- Email: [email protected]
Cross resistance of Escherichia coli B ribosomes to inhibition of the puromycin reaction by erythromycin, spiramycin and chloramphenicol
43b
PRELIMINARY NOTES
BBA 912.07
Cross resistance of Escherichia coli B ribosomes to inhibition of the puromycin reaction by erythromycin, spiramycin and chloramphenicol Erythromycin is known to inhibit protein biosynthesis in bacteria z-a. As has been shown recently, this inhibition is associated with binding of erythromycin to the ribosomes 4& Erythromycin is bound to ribosomes from Bacillus megaterium ~, Escherichia coli 6 and Bacillus subtilis 7,8. Erythromycin and other antibiotics inhibit the puromycin reaction on E. coli ribosomes 9,zo. In the present communication we describe a mutant of E. coli B resistant to erythromycin and the effect of antibiotics inhibiting protein synthesis (erythromycin, spiramycin, chloramphenicol, chlortetracycline, neomycin) on the puromycin reaction on ribosomes of this mutant. Growth of the parent strain of E. coli B was completely inhibited by IiO fig erythromycin per ml. A resistant strain of E. coli B capable of growth even at a concentration of 5.8 4 mg erythromycin per ml was prepared by cultivation of the sensitive strain on meat peptone broth with increasing concentrations of erythromycin. The resistance remained unchanged after IO transfers in erythromycin-free broth. Erythromycin-resistant bacteria are still prototrophic and the bacterial colonies have the same appearance as the colonies of the parent strain. Ribosomes were prepared both from the resistant and from the sensitive strain of " T
. . . . .
~
-
-
-
-
T
-
-
-
-
IoooI
-
t000I
r
[
r~
30
40
. . . . . . . -2..---4l_
6O0
'~
,
3
!
-
. .¢::CS---""
2001
J----~ 10
0
rn~n
I 30
L 40
10
20 rNn
Fig. I. Tile effect of e r y t h r o m y c i n on t h e t i m e course of t h e release of lysine p e p t i d e s b y p u r o m y c i n . [ z ' C ] P o l y l y s y l - t R N A (188o c o u n t s / m i n p e r io/~g) w a s i n c u b a t e d w i t h i o / ~ g p o l y a d e n y l i c acid a n d r i b o s o m e s ( i o o / z g p r o t e i n ) in o.i M T r i s - a c e t a t e b u f f e r (pH 7.2), o.I M a m m o n i u m a c e t a t e a n d o.oi M m a g n e s i u m a c e t a t e in a t o t a l vol. of o.2 m l for 15 m i n a t 35 °. P u r o m y c i n (IO -4 M) w a s t h e n a d d e d a n d , in t h e course of s u b s e q u e n t i n c u b a t i o n , s a m p l e s were w i t h d r a w n a f t e r o, IO, 25 a n d 4 ° m i n , p r e c i p i t a t e d w i t h 5 % trichloroacetic acid, filtered t h r o u g h m e m b r a n e filters a n d t h e i r r a d i o a c t i v i t y w a s m e a s u r e d . I, r a d i o a c t i v i t y in c o u n t s / m i n released b y p u r o m y c i n f r o m t h e r i b o s o m e s of a s e n s i t i v e s t r a i n of E. coli t3; 2, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m r i b o s o m e s of a n E. coli B s t r a i n r e s i s t a n t to 5840/zg e r y t h r o m y c i n p e r ml; 3, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m r i b o s o m e s of t h e r e s i s t a n t s t r a i n in t h e p r e s e n c e of IO -s M e r y t h r o m y c i n ; 4, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m r i b o s o m e s of t h e s e n s i t i v e s t r a i n in t h e p r e s e n c e of lO -41~I e r y t h r o m y c i n . Fig. 2. T h e effect of c h l o r a m p h e n i c o l o n t h e t i m e course of t h e release of lysine p e p t i d e s b y p u r o m y c i n . Tile i n c u b a t i o n m i x t u r e is d e s c r i b e d in t h e legend to Fig. I. i, r a d i o a c t i v i t y in c o u n t s / m i n released b y p u r o m y c i n f r o m E. coli B r i b o s o m e s ; 2, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m E. coli B r i b o s o m e s r e s i s t a n t to e r y t h r o m y c i n ; 3, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m ribos o m e s of t h e r e s i s t a n t s t r a i n in t h e p r e s e n c e of 5" lO-4 M c h l o r a m p h e n i c o l ; 4, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m t h e r i b o s o m e s of E. coli 13 in t h e p r e s e n c e of 5" io-* M c h l o r a m p h e n i c o l . Biochim. Biophys. Acta, 157 (1968) 436--438
