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Sensitivity and resistance to erythromycin in Bacillus subtilis 168: The ribosomal binding of erythromycin and chloramphenicol
PRELIMINARY NOTES
438
BBA 91122
Sensitivity ond resistance to erythromycin in Bacillus subtilis 168: the ribosomal bindin 9 of erythromycin ond chloramphenicol Erythromycin-A (erythromycin), one of the macrolide group of antibiotics, inhibits bacterial synthesis of polypeptides, both in vivol, ~ and in vitro2, 3,4. It does not inhibit amino acid activation to amino acyl-tRNA, and, therefore, most probably acts on either the assemblage of the polysome or on some aspect of its function. In the studies reported here, biologically active tritiated erythromycin was incubated with 2 different cell-free systems capable of making polypeptides from free amino acids. One system was derived from erythromycin-sensitive Bacillus subtilis 168, and the other from an erythromycin-resistant mutant. Biologically active [3H]erythromycin, added to a cell-free system derived from the erythromycin-sensitive strain of B. subtilis, is found to associate with the ribosomal components. Furthermore, a greatly enhanced concentration of the antibiotic is found in the fractions which contain 5o-S ribosomal units (Fig. IA). This association with 5o-S particles is time dependent, but attains the maximal level by 2-3 min. The erythromycin-resistant mutant of B. subtilis 168 was studied in the same way as the parent strain. The associative pattern of 73H ]erythromycin with the ribosomal particles in a cell-free extract (shown as Fig. IB) is not tile same as that described for the antibiotic-sensitive bacteria. The binding of the erythromycin with the particle is reduced, and no specific accumulation of radioactivity in the 5o-S fraction is seen. The difference in the relative abundance of To-S, 5o-S and 3o-S particles between the experiments shown as Figs. IA and B is within normal limits for different preparations of cell-free extracts. According to several reports, chloramphenicol binds to 5o-S ribosomal particles in cell-free extracts of Staphylococcus aureus, Bacillus megaterium and Escherichia coli 5-8. The binding is not time, energy and temperature dependent, and it is readily reversible. Chloramphenicol binding to ribosomes is effectively inhibited by erythromycin, and this has prompted speculation on the probability that both antibiotics bind or act at the same ribosomal site(s)5,v, s. The results of our studies with B. subtilis extracts are of interest in evaluating this hypothesis, since it was found that there is a major difference between the binding of erythromycin and chloramphenicol to 5o-S ribosomal particles. Erythromycin, when bound, is not lost from the 5o-S units during ultracentrifugation through a sucrose density-gradient system. Furthermore, a large molar excess of chloramphenicol (I58-fold) does not affect the asst~ciation of erythromycin and the 5o-S ribosomal particles (Fig. IC). In confirmation of the results using E. coli ribosomes 8, it was demonstrated that any chloramphenicol that m a y be bound to the 5o-S ribosomal units from B. subtilis does not survive fractionation by the ultracentrifugal method (Fig. I D ) . These experiments prove in a direct way that erythromycin has a much greater affinity for B. sublilis ribosomes than does chloramphenicol. The antibiotics, both individually and together, were active in inhibiting protein synthesis, although in these and similar A b b r e v i a t i o n : tRN,,\, t r a n s f e r I¢,NA.
