PRELIMINARY NOTES

PN 41 020

Inhibition of greening of heat-bleached Euglena by streptomycin

663

The normally green autotrophic flagellate, Euglena gracilis, can be transformed to a bleached heterotroph by several agents. Growth at 34.5 ° (ref. I), in the presence of streptomycin * or other chemicals 3,4, or ultraviolet light s results in a permanently colorless organism which still retains fluorescent proplastid-like structures e. Strepto- mycin does not inhibit the greening of dark-grown Euglena gracilis, strain Z, under resting conditions at pH 5; the antibiotic must be present during cell division for maximum effect 7. If Euglena is returned to lower temperatures after less than about 200 h growth at 34.5 ° the organism will revert to a green state after a lag period s. 34.5 ° apparently inhibits the development of a factor responsible for normal chloro- plast growth and chlorophyll synthesis s. The factor can be repleted at lower tempera- tures providing it has not been completely eliminated by growth at 34.5 ° . This suggests some replicating system which becomes diluted out by growth at elevated temperature or in the presence of streptomycin.

If Euglena is grown at 34.5 ° and then transferred to room temperature in a resting medium (no nitrogen or carbon source), the synthesis of chlorophyll under these conditions would indicate the replication of the temperature-sensitive system in the absence of extensive cell division. If the temperature-sensitive system is the same as the one which becomes deleted by growth in the presence of streptomycin, or if it occurs before the streptomycin block, then the antibiotic should inhibit greening in the resting medium.

Euglena gracilis, strain Z, was used in this study. The medium used for growth, composition of the resting medium, chlorophyll assay, etc. were the same as described previously 9. The organisms were grown aseptically at 34-35 ° in 5oo-ml quantities,

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Fig. I. Inhib i t ion of greening at room tempera- ture by s t rep tomycin of a culture grown for 3 days at 34.5 ° . 0 - - O , no s t rep tomycin ;

O - - - O , s t rep tomycin , io/~g/ml.

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/X S / Time at room temperature(days)

Fig. 2. Inhib i t ion of greening at room tempera- ture by s t rep tomycin of a culture grown for 4 days at 34.5 ° . O - - O , no s t rep tomycin ;

• - - - • , s t reptomycin , Io/zg/ml.

Biochim. Biophys. Acta, 88 (1964) 663-665

664 PRELIMINARY NOTES

centrifuged and washed twice in IO ml resting medium, and suspended in 5 ml resting medium with and without s treptomycin.

Figs. I, 2, and 3 illustrate the inhibition of greening by i o / , g / m l s treptomycin of organisms grown for 3, 4, and 5 days at 34.5 °, respectively. This concentrat ion of s t reptomycin does not inhibit cell division but is sufficient to completely bleach Euglena gracilis when grown in the presence of the antibiotic ~. The inhibition was

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Fig. 3. Inhibition of greening at room tempera- ture by streptomycin of a culture grown for 5 days at 34.5 ° , 0 - - 0 , no streptomycin;

• --- 0 , streptomycin, IO #g/ml.

temperature for 3 days, and then transferred to the light, io ffg/ml streptomycin was added to one culture at the beginning of the dark period (Curve 2), to another after the dark period (Curve i), and

the control received no streptomycin (Curve 3)-

0 1 2 3 4 5 Time in light at r o o m temperature(days)

Fig. 4. Reversal of the effect of s t rep tomycin on greening of 34.5 ° culture by a 3-day-dark period. All cultures were grown for 4 days at the elevated tempera ture , washed twice in rest ing medium, placed in the dark at room

vir tual ly complete in the culture grown for 5 days and was progressively less in the 4-day and 3-day cultures. The lag period before chlorophyll synthesis commenced also appeared to be longer with increasing t ime at 34.5 ° . These results suggest tha t the factor responsible for chloroplast development and chlorophyll synthesis became progressively more depleted with increasing time at the high temperature. After 3 days growth, for instance, enough factor remained to allow for about 50 % of the chloro- phyll to be synthesized. Fig. 4 shows tha t the temperature-sensit ive component could be restored in the dark at room temperature, but a lag period in the light was still required for chlorophyll synthesis to occur.

These experiments strongly suggest tha t the step inhibited by s t reptomycin in the pa thway leading to the formation of green chloroplasts is the same as, or occurs after the one which is inhibited by growth at 34.5 ° . I f s t reptomycin inhibited some step before the one blocked by growth at 34.5 ° the antibiotic would not have inhibited

Biochim. Biophys. Acta, 88 (1964) 663-665

PRELIMINARY NOTES 665

greening at room temperature. Since tile cells were allowed to green in a medium lacking a nitrogen source, restoration of the temperature-sensitive step occurred in the absence of extensive cell division.

FLAKS AND WITTING TM have recently proposed that streptomycin acts by pre- venting the formation of ribosomes. The effect of elevated temperature or strepto- mycin on greening in Euglena may be to inhibit the replication of ribosomes speci- fically involved in chloroplast development.

This work was supported in part by grant C-3256, National Institutes of Health, U.S. Public Health Service; and by Contract AT (3o-I), U.S. Atomic Energy Com- mission.

Baltimore City Hospitals, JOHN L. MEGO Baltimore, Md. (U.S.A.)

1 E. G. PRINGSHEIM AND O. PRINGSHEIM, New Phytologist, 51 (1952) 65. 2 M. BRACCO AND H. VON EULER, Kern. Arb., 2 (1947) IO.

J. A. GROSS, T. L. JAHN AND E. BERNSTEIN, J. Pro/ozool., 2 (1955) 71. 4 S. AARONSON AND B. BENSKY, J. Gen. Microbiol., 27 (1962) 75. 5 H. LYMAN, H. T. EPSTEIN AND J. A. SCHIFF, Biochim. Biophys. Acta, 5 ° (1961) 3Ol. ¢ A. GIBOR AND S. GRANICK, J. Protozool., 9 (1962) 327 . 7 j . L. 1V[EGO ANn D. E. BUETOW, Abstr., Am. Soc. Cell Biol., 4th Ann. Meeting, z964, J. Cell Biol.,

23 (1964) 58A. 8 G . BRAWERMAN AND E. CHARGAFF, Biochim. Biophys. Acta, 31 (1959) 178. 9 j . L. MEGO, Biochim. Biophys. Acta, 79 (1964) 221.

10 j . G. FLAKS AND M. L. WITTING, Bacteriol. Proc., (1963) 118.

Received September 28th, 1964

Biochim. Biophys. Acta, 88 (1964) 663-665