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A ribosomal mechanism for synthesis of peptides related to nisin
263
SHORT COMMUNICATIONS BBA 93543
A ribosomol mechonism for synthesis of peptides reloted to nisin Nisin is a large polypeptide antibiotic (molecular weight 7ooo) synthesised by Streptococcus lactis. Nisin contains two unsaturated amino acids, dehydroalanine and $-methyldehydroalanine1 and also two thioether amino acids, lanthionine and /5-methyllanthionine2. BODANSZKYAND PERLMANa suggested that all polypeptide antibiotics are made by a nonribosomal mechanism. They considered that an invariant genetic code does not permit ribosomal synthesis of antibiotics containing unusual amino acids. Peptide n
o
200, E
" " 16C
IV
L
i ~2c
Q-~-~ 0
>
L V>,
u
80
40
d2 b
&
&
E lec t r o p h o r e t i c
08
62 d4 66 08
1.0
Mobility relative to dye
mobility
.E E
E
3
o 6
8C
>
2
Z
o o o rr
d2 Mobili%y
de