PRELIMINARY
437
NOTES
E. coli B according to the method of NISHIMURA et al. n. The puromycin reaction was followed by the technique described in the legend to Fig. I (see ref. 9), estimating the amount of lysine peptides split off from polylysyl-tRNA bound to ribosomes b y the action of puromycin. The ribosomes from the sensitive strain are more active in the puromycin reaction than the ribosomes from the resistant strain; the former ribosomes release 46 % of the radioactivity of added [x~C]polylysyl-tRNA, the latter ribosomes, 3 1 % under standard assay conditions. Fig. I shows the effect of erythromycin on the course of the puromycin reaction on E. coli ribosomes sensitive and resistant to erythromycin. The splitting of lysine peptides from ribosomes of the sensitive strain is inhibited b y erythromycin to a higher degree than from ribosomes of the resistant strain. At io -s M erythromycin, the puromycin reaction on ribosomes from the sensitive strain is inhibited b y 85 %, that on ribosomes from the resistant strain by only 3 ° %. Our results are in agreement with the recent paper by WILHELM AND CORCORANTM who studied the effect of erythromycin on polypeptide synthesis in a cell-free system from B. subtilis 168 (both an erythromycin-sensitive and -resistant strain) and found the same difference in sensitivity to erythromycin as we observed in the puromycin reaction in tbe present paper. Ribosomes from E. coli B, resistant to erythromycin, exhibited cross resistance to another macrolide antibiotic, spiramycin, and even to chloramphenicol (Figs. 2 and 3)*. These antibiotics, similarly to erythromycin, inhibited the puromycin reaction on the ribosomes from the erythromycin-resistant mutant at concentrations higher by one or two orders of magnitude, than for ribosomes of the sensitive strain. On the other hand, ribosomes from E. coli B that are resistant to erythromycin remain sensitive to the effect of chlortetracycline or neomycin. These antibiotics inhibited the puromycin reaction with the same intensity on erythromycin-resistant and -sensitive ribosomes (Fig. 3)100
F
oo
I
I
-6
-5 10g M
1/o2/
//
7/o;,
~
I'
-4
-3
20
-7
Fig. 3. Concentration dependence of the antibiotic effect on the release of lysine peptides b y puromycin. The effect is expressed in % inhibition of the puromycin reaction. The incubation mixture and conditions are as described for Fig. I. The puromycin reaction was stopped after 20 rain of incubation. Inhibitors of the puromycin reaction in the sensitive strain: i, spiramycin; z, erythromycin; 3, neomycin; 4, ch]ortetracycline; 5, chloramphenicol; inhibitors in the resistant strain: 6, spiramycin; 7, erythromycin; 8, neomycin, 9, chlortetracyclJne, io, chloramphenicol. " lO -4 M chloramphenicol was foundiS, 14 to inhibit the puromycin reaction in the cell-free system from the sensitive strain of E. coli b y 5 ° %.
Biochim. Biophys. Acla, 1 5 7 ( 1 9 6 8 ) 4 3 6 - 4 3 8
438
PRELIMINARY NOTES
The puromycin reaction, which we used to test the effect of the antibiotics, is equivalent to a single-step transfer of the peptide chain from peptidyl-tRNA to aminoacyl-tRNA is. The finding that ribosomes that are resistant to erythromycin are simultaneously resistant to spiramycin and chloramphenicol, but not to chlortetracycline or neomycin, indicates that the first three antibiotics bring about inhibition of formation of the peptide chain on the ribosomes by a similar mechanism and that the same ribosomal site is responsible for their effect. On the other hand, chlortetracycline and neomycin which also inhibit the puromycin reaction, apparently act differently and at different sites on the ribosome. This is in agreement with the finding of VAZQUEZ5 that erythromycin, spiramycin and chloramphenicol are competing for the same site on the ribosome. We assume that resistance to erythromycin is due to a modification at that site. The results also indicate a structural and functional heterogeneity of the ribosome. In the same way we have prepared mutants of E. coli ribonuclease-less strain A19 and E. coli Hfr H with resistant ribosomes which are now being studied in our laboratory.
