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Studies on the mode of action of streptogramin
PRELIMINARY NOTES
849
PN 6074
Studies on the mode of action of streptogramin The antibiotic streptogramin was first isolated from culture filtrates of Streptornyces gramino/aciens b y CHARNEY et al. 1. The antibiotic is bactericidal. Growing cultures of Gram-positive organisms are inhibited at concentrations of o.o2-I.8o/,g streptogramin per ml whereas most Gram-negative organisms are inhibited at i o - I o o t,g streptogramin per Inl. The antibiotic has no inhibitory action on fungi, actinomycetes or protozoa 2. The present work was carried out with Staphylococcus aureus strain Duncan for which the minimum growth-inhibitory concentration of streptogramin in vitro was 0.6/~g/ml. The size of the bacterial inoculum and the composition of the growth medium had no marked effect on the minimum inhibitory concentration. The absorbancy of growing cultures of S. aureus increased during incubation in presence of growth-limiting concentrations of streptogramin although at a slower rate than the control, but the number of viable bacteria determined by the method of MILES AI~D MISRA3 decreased. The m a x i m u m rate of killing occurred during the first 2 h in the presence of the antibiotic. Streptogramin even at a concentration of 60 #g/ml was found to have no effect on endogenous respiration, oxidation of ethanol or glucose, or anaerobic fermentation of glucose. Suspensions of S. aureus were incubated in the defined medium of GALE4 and the action of the antibiotic was studied on the net increase of the following fractions: (I) "Pool". This was the material extractable with either cold 5 9o trichloroacetic acid or 0.2 N HC1Oa. (2) "Nucleic acid fraction". Material extracted with either 5 ~o trichloroacetic acid at 9 °0 for 15 rain or 0 5 N HC104 at 7 °° for 30 rain. (3) "Protein fraction". This was the acid-insoluble residue. The fractionation procedure was based on that described for Escherichia coli by ROBERTS et al. 5 Confirmatory experiments were carried out using the incorporation of 14C-labelled compounds as a measure of synthesis of these fractions and also of the cell-wall fraction prepared b y the method of HANCOCK AND PARK6. Streptogramin had no effect on accumulation of nucleic acid derivatives (material having an absorption m a x i m u m at 260 m/~) in the "pool" or on the incorporation of laC-labelled glycine into this fraction, but the accumulation of ~*C-labelled glutamic or aspartic acids in the "pool" was increased b y 35 % in the presence of 3/~g streptogramin per rnl when compared with the control in the absence of antibiotic. Nucleic acid synthesis was stimulated in the presence of streptogramin. This stimulation was due to an increase in the rate of RNA formation, the synthesis of DNA being unaffected (Fig. I). At the minimum growth-inhibitory concentration there was a 7 ° ~o inhibition of net synthesis of protein (Fig. 2) and of incorporation of 14C-labelled amino acids into this fraction. GALE9 found similar effects of chloramphenicol on protein and RNA synthesis b y S. aureus. Streptogramin at concentrations of 0.6 or 3/~g/ml inhibited b y 70 % and 9 ° °/o, respectively, the synthesis of /~-galactosidase studied b y the method of LEDERBERG10. CREASER11 found an inhibition of ~galactosidase formation by S. aureus in the presence of chloramphenicol or other inhibitors of protein synthesis. Streptogramin had no effect on the incorporation of 14C-labelled glucose, glutamic acid or glycine into the cell-wall fraction of S. aureus. Biochim. Biophys..4cla, 61 (1962) 849-85z
850
PRELIMINARY NOTES 80
6O
"~ 40
2C
3'0
°o
(~o
~o
Time (min) a f t e r add ition of streptogramin
Fig. I. T i m e course of t h e effect of s t r e p t o g r a m i n on n e t nucleic acid a n d D N A s y n t h e s i s in S. n e t nucleic acid s y n t h e s i s in t h e control; Q - O , n e t n u cleic acid s y n t h e s i s in presence of 0.6 # g s t r e p t o g r a m i n p e r ml; A - A n e t D N A s y n t h e s i s in t h e control; A - A , n e t D N A s y n t h e s i s in p r e s e n c e of 0.6 # g s t r e p t o g r a m i n p e r ml. S u s p e n s i o n s of I m g d r y wt. o r g a n i s m s p e r m l were i n c u b a t e d in t h e defined m e d i u m of GALE4. S a m p l e s were t a k e n at i n t e r v a l s a n d nucleic acid w a s e x t r a c t e d w i t h 0. 5 N h o t HC104 after p r e v i o u s e x t r a c t i o n of t h e " p o o l " w i t h 0.2 N cold HC104. Nucleic acid w a s e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y a t 260 m/~ and D N A b y t h e m e t h o d of ]3URTON7. a u r e u s D u n c a n i n c u b a t e d a t 37 °. 0 - ( ~
3O
2C
8L•~ 1(3
0
0
30 60 90 Time (min) a f t e r addition o f streptogromin
Fig. 2. T i m e course of t h e effect of s t r e p t o g r a m i n on n e t p r o t e i n s y n t h e s i s in S. a u r e u s D u n c a n inc u b a t e d a t 37 °. O - O , n e t p r o t e i n s y n t h e s i s in t h e control; S - Q , n e t p r o t e i n s y n t h e s i s in presence of 0.6/~g s t r e p t o g r a m i n p e r m l . S u s p e n s i o n s of I m g d r y wt. o r g a n i s m s p e r m l were i n c u b a t e d in t h e d e f i n e d m e d i u m of GALE. S a m p l e s were t a k e n a t i n t e r v a l s a n d p r o t e i n w a s e s t i m a t e d b y t h e m e t h o d of L o w r y et al. 8 in t h e m a t e r i a l insoluble in h o t 0. 5 N HC104.
