PN 21 o66
Studies on the mode of action of etamycin (viridogrisein) The antibiotic etamycin studied here was isolated from culture filtrates of an unidentified species of Streptomyces and of a strain of Streptomyces griseus independently by HEINEMANN et al. 1, and BARTZ et al. ~', who named it etamycin and viridogrisein respectively. The chemical structure and hence the identity of both antibiotics was later established in the same laboratories and also by SHEEHAN et al. a. Etamycin was included by LESTER SMITH4 within group B of the ostreogrycin family; any member of this group strongly potentiates the antimicrobial action of any member of group A. The antibiotic exhibits considerable activity against a range of grampositive organisms (minimum growth inhibitory concentrations o.1-5/zg/ml) but has little or no activity against gram-negative bacteria, yeasts, fungi or protozoa 5. Studies on the mode of action of etamycin have been carried out with Staphylococcus aureus strain Duncan, and Bacillus megaterium strain KM (minimum growth inhibitory concentrations are 3 and 5/,g/ml respectively). For both organisms, the antibiotic is bacteriostatic and shows a marked synergism with ostreogryein A (synonyms P A - I I 4 A-I, staphylomycin M1, streptogralnin A) comparable to that found by VANDERHAEGHE AND PARMENTIER6 with staphylomycin Mr. The synergistic mixture of etamycin and ostreogrycin A is bactericidal. This last finding is consistent with the results obtained by VAZQUEZ7 for streptogramin, a bactericidal antibiotic complex in which the separate purified compounds are bacteriostatic. Etamycin at ten times the minimum growth inhibitory concentration had no effect on endogenous respiration, oxidation or fermentation of glucose by S. aureus and even at concentrations of IOO/,g/ml showed no surface activity for this bacterium. The effects of etamycin on net synthesis of the various cellular constituents of S. aureus and B. megaterium incubated in the defined medium of GALE AND FOLKESs were studied. The bacteria were fractionated by a method based on that described by ROBERTS et al. 9 The "pool" was extracted with cold 0.2 N perchloric acid; nucleic acid was extracted with 0.5 N perchloric acid at 70 ° for 30 rain. Protein was estimated in the insoluble residue by the method of LowRY et al. 1°. The incorporation of 14Clabelled compounds was also used to follow macromolecular synthesis and in this case the bacteria were fractionated into "pool" (extracted with 5 % (w/v) trichloroacetic acid at 4 ° for 15 rain), nucleic acid (extracted with 5 % (w/v) trichloroacetic acid at 9 °0 for 15 min), protein and cell wall by the method of HANCOCKAND PARK11. Etamycin at the minimum growth inhibitory concentration had no significant effect on the accumulation of ninhydrin-positive material but showed some inhibitory effect on the accumulation of nucleic acid precursors (material having an absorption m a x i m u m at 260 m/~) in the pool fraction. The uptake into the pool of l~C-labelled amino acids was unaffected, while uptake of laC-labelled adenine was partially inhibited as was its incorporation into nucleic acid (Table I). Net synthesis of deoxyribonucleic acid was more sensitive to etamycin than was synthesis of ribonucleic acid. At the minimum growth inhibitory concentration etamycin inhibited net protein synthesis by 9 ° %. A similar degree of inhibition was observed for the incorporation of [14C~amino acids into the protein fraction. The inhibitory effect of etamycin on amino acid incorporation could be detected within one minute of adding the antibiotic. The incorporation of [14C]glycine and [14Cjglutamic acid into the cell wall fraction Biochim. Biophys. Acla, 97 (1965) 394-396
PRELIMINARY NOTES TABLE I THE
OF ETAMYCIN OF
n . megaterium
Suspensions of B. megaterium (i m g dry wt. o r g a n i s m s per ml) were i n c u b a t e d at 37 ° in the defined m e d i u m of GALE A N D F O L K E S 8, s u p p l e m e n t e d w i t h a 14C-labelled c o m p o u n d . Samples were taken at intervals a n d the radioactivity of the various fractions determined. Concentration of e t a m y c i n 5 #g/ml.
% inhibition of incorporation
Incubation time (rain)
G l u t a m i c acid
* NA = nucleic acid.