Institute o/Organic Chemistry and Biochemistry, Czechoslovak Academy o/Sciences, Praha (Czechoslovakia) i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15
J, CERNA I. R Y C H L f K
T. D. BROCK AND M. L. ]3ROCK, Biochim. Biophys. Acta. 33 (1959) 274. S. ]3. TAUBMAN, F. E. YOUNG AND J. W . CORCORAN, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 955. A. D. WOLFE AND V. 1~. HAHN, Science, 143 (1964) 1445. K. TANAKA AND H. TI~RAOKA, Biochim. Biophys. Acta, 114 (1966) 2o 4. D. VAZQUEZ, Biochim. Biophys. Acta, 114 (1966) 277. K. TANAKA, H. TERAOKA, T. I%IAGIRAAND 1~¢[.TAMAKI, Biochim. Biophys. Acta, 123 (1966) 435. S. ]3. TAUBMAN, 1NT.]:{., JONES, F. E. YOUNG AND J. W . CORCORAN, Biochim. Biophys. Acta, 123 (1966) 438 . N. L. OLEINICK AND ]. W. CORCORAN, Federation Proc., 26 (1967) 285. I. RYCHLfK, Biochim. Biophys. Acta, 114 (1966) 425 . J. ~ERN~, I. RX'CHLIK AND F. ~ORM, in p r e p a r a t i o n . S. ~[ISHIMURA, D, S. JONES, E. OHTSUKA, H. HAYATSU, T. M, JACOB AND I'{. G. KHORANA, J. Mol. Biol., 13 (1965) 283. J. M. WILHELM AND W . CORCORAN, Biochemistry, 6 (1967) 2578. C. COUTSOGEORGOPOULOS, Biochem. Biophys. Res. Commun., 27 (1967) 46. I. H. GOLDBERG AND K. MITSUGI, Biochemistry, 6 (1967) 383. R. E. MONRO, ]3. E. H. 1ViADENAND R. R. TRAUT, ill D. SHUGAR, Genetic Elements -- Properties and Function, A c a d e m i c Press, L o n d o n , 1967, p. 179.
Received February 7th, 1968 Biochim. Biophys. Acta, 157 (1968) 436-438
PRELIMINARY NOTES
BBA 912.07
Cross resistance of Escherichia coli B ribosomes to inhibition of the puromycin reaction by erythromycin, spiramycin and chloramphenicol Erythromycin is known to inhibit protein biosynthesis in bacteria z-a. As has been shown recently, this inhibition is associated with binding of erythromycin to the ribosomes 4& Erythromycin is bound to ribosomes from Bacillus megaterium ~, Escherichia coli 6 and Bacillus subtilis 7,8. Erythromycin and other antibiotics inhibit the puromycin reaction on E. coli ribosomes 9,zo. In the present communication we describe a mutant of E. coli B resistant to erythromycin and the effect of antibiotics inhibiting protein synthesis (erythromycin, spiramycin, chloramphenicol, chlortetracycline, neomycin) on the puromycin reaction on ribosomes of this mutant. Growth of the parent strain of E. coli B was completely inhibited by IiO fig erythromycin per ml. A resistant strain of E. coli B capable of growth even at a concentration of 5.8 4 mg erythromycin per ml was prepared by cultivation of the sensitive strain on meat peptone broth with increasing concentrations of erythromycin. The resistance remained unchanged after IO transfers in erythromycin-free broth. Erythromycin-resistant bacteria are still prototrophic and the bacterial colonies have the same appearance as the colonies of the parent strain. Ribosomes were prepared both from the resistant and from the sensitive strain of " T
. . . . .
~
-
-
-
-
T
-
-
-
-
IoooI
-
t000I
r
[
r~
30
40
. . . . . . . -2..---4l_
6O0
'~
,
3
!