Although streptogramin has an effect on net synthesis of protein and nucleic acid similar to that of chloramphenicol, the antibiotics showed no additive or synergistic effect when present together and also streptogramin, unlike chloramphenicol, is bactericidal. These results suggest that the primary point of action is not identical for the two compounds. Chloramphenicol at concentrations less than the growthlimiting concentration gives partial inhibition of protein synthesis in suspensions of S . a u r e u s but there is no increase in this inhibition on the further addition after IO rain of streptogramin. Assays on agar showed no antagonism between these antiBiochim.
Biophys.
A c t a , 61 (1962) 849-851
PRELIMINARY NOTES
85I
biotics. Addition of chloramphenicol at the minimum growth-inhibitory concentration (IO #g/ml) to a culture of S. aureus completely prevented the bactericidal action of streptogramin. The protection of the bacteria from the bactericidal effect of streptogramin occurred to the same extent when the chloramphenicol concentration was reduced to 3/~g/ml. This protective effect was obtained when chloramphenicol was added before or at the same time as streptogramin and also to some extent when added io rain after the streptogramin. It has been suggested that the protective effect of chloramphenicol against the bactericidal action of penicillin and streptomycin could be due to the inhibition of bacterial growth b y chloramphenicol 1~, but the bactericidal effect of streptogramin is prevented by concentrations of chloramphenicol which inhibit protein synthesis by 60 °/oonly. Omitting glutamic acid from the incubation medium caused a 7 ° % reduction in protein synthesis and under these conditions there was some protection against the bactericidal effect of streptogramin. The addition of 80/,g puromycin per ml which produces a 70 % inhibition of protein synthesis, did not protect the bacteria against streptogramin. The author wishes to thank Merck Dohme Research Laboratories for a gift of streptogramin and The British Council for a Scholarship.
Medical Research Council Unit /or Chemical Microbiology, Department o] Biochemistry, University o/ Cambridge, Cambridge (Great Britain)
D. VAZQUEZ
1 j . CHARNEY, W. P. FISHER, CH. CURRAN, t{. A. MACHLOWlTZ AND A. A.. TYTELI, Antibiot. Chemotherapy, 3 (1953) 1283. 2 W. F. VER'WEY, 3/I. I~. WEST AND A. K. MILLER, Antibiot. Chemotherapy, 8 (1958) 500. 3 A. A. MILES AND S. S. MISRA, J . Hyg., 38 (1938) 732. 4 E. F. GALE AND J. P. FOLKES, Biochem. J., 53 (1953) 483 • 5 R. ]~. ROBERTS, D. B. COWlE, P. H. ABELSON, E. T. BOLTON AND B. J. BRITTEN, Studies o/ Biosynthesis in Escherichia coli, 1955, Carnegie Inst., W a s h i n g t o n , Publ. No. 607. 6 ]~. HANCOCK AND J. T. PARK, Nature, 181 (1958) lO5O. 7 K. BURTON, Biochem. J., 62 (1956 ) 315 . s O. I-i. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, dr. Biol. Chem., 193 (1951) 265 . 9 E. F. GALE AND J. P. FOLKES, Biochem. J., 53 (1953) 493. 10 j . LEDERBERG, J. Bacteriol., 60 (I95o) 381. ll E. l-~. CREASER, J. Gen. Microbiol., 12 (1955) 288. 12 T. D. BROCK, Bacteriol. Rev., 25 (1961) 32.