Biochin,. 13iophys. Acta, 123 (196~)) 438-44 °
PRELIMINARY NOTES
o.*
439
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8O
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20 Fraction
/
20
r¢
.,,.2'. . . . . ~s..~?s. ?p.s.. I
5 number
I0
15
20
Fig. i. Ultracentrifugal analysis of cell-free s y s t e m s from B. subtilis. The strains of B. subtilis used a n d the preparation of cell-free e x t r a c t s were described earlier=, 3. Sucrose d e n s i t y - g r a d i e n t centrifugation was carried out using 4.4 ml of a 25-1o % continuous linear sucrose gradient buffered with Tris-HC1 (pH 8.I, o.I M), a n d containing m a g n e s i u m acetate (o.o12 M) and NHiC1 (o.o2 M). After incubation, a reaction m i x t u r e was chilled and a o.2-ml aliquot layered on top of the sucrose gradient. The sample was centrifuged at 4 ° in a Spinco L-2 ultracentrifuge for 135 m i n at 35 ooo r e v . / m i n in an SW 39 rotor. Sequential 15-drop fractions (approx. o.2 ml each) were collected after p u n c t u r i n g the b o t t o m of the centrifuge tube. Analysis, b o t h for light absorbance at 260 m/~ and radioactivity, was carried out on an aliquot of each fraction 3. The preparation of [SH]erythromycin h a s been described 2. Tritiated chloramphenicol, prepared b y t h e Wilzbach technique, was purchased from New E n g l a n d Nuclear Corporation, as was u n i f o r m l y labeled L-[liC]arginine. The effect of the antibiotics on t h e incorporation of arginine into h o t trichloroacetic acid-precipitable material was determined directly on o . i - m l aliquots of unfractionated reaction m i x t u r e s 1.. E a c h experimental sample contained t h e following (ffmoles/ml, unless otherwise indicated): Tris-HC1, ioo (pH 8.I); m a g n e s i u m acetate, 12; NH4C1, 40; 2-thioethanol, 3.3; ATP, i; phosphoenolpyruvate, 5, GTP, UTP, CTP, o.I each; L-amino acid m i x t u r e lacking L-arginine, 0.067 each, L-arginine, o.o13; p y r u v a t e kinase (EC 2. 7. E.4o), 3.3 ffg/ml. In each experiment a duplicate incubation was performed, in which L-[14Clarginine (O.Ol3, 0.33/*C/ml) was s u b s t i t u t e d for the non-radioactive arginine. In addition, the individual reaction m i x t u r e s were characterized by the following: A. E r y t h r o m y c i n - s e n s i t i v e strain: protein, 1. 7 mg/ml; RNA, 384/~g/ml; DNA, 125/zg/ ml; [3H]erythromycin, 3 . i . i o - l / z m o l e / m l , o.oi/zC/ml; 3-min incubation. B. E r y t h r o m y c i n r e s i s t a n t strain: protein, 4.7 mg/ml; RNA, 3o4ffg/ml; DNA, 4o3/zg/ml; [3H]erythromycin, 3.I • 1o -4/~mole/ml, o.oi ffC/ml; 5-min incubation. C. E r y t h r o m y c i n - s e n s i t i v e strain: protein, 1. 7 mg/ml; 1RNA, 3 8 4 # g / m l ; DNA, I25/zg/ml; [3H]erythromycin, 3 . I - i o - i / z m o l e / m l , o.oi/*C/ ml; chloramphenicol, o.o5/~mole/ml; Io-min incubation. D. E r y t h r o m y c i n - s e n s i t i v e strain: protein, 1. 7 mg/ml; RNA, 384ffg/ml; DNA, I25/zg/ml; [3HJchloramphenicol, o.o5/~mole/ml, 5.8~C/ml; Io-min incubation. All the i n c u b a t i o n s were done a t 37 ° and t e r m i n a t e d b y chilling to 4 °, E r y t h r o m y c i n , alone, reduced the incorporation of L-arginine into polypeptide linkage b y 19 % (A), while chloramphenicol, alone, caused a 76 % inhibition (D). The combination of e r y t h r o m y c i n and chloramphenicol produced a 63 % d i m i n u t i o n in L-arginine utilization (C). W i t h the s y s t e m from t h e e r y t h r o m y c i n - r e s i s t a n t B. subtilis, e r y t h r o m y c i n lowered L-arginine utilization b y only
2 % (B).