-
. .¢::CS---""
2001
J----~ 10
0
rn~n
I 30
L 40
10
20 rNn
Fig. I. Tile effect of e r y t h r o m y c i n on t h e t i m e course of t h e release of lysine p e p t i d e s b y p u r o m y c i n . [ z ' C ] P o l y l y s y l - t R N A (188o c o u n t s / m i n p e r io/~g) w a s i n c u b a t e d w i t h i o / ~ g p o l y a d e n y l i c acid a n d r i b o s o m e s ( i o o / z g p r o t e i n ) in o.i M T r i s - a c e t a t e b u f f e r (pH 7.2), o.I M a m m o n i u m a c e t a t e a n d o.oi M m a g n e s i u m a c e t a t e in a t o t a l vol. of o.2 m l for 15 m i n a t 35 °. P u r o m y c i n (IO -4 M) w a s t h e n a d d e d a n d , in t h e course of s u b s e q u e n t i n c u b a t i o n , s a m p l e s were w i t h d r a w n a f t e r o, IO, 25 a n d 4 ° m i n , p r e c i p i t a t e d w i t h 5 % trichloroacetic acid, filtered t h r o u g h m e m b r a n e filters a n d t h e i r r a d i o a c t i v i t y w a s m e a s u r e d . I, r a d i o a c t i v i t y in c o u n t s / m i n released b y p u r o m y c i n f r o m t h e r i b o s o m e s of a s e n s i t i v e s t r a i n of E. coli t3; 2, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m r i b o s o m e s of a n E. coli B s t r a i n r e s i s t a n t to 5840/zg e r y t h r o m y c i n p e r ml; 3, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m r i b o s o m e s of t h e r e s i s t a n t s t r a i n in t h e p r e s e n c e of IO -s M e r y t h r o m y c i n ; 4, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m r i b o s o m e s of t h e s e n s i t i v e s t r a i n in t h e p r e s e n c e of lO -41~I e r y t h r o m y c i n . Fig. 2. T h e effect of c h l o r a m p h e n i c o l o n t h e t i m e course of t h e release of lysine p e p t i d e s b y p u r o m y c i n . Tile i n c u b a t i o n m i x t u r e is d e s c r i b e d in t h e legend to Fig. I. i, r a d i o a c t i v i t y in c o u n t s / m i n released b y p u r o m y c i n f r o m E. coli B r i b o s o m e s ; 2, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m E. coli B r i b o s o m e s r e s i s t a n t to e r y t h r o m y c i n ; 3, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m ribos o m e s of t h e r e s i s t a n t s t r a i n in t h e p r e s e n c e of 5" lO-4 M c h l o r a m p h e n i c o l ; 4, r a d i o a c t i v i t y released b y p u r o m y c i n f r o m t h e r i b o s o m e s of E. coli 13 in t h e p r e s e n c e of 5" io-* M c h l o r a m p h e n i c o l . Biochim. Biophys. Acta, 157 (1968) 436--438
PRELIMINARY
437
NOTES
E. coli B according to the method of NISHIMURA et al. n. The puromycin reaction was followed by the technique described in the legend to Fig. I (see ref. 9), estimating the amount of lysine peptides split off from polylysyl-tRNA bound to ribosomes b y the action of puromycin. The ribosomes from the sensitive strain are more active in the puromycin reaction than the ribosomes from the resistant strain; the former ribosomes release 46 % of the radioactivity of added [x~C]polylysyl-tRNA, the latter ribosomes, 3 1 % under standard assay conditions. Fig. I shows the effect of erythromycin on the course of the puromycin reaction on E. coli ribosomes sensitive and resistant to erythromycin. The splitting of lysine peptides from ribosomes of the sensitive strain is inhibited b y erythromycin to a higher degree than from ribosomes of the resistant strain. At io -s M erythromycin, the puromycin reaction on ribosomes from the sensitive strain is inhibited b y 85 %, that on ribosomes from the resistant strain by only 3 ° %. Our results are in agreement with the recent paper by WILHELM AND CORCORANTM who studied the effect of erythromycin on polypeptide synthesis in a cell-free system from B. subtilis 168 (both an erythromycin-sensitive and -resistant strain) and found the same difference in sensitivity to erythromycin as we observed in the puromycin reaction in tbe present paper. Ribosomes from E. coli B, resistant to erythromycin, exhibited cross resistance to another macrolide antibiotic, spiramycin, and even to chloramphenicol (Figs. 2 and 3)*. These antibiotics, similarly to erythromycin, inhibited the puromycin reaction on the ribosomes from the erythromycin-resistant mutant at concentrations higher by one or two orders of magnitude, than for ribosomes of the sensitive strain. On the other hand, ribosomes from E. coli B that are resistant to erythromycin remain sensitive to the effect of chlortetracycline or neomycin. These antibiotics inhibited the puromycin reaction with the same intensity on erythromycin-resistant and -sensitive ribosomes (Fig. 3)100
F
oo
I
I
-6
-5 10g M
1/o2/
//
7/o;,
~
I'
-4
-3
20
-7
Fig. 3. Concentration dependence of the antibiotic effect on the release of lysine peptides b y puromycin. The effect is expressed in % inhibition of the puromycin reaction. The incubation mixture and conditions are as described for Fig. I. The puromycin reaction was stopped after 20 rain of incubation. Inhibitors of the puromycin reaction in the sensitive strain: i, spiramycin; z, erythromycin; 3, neomycin; 4, ch]ortetracycline; 5, chloramphenicol; inhibitors in the resistant strain: 6, spiramycin; 7, erythromycin; 8, neomycin, 9, chlortetracyclJne, io, chloramphenicol. " lO -4 M chloramphenicol was foundiS, 14 to inhibit the puromycin reaction in the cell-free system from the sensitive strain of E. coli b y 5 ° %.