Received October 5th, 1962 Biochim. Biophys. dcta, 61 (1962) 849-851
849
PN 6074
Studies on the mode of action of streptogramin The antibiotic streptogramin was first isolated from culture filtrates of Streptornyces gramino/aciens b y CHARNEY et al. 1. The antibiotic is bactericidal. Growing cultures of Gram-positive organisms are inhibited at concentrations of o.o2-I.8o/,g streptogramin per ml whereas most Gram-negative organisms are inhibited at i o - I o o t,g streptogramin per Inl. The antibiotic has no inhibitory action on fungi, actinomycetes or protozoa 2. The present work was carried out with Staphylococcus aureus strain Duncan for which the minimum growth-inhibitory concentration of streptogramin in vitro was 0.6/~g/ml. The size of the bacterial inoculum and the composition of the growth medium had no marked effect on the minimum inhibitory concentration. The absorbancy of growing cultures of S. aureus increased during incubation in presence of growth-limiting concentrations of streptogramin although at a slower rate than the control, but the number of viable bacteria determined by the method of MILES AI~D MISRA3 decreased. The m a x i m u m rate of killing occurred during the first 2 h in the presence of the antibiotic. Streptogramin even at a concentration of 60 #g/ml was found to have no effect on endogenous respiration, oxidation of ethanol or glucose, or anaerobic fermentation of glucose. Suspensions of S. aureus were incubated in the defined medium of GALE4 and the action of the antibiotic was studied on the net increase of the following fractions: (I) "Pool". This was the material extractable with either cold 5 9o trichloroacetic acid or 0.2 N HC1Oa. (2) "Nucleic acid fraction". Material extracted with either 5 ~o trichloroacetic acid at 9 °0 for 15 rain or 0 5 N HC104 at 7 °° for 30 rain. (3) "Protein fraction". This was the acid-insoluble residue. The fractionation procedure was based on that described for Escherichia coli by ROBERTS et al. 5 Confirmatory experiments were carried out using the incorporation of 14C-labelled compounds as a measure of synthesis of these fractions and also of the cell-wall fraction prepared b y the method of HANCOCK AND PARK6. Streptogramin had no effect on accumulation of nucleic acid derivatives (material having an absorption m a x i m u m at 260 m/~) in the "pool" or on the incorporation of laC-labelled glycine into this fraction, but the accumulation of ~*C-labelled glutamic or aspartic acids in the "pool" was increased b y 35 % in the presence of 3/~g streptogramin per rnl when compared with the control in the absence of antibiotic. Nucleic acid synthesis was stimulated in the presence of streptogramin. This stimulation was due to an increase in the rate of RNA formation, the synthesis of DNA being unaffected (Fig. I). At the minimum growth-inhibitory concentration there was a 7 ° ~o inhibition of net synthesis of protein (Fig. 2) and of incorporation of 14C-labelled amino acids into this fraction. GALE9 found similar effects of chloramphenicol on protein and RNA synthesis b y S. aureus. Streptogramin at concentrations of 0.6 or 3/~g/ml inhibited b y 70 % and 9 ° °/o, respectively, the synthesis of /~-galactosidase studied b y the method of LEDERBERG10. CREASER11 found an inhibition of ~galactosidase formation by S. aureus in the presence of chloramphenicol or other inhibitors of protein synthesis. Streptogramin had no effect on the incorporation of 14C-labelled glucose, glutamic acid or glycine into the cell-wall fraction of S. aureus. Biochim. Biophys..4cla, 61 (1962) 849-85z
850
PRELIMINARY NOTES 80
6O
"~ 40
2C
3'0
°o
(~o
~o
Time (min) a f t e r add ition of streptogramin
Fig. I. T i m e course of t h e effect of s t r e p t o g r a m i n on n e t nucleic acid a n d D N A s y n t h e s i s in S. n e t nucleic acid s y n t h e s i s in t h e control; Q - O , n e t n u cleic acid s y n t h e s i s in presence of 0.6 # g s t r e p t o g r a m i n p e r ml; A - A n e t D N A s y n t h e s i s in t h e control; A - A , n e t D N A s y n t h e s i s in p r e s e n c e of 0.6 # g s t r e p t o g r a m i n p e r ml. S u s p e n s i o n s of I m g d r y wt. o r g a n i s m s p e r m l were i n c u b a t e d in t h e defined m e d i u m of GALE4. S a m p l e s were t a k e n at i n t e r v a l s a n d nucleic acid w a s e x t r a c t e d w i t h 0. 5 N h o t HC104 after p r e v i o u s e x t r a c t i o n of t h e " p o o l " w i t h 0.2 N cold HC104. Nucleic acid w a s e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y a t 260 m/~ and D N A b y t h e m e t h o d of ]3URTON7. a u r e u s D u n c a n i n c u b a t e d a t 37 °. 0 - ( ~
3O
2C
8L•~ 1(3
0
0
30 60 90 Time (min) a f t e r addition o f streptogromin
Fig. 2. T i m e course of t h e effect of s t r e p t o g r a m i n on n e t p r o t e i n s y n t h e s i s in S. a u r e u s D u n c a n inc u b a t e d a t 37 °. O - O , n e t p r o t e i n s y n t h e s i s in t h e control; S - Q , n e t p r o t e i n s y n t h e s i s in presence of 0.6/~g s t r e p t o g r a m i n p e r m l . S u s p e n s i o n s of I m g d r y wt. o r g a n i s m s p e r m l were i n c u b a t e d in t h e d e f i n e d m e d i u m of GALE. S a m p l e s were t a k e n a t i n t e r v a l s a n d p r o t e i n w a s e s t i m a t e d b y t h e m e t h o d of L o w r y et al. 8 in t h e m a t e r i a l insoluble in h o t 0. 5 N HC104.