of B. megaterium was lO-2O % inhibited by etamycin at a concentration producing 9° % inhibition of protein synthesis (Table I). From these results it appears that the overall effects of etamycin on sensitive bacteria are similar to those found by VAZQUEZ7 with PA-II 4 B-I. Confirmation of the inhibitory effect of etamycin on protein synthesis is the finding that synthesis of an inducible ~-galactosidase TMby S. aureus was 9 ° % inhibited by etamycin at the growth inhibitory concentration. As the primary site of action of etamycin appeared to be in the sequence of reactions leading to the formation of protein, a study was made of the effect of etamycin on each of the intermediate steps in the pathway of protein synthesis. Etamycin at the minimum growth inhibitory or higher concentrations had no effect on the activation of amino acids when studied by the formation of aminoacyl hydroxamates 13. TABLE II THE
IN A CELL-FREE
OF B. megaterium Complete s y s t e m contained in a volume of i ml: Tris, 35 # m o l e s (pH 7.4); KC1, 5 °/~moles; MgC12, 13 p m o l e s ; creatine p h o s p h a t e , 5 / , m o l e s ; creatine kinase, i o o / z g ; ATP, 3 / , m o l e s ; CTP, o . 6 / , m o l e ; m e r c a p t o e t h a n o l , io/zmoles; poly-U, 5o/~g; L-[XlC]phenylalanine, 0.5 /zC (o.oo35 /zmole]/~C) ; i m g of ribosomes a n d soluble fraction (i m g of protein). I n c u b a t i o n was carried out a t 37 ° a n d after 3o min, the reaction was s t o p p e d b y the addition of t ml of Io % (w/v) trichloroacetic acid. P r o t e i n p r e p a r a t i o n was o b t a i n e d b y the m e t h o d of SIEKEVITZ TM.
Complete + e t a m y c i n (5° / t g / m l )
Complete + e t a m y c i n (ioo #g/ml)
Complete + chloramphenicol (i oo/zg/ml) 291
Biochim. Biophys. Acta, 97 (1965) 394-396
The formation of aminoacyl s-RNA was studied in a cell-free system from B. megaterium using the method described by NATtIANS AND LIPMANN14, and it was found that etamycin at a concentration of 50 t~g/ml inhibited the formation of leucyl s-RNA by about 15-2o %. The transfer of [14Clleucine from leucyl s-RNA to the protein fraction was studied in the same cell-free system and was not inhibited by etamycin at a concentration of 5 °/zg/ml. Confirmation of these findings was obtained by studying the incorporation of L14Clphenylalanine by a cell-free system of B. megaterium using the incubation conditions described by NIRENBERG AND MATTHAE115. It was found that [14C]phenylalanine incorporation into protein although sensitive to chloramphenicol under the experimental conditions used, was not significantly inhibited by etamycin even at ten times the minimum growth inhibitory concentration (Table II). These results suggest that the failure of etamycin to inhibit amino acid incorporation into protein in these cell-free systems could be due either to the necessity for some modification of the antibiotic to a more active form which occurs only in whole cells, or the possibility that the internal concentration of etamycin inhibited cells is very much greater than the concentration used in the broken cells preparations. The author wishes to thank Dr. J. EI~RLICI-I (Parke, Davis & Co. Michigan, U.S.A.) and Dr. B. HEINEMANN (Bristol Laboratories, New York, U.S.A.) for gifts of viridogrisein and etamycin, respectively.
Sub-department of Chemical Microbiology, Department of Biochemistry, The University, Cambridge (Great Britain)
I B. HEINEMANN, A. GOUREVITCH, J. LEIN, D. L. JohNSON, M. A. KAPLAN, D. VANAS AND I. R. HOOPER, Antibiot. Ann., Medical Encyclopedia Inc., 1954-55, New York, 728. 2 Q. R. BARTZ, J. STANDIFORD, J. D. MOLD, D. W. JOHANNESSEN, A. RYDER, A. MARETZKI AND T. H. HASKELL, Mntibiot. Ann., Medical Encyclopedia Inc., New York, I954-55, p. 7773 J. C. SHEEHAN, H. G. ZACHAU AND W. B. LAWSON, J. Am. Chem. Soc., 80 (1958) 3349. 4 E. LESTER SMITH, J . Gen. Microbiol., 33 (1963) iii. 5 J. EHRLICH, G. L. COFFEV, M. M. GALBRAITH, M. P. KNUDSEN, A. S. SCHLINGMAN AND R. M. SMITH, Antibiot. Ann., Medical Encyclopedia Inc., New York, 1954-55, p. 79o. 6 H. VANDERHAI?.GHEAND G. ]9ARMENTIER, J. 2Jm. Chem. Soc., 82 (196o) 4414 . 7 D. VAZQUEZ, J. Gen. Mierobiol., 33 (1963) ix. 8 E. F. GALE AND J. P. FOLKES, Biochem. f . , 53 (I953) 483 . 9 R. B. ROBERTS, D. B. COWlE, P. H. ABELSON, E. T. BOLTON AND R. BRITTEN, Carnegie Inst. Wash., Publ., 607 (I955). IO O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. i i 1~. HANCOCK AND J. T. PARK, Nature, 181 (1958) lO5O. I2 E. H. CREASER, J. Gen. Microbiol., 12 (1955) 288. 13 M. B. HOAGLAND, Biochim. Biophys. Acta, 16 (1955) 288. 14 D. NATHANS AND V. LIPMANN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 497. 15 M. VV. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (1961) 158. 16 ]9. SIEKEVlTZ, J. Biol. Chem., 195 (1952) 549-
Received October 28th, 1964 * Present address: Instituto "Jaime Ferran" de Microbiologia, Velazquez 138, Madrid, Spain.
Biochim. Biophys. Acta, 97 (1965) 394-396