experiments the inhibitory effect of erythromycin and chloramphenicol has never been additive (see legend Fig. I). Nevertheless, until further studies are made of the effect of varying their amounts and molar ratios, it seems impossible to judge if erythromycin and chloramphenicol are competing for the exact same ribosomal site (s) Biochim. Biophys. Acta, 123 (1966) 438-44 o
44 °
PRELIMINARY NOTES
Until now the only evidence of a primary effect of erythromycin on protein synthesis in bacteria was the inhibition exerted by this antibiotic on amino acid utilization, by whole cells and cell-free extracts. It is most important to have other lines of evidence which also suggest that protein synthesis is the primary target of erythromycin action. Recently TANAKA AND TERAOKA ° have shown that a preincubation with erythromycin affects the products produced in a cell-free system from E. coli, when l.-lvsine and an artifical message-RNA (poly A) are added. A further argument for the 5o-S ribosomal unit being the site of some biochemical difference related to erythromycin resistance has resulted from studies of DUBNAU et al. 1°, n. From this work it appears that the chromosomal locus of mutations leading to erythromycin resistance in B. subtilis is close to that controlling the formation of the RNA found in 5o-S ribosomal particles. All of this evidence is consistent with the hy,pothesis that the binding in vitro of erythromycin to 5o-S partMes in B. subtilis 168 is an important aspect of the primary mechanism of action of ervthromycin in whole bacteria. The reduced binding of erythromyein by 5o-S ribosomal particles of the resistant mutant, together with a diminished uptake of the antibiotic in vivo 2, also may be the basis of acquired, genetically stable, resistance to erythromycin in t3. subtilis. One of the authors (.].W.C.) was a Career Deveh)pment Awardee of the U.S. Public Health Service (5K3-GM-2545). The research support of the U.S. Public Health Service (AI-o6758, Training Grant 5-T1-GM-35), the American Heart Association, Inc. (64Gr55) and Abbott Laboratories is gratefully acknowledged.
Departments o~ Biochemistry and Pathology, Western Reserve University School o/ Medicine, Cleveland, Ohio (U.S.A.)
StIELDON B. TAUBMAN NICHOLAS R. ]:RANK E .
JONES
YOUNG*
JOHN \V. ('ORCORAN
t T. D. BROCK AND M. L. BROCK, Biochim. Biophys. Acta, 33 (1959) 274. 2 S. l'l. TAUBMAN, F. E. YOUNG AND J. W. CORCORAN, Pvoc. Natl. Acad. Sci. U.S., 5 o (191")3) 955. 3 S. B. TAUBMAN, A. G. So, F. E. YOUNG, E. \V. ]_)AVIEAND J. W. CORCORAN, in J. C. SYI.VVS'rER, Antimicrobial Agents and Chemotherapy, Ann Arbor, Mich., 1964, p. 395. 4 A. D. \VoLvE AND F. E. HAH:q, Science, I43 (1964) 1445 . 5 l). VAZQUEZ, Biochem. Biophys. Res. Commun., I2 (I903) 4o9 . 6 D. VAZ(2UEZ, Biochem. Biophys. Res. Commun., 15 (1964) 464 . 7 D. VAZQUEZ, Biochim. Biophys. Acta, I 14 (1966) 277, 289. 8 A. D. WOLFE AND F. E. IlAHN, Biochim. Biophys. Acta, 95 (1965) I40. 9 K. TANAKA AND H. TERAOKA, Biochim. Biophys. Acta, 1I 4 (I966) 2o 4. Io 1). I)t:~NAU, I. S.~UTH, P. MORELL AND J. MAR.~IUR, Proc. Natl. Acad. Sci. U.S., 54 (1965) 49I. I I D. DOBNAU, I. SMITH ANt) J. MARMUR, Proc. Natl. Acad. Sci. U.S., 54 (1905) 724 • I2 R. J. MA.'qS AND (;. D. NOW'LUL Biochem. Biophys. Res Commun., 3 (196o) 54 °.
Received May 9th, I966 * F a c u l t y Research Associate, Anlerican Cancer Society, Present address: Scripps Clinic and Research l"oundation, I.a Jolla, Calif., U.S.A.