Biochim. Biophys. Acla, 1 5 7 ( 1 9 6 8 ) 4 3 6 - 4 3 8
438
PRELIMINARY NOTES
The puromycin reaction, which we used to test the effect of the antibiotics, is equivalent to a single-step transfer of the peptide chain from peptidyl-tRNA to aminoacyl-tRNA is. The finding that ribosomes that are resistant to erythromycin are simultaneously resistant to spiramycin and chloramphenicol, but not to chlortetracycline or neomycin, indicates that the first three antibiotics bring about inhibition of formation of the peptide chain on the ribosomes by a similar mechanism and that the same ribosomal site is responsible for their effect. On the other hand, chlortetracycline and neomycin which also inhibit the puromycin reaction, apparently act differently and at different sites on the ribosome. This is in agreement with the finding of VAZQUEZ5 that erythromycin, spiramycin and chloramphenicol are competing for the same site on the ribosome. We assume that resistance to erythromycin is due to a modification at that site. The results also indicate a structural and functional heterogeneity of the ribosome. In the same way we have prepared mutants of E. coli ribonuclease-less strain A19 and E. coli Hfr H with resistant ribosomes which are now being studied in our laboratory.
Institute o/Organic Chemistry and Biochemistry, Czechoslovak Academy o/Sciences, Praha (Czechoslovakia) i 2 3 4 5 6 7 8 9 IO ii 12 13 14 15
J, CERNA I. R Y C H L f K
T. D. BROCK AND M. L. ]3ROCK, Biochim. Biophys. Acta. 33 (1959) 274. S. ]3. TAUBMAN, F. E. YOUNG AND J. W . CORCORAN, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 955. A. D. WOLFE AND V. 1~. HAHN, Science, 143 (1964) 1445. K. TANAKA AND H. TI~RAOKA, Biochim. Biophys. Acta, 114 (1966) 2o 4. D. VAZQUEZ, Biochim. Biophys. Acta, 114 (1966) 277. K. TANAKA, H. TERAOKA, T. I%IAGIRAAND 1~¢[.TAMAKI, Biochim. Biophys. Acta, 123 (1966) 435. S. ]3. TAUBMAN, 1NT.]:{., JONES, F. E. YOUNG AND J. W . CORCORAN, Biochim. Biophys. Acta, 123 (1966) 438 . N. L. OLEINICK AND ]. W. CORCORAN, Federation Proc., 26 (1967) 285. I. RYCHLfK, Biochim. Biophys. Acta, 114 (1966) 425 . J. ~ERN~, I. RX'CHLIK AND F. ~ORM, in p r e p a r a t i o n . S. ~[ISHIMURA, D, S. JONES, E. OHTSUKA, H. HAYATSU, T. M, JACOB AND I'{. G. KHORANA, J. Mol. Biol., 13 (1965) 283. J. M. WILHELM AND W . CORCORAN, Biochemistry, 6 (1967) 2578. C. COUTSOGEORGOPOULOS, Biochem. Biophys. Res. Commun., 27 (1967) 46. I. H. GOLDBERG AND K. MITSUGI, Biochemistry, 6 (1967) 383. R. E. MONRO, ]3. E. H. 1ViADENAND R. R. TRAUT, ill D. SHUGAR, Genetic Elements -- Properties and Function, A c a d e m i c Press, L o n d o n , 1967, p. 179.
Received February 7th, 1968 Biochim. Biophys. Acta, 157 (1968) 436-438