Although streptogramin has an effect on net synthesis of protein and nucleic acid similar to that of chloramphenicol, the antibiotics showed no additive or synergistic effect when present together and also streptogramin, unlike chloramphenicol, is bactericidal. These results suggest that the primary point of action is not identical for the two compounds. Chloramphenicol at concentrations less than the growthlimiting concentration gives partial inhibition of protein synthesis in suspensions of S . a u r e u s but there is no increase in this inhibition on the further addition after IO rain of streptogramin. Assays on agar showed no antagonism between these antiBiochim.
Biophys.
A c t a , 61 (1962) 849-851
PRELIMINARY NOTES
85I
biotics. Addition of chloramphenicol at the minimum growth-inhibitory concentration (IO #g/ml) to a culture of S. aureus completely prevented the bactericidal action of streptogramin. The protection of the bacteria from the bactericidal effect of streptogramin occurred to the same extent when the chloramphenicol concentration was reduced to 3/~g/ml. This protective effect was obtained when chloramphenicol was added before or at the same time as streptogramin and also to some extent when added io rain after the streptogramin. It has been suggested that the protective effect of chloramphenicol against the bactericidal action of penicillin and streptomycin could be due to the inhibition of bacterial growth b y chloramphenicol 1~, but the bactericidal effect of streptogramin is prevented by concentrations of chloramphenicol which inhibit protein synthesis by 60 °/oonly. Omitting glutamic acid from the incubation medium caused a 7 ° % reduction in protein synthesis and under these conditions there was some protection against the bactericidal effect of streptogramin. The addition of 80/,g puromycin per ml which produces a 70 % inhibition of protein synthesis, did not protect the bacteria against streptogramin. The author wishes to thank Merck Dohme Research Laboratories for a gift of streptogramin and The British Council for a Scholarship.
Medical Research Council Unit /or Chemical Microbiology, Department o] Biochemistry, University o/ Cambridge, Cambridge (Great Britain)
D. VAZQUEZ
1 j . CHARNEY, W. P. FISHER, CH. CURRAN, t{. A. MACHLOWlTZ AND A. A.. TYTELI, Antibiot. Chemotherapy, 3 (1953) 1283. 2 W. F. VER'WEY, 3/I. I~. WEST AND A. K. MILLER, Antibiot. Chemotherapy, 8 (1958) 500. 3 A. A. MILES AND S. S. MISRA, J . Hyg., 38 (1938) 732. 4 E. F. GALE AND J. P. FOLKES, Biochem. J., 53 (1953) 483 • 5 R. ]~. ROBERTS, D. B. COWlE, P. H. ABELSON, E. T. BOLTON AND B. J. BRITTEN, Studies o/ Biosynthesis in Escherichia coli, 1955, Carnegie Inst., W a s h i n g t o n , Publ. No. 607. 6 ]~. HANCOCK AND J. T. PARK, Nature, 181 (1958) lO5O. 7 K. BURTON, Biochem. J., 62 (1956 ) 315 . s O. I-i. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, dr. Biol. Chem., 193 (1951) 265 . 9 E. F. GALE AND J. P. FOLKES, Biochem. J., 53 (1953) 493. 10 j . LEDERBERG, J. Bacteriol., 60 (I95o) 381. ll E. l-~. CREASER, J. Gen. Microbiol., 12 (1955) 288. 12 T. D. BROCK, Bacteriol. Rev., 25 (1961) 32.
Received October 5th, 1962 Biochim. Biophys. dcta, 61 (1962) 849-851