Biochon. Biophys. Acta, 123 (1966) 438-44 °
438
BBA 91122
Sensitivity ond resistance to erythromycin in Bacillus subtilis 168: the ribosomal bindin 9 of erythromycin ond chloramphenicol Erythromycin-A (erythromycin), one of the macrolide group of antibiotics, inhibits bacterial synthesis of polypeptides, both in vivol, ~ and in vitro2, 3,4. It does not inhibit amino acid activation to amino acyl-tRNA, and, therefore, most probably acts on either the assemblage of the polysome or on some aspect of its function. In the studies reported here, biologically active tritiated erythromycin was incubated with 2 different cell-free systems capable of making polypeptides from free amino acids. One system was derived from erythromycin-sensitive Bacillus subtilis 168, and the other from an erythromycin-resistant mutant. Biologically active [3H]erythromycin, added to a cell-free system derived from the erythromycin-sensitive strain of B. subtilis, is found to associate with the ribosomal components. Furthermore, a greatly enhanced concentration of the antibiotic is found in the fractions which contain 5o-S ribosomal units (Fig. IA). This association with 5o-S particles is time dependent, but attains the maximal level by 2-3 min. The erythromycin-resistant mutant of B. subtilis 168 was studied in the same way as the parent strain. The associative pattern of 73H ]erythromycin with the ribosomal particles in a cell-free extract (shown as Fig. IB) is not tile same as that described for the antibiotic-sensitive bacteria. The binding of the erythromycin with the particle is reduced, and no specific accumulation of radioactivity in the 5o-S fraction is seen. The difference in the relative abundance of To-S, 5o-S and 3o-S particles between the experiments shown as Figs. IA and B is within normal limits for different preparations of cell-free extracts. According to several reports, chloramphenicol binds to 5o-S ribosomal particles in cell-free extracts of Staphylococcus aureus, Bacillus megaterium and Escherichia coli 5-8. The binding is not time, energy and temperature dependent, and it is readily reversible. Chloramphenicol binding to ribosomes is effectively inhibited by erythromycin, and this has prompted speculation on the probability that both antibiotics bind or act at the same ribosomal site(s)5,v, s. The results of our studies with B. subtilis extracts are of interest in evaluating this hypothesis, since it was found that there is a major difference between the binding of erythromycin and chloramphenicol to 5o-S ribosomal particles. Erythromycin, when bound, is not lost from the 5o-S units during ultracentrifugation through a sucrose density-gradient system. Furthermore, a large molar excess of chloramphenicol (I58-fold) does not affect the asst~ciation of erythromycin and the 5o-S ribosomal particles (Fig. IC). In confirmation of the results using E. coli ribosomes 8, it was demonstrated that any chloramphenicol that m a y be bound to the 5o-S ribosomal units from B. subtilis does not survive fractionation by the ultracentrifugal method (Fig. I D ) . These experiments prove in a direct way that erythromycin has a much greater affinity for B. sublilis ribosomes than does chloramphenicol. The antibiotics, both individually and together, were active in inhibiting protein synthesis, although in these and similar A b b r e v i a t i o n : tRN,,\, t r a n s f e r I¢,NA.
