.

PrOlOZOOlOgy 12, 34-41.

52 L. EBRINGER

WILHELM, J. M., OLEINICK, N. L. & CORCORAN, J. W. (1968). Interaction of antibiotics with ribosomes: structure-function relationships and a possible common mechanism for the antimicrobial action of the macrolides and lincomycin. In Antimicrobial Agents and Chemotherapy, I 967, pp. 236-250. Edited by G. L. Hobby. Ann Arbor: American Society for Microbiology.

ZAHALSKY, A. C., HUTNER, S. H., KEANE, M. & BURGER, R. M. (1962). Bleaching of Euglena gracilis with an ti his t mines and streptomycin- type antibiotics. A rchiv fiir Mikrobio Zogie 4 2 , 4 6 3 5 .

ZIMMER, C., TRIEBEL, H. & THRUM, H. (1967). Interaction of streptothricin and related antibiotics with nucleic acids. Biochimica et biophysica acta 145, 742-75 I .