Biochin,. 13iophys. Acta, 123 (196~)) 438-44 °
PRELIMINARY NOTES
o.*
439
I~
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A
i I Ii 0.3
I
B
/~A
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i
60
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20
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15
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8O
.c_ E
20 Fraction
/
20
r¢
.,,.2'. . . . . ~s..~?s. ?p.s.. I
5 number
I0
15
20
Fig. i. Ultracentrifugal analysis of cell-free s y s t e m s from B. subtilis. The strains of B. subtilis used a n d the preparation of cell-free e x t r a c t s were described earlier=, 3. Sucrose d e n s i t y - g r a d i e n t centrifugation was carried out using 4.4 ml of a 25-1o % continuous linear sucrose gradient buffered with Tris-HC1 (pH 8.I, o.I M), a n d containing m a g n e s i u m acetate (o.o12 M) and NHiC1 (o.o2 M). After incubation, a reaction m i x t u r e was chilled and a o.2-ml aliquot layered on top of the sucrose gradient. The sample was centrifuged at 4 ° in a Spinco L-2 ultracentrifuge for 135 m i n at 35 ooo r e v . / m i n in an SW 39 rotor. Sequential 15-drop fractions (approx. o.2 ml each) were collected after p u n c t u r i n g the b o t t o m of the centrifuge tube. Analysis, b o t h for light absorbance at 260 m/~ and radioactivity, was carried out on an aliquot of each fraction 3. The preparation of [SH]erythromycin h a s been described 2. Tritiated chloramphenicol, prepared b y t h e Wilzbach technique, was purchased from New E n g l a n d Nuclear Corporation, as was u n i f o r m l y labeled L-[liC]arginine. The effect of the antibiotics on t h e incorporation of arginine into h o t trichloroacetic acid-precipitable material was determined directly on o . i - m l aliquots of unfractionated reaction m i x t u r e s 1.. E a c h experimental sample contained t h e following (ffmoles/ml, unless otherwise indicated): Tris-HC1, ioo (pH 8.I); m a g n e s i u m acetate, 12; NH4C1, 40; 2-thioethanol, 3.3; ATP, i; phosphoenolpyruvate, 5, GTP, UTP, CTP, o.I each; L-amino acid m i x t u r e lacking L-arginine, 0.067 each, L-arginine, o.o13; p y r u v a t e kinase (EC 2. 7. E.4o), 3.3 ffg/ml. In each experiment a duplicate incubation was performed, in which L-[14Clarginine (O.Ol3, 0.33/*C/ml) was s u b s t i t u t e d for the non-radioactive arginine. In addition, the individual reaction m i x t u r e s were characterized by the following: A. E r y t h r o m y c i n - s e n s i t i v e strain: protein, 1. 7 mg/ml; RNA, 384/~g/ml; DNA, 125/zg/ ml; [3H]erythromycin, 3 . i . i o - l / z m o l e / m l , o.oi/zC/ml; 3-min incubation. B. E r y t h r o m y c i n r e s i s t a n t strain: protein, 4.7 mg/ml; RNA, 3o4ffg/ml; DNA, 4o3/zg/ml; [3H]erythromycin, 3.I • 1o -4/~mole/ml, o.oi ffC/ml; 5-min incubation. C. E r y t h r o m y c i n - s e n s i t i v e strain: protein, 1. 7 mg/ml; 1RNA, 3 8 4 # g / m l ; DNA, I25/zg/ml; [3H]erythromycin, 3 . I - i o - i / z m o l e / m l , o.oi/*C/ ml; chloramphenicol, o.o5/~mole/ml; Io-min incubation. D. E r y t h r o m y c i n - s e n s i t i v e strain: protein, 1. 7 mg/ml; RNA, 384ffg/ml; DNA, I25/zg/ml; [3HJchloramphenicol, o.o5/~mole/ml, 5.8~C/ml; Io-min incubation. All the i n c u b a t i o n s were done a t 37 ° and t e r m i n a t e d b y chilling to 4 °, E r y t h r o m y c i n , alone, reduced the incorporation of L-arginine into polypeptide linkage b y 19 % (A), while chloramphenicol, alone, caused a 76 % inhibition (D). The combination of e r y t h r o m y c i n and chloramphenicol produced a 63 % d i m i n u t i o n in L-arginine utilization (C). W i t h the s y s t e m from t h e e r y t h r o m y c i n - r e s i s t a n t B. subtilis, e r y t h r o m y c i n lowered L-arginine utilization b y only
2 % (B).
experiments the inhibitory effect of erythromycin and chloramphenicol has never been additive (see legend Fig. I). Nevertheless, until further studies are made of the effect of varying their amounts and molar ratios, it seems impossible to judge if erythromycin and chloramphenicol are competing for the exact same ribosomal site (s) Biochim. Biophys. Acta, 123 (1966) 438-44 o
44 °
PRELIMINARY NOTES
Until now the only evidence of a primary effect of erythromycin on protein synthesis in bacteria was the inhibition exerted by this antibiotic on amino acid utilization, by whole cells and cell-free extracts. It is most important to have other lines of evidence which also suggest that protein synthesis is the primary target of erythromycin action. Recently TANAKA AND TERAOKA ° have shown that a preincubation with erythromycin affects the products produced in a cell-free system from E. coli, when l.-lvsine and an artifical message-RNA (poly A) are added. A further argument for the 5o-S ribosomal unit being the site of some biochemical difference related to erythromycin resistance has resulted from studies of DUBNAU et al. 1°, n. From this work it appears that the chromosomal locus of mutations leading to erythromycin resistance in B. subtilis is close to that controlling the formation of the RNA found in 5o-S ribosomal particles. All of this evidence is consistent with the hy,pothesis that the binding in vitro of erythromycin to 5o-S partMes in B. subtilis 168 is an important aspect of the primary mechanism of action of ervthromycin in whole bacteria. The reduced binding of erythromyein by 5o-S ribosomal particles of the resistant mutant, together with a diminished uptake of the antibiotic in vivo 2, also may be the basis of acquired, genetically stable, resistance to erythromycin in t3. subtilis. One of the authors (.].W.C.) was a Career Deveh)pment Awardee of the U.S. Public Health Service (5K3-GM-2545). The research support of the U.S. Public Health Service (AI-o6758, Training Grant 5-T1-GM-35), the American Heart Association, Inc. (64Gr55) and Abbott Laboratories is gratefully acknowledged.
Departments o~ Biochemistry and Pathology, Western Reserve University School o/ Medicine, Cleveland, Ohio (U.S.A.)
StIELDON B. TAUBMAN NICHOLAS R. ]:RANK E .
JONES
YOUNG*
JOHN \V. ('ORCORAN
t T. D. BROCK AND M. L. BROCK, Biochim. Biophys. Acta, 33 (1959) 274. 2 S. l'l. TAUBMAN, F. E. YOUNG AND J. W. CORCORAN, Pvoc. Natl. Acad. Sci. U.S., 5 o (191")3) 955. 3 S. B. TAUBMAN, A. G. So, F. E. YOUNG, E. \V. ]_)AVIEAND J. W. CORCORAN, in J. C. SYI.VVS'rER, Antimicrobial Agents and Chemotherapy, Ann Arbor, Mich., 1964, p. 395. 4 A. D. \VoLvE AND F. E. HAH:q, Science, I43 (1964) 1445 . 5 l). VAZQUEZ, Biochem. Biophys. Res. Commun., I2 (I903) 4o9 . 6 D. VAZ(2UEZ, Biochem. Biophys. Res. Commun., 15 (1964) 464 . 7 D. VAZQUEZ, Biochim. Biophys. Acta, I 14 (1966) 277, 289. 8 A. D. WOLFE AND F. E. IlAHN, Biochim. Biophys. Acta, 95 (1965) I40. 9 K. TANAKA AND H. TERAOKA, Biochim. Biophys. Acta, 1I 4 (I966) 2o 4. Io 1). I)t:~NAU, I. S.~UTH, P. MORELL AND J. MAR.~IUR, Proc. Natl. Acad. Sci. U.S., 54 (1965) 49I. I I D. DOBNAU, I. SMITH ANt) J. MARMUR, Proc. Natl. Acad. Sci. U.S., 54 (1905) 724 • I2 R. J. MA.'qS AND (;. D. NOW'LUL Biochem. Biophys. Res Commun., 3 (196o) 54 °.
Received May 9th, I966 * F a c u l t y Research Associate, Anlerican Cancer Society, Present address: Scripps Clinic and Research l"oundation, I.a Jolla, Calif., U.S.A.
Biochon. Biophys. Acta, 123 (1966) 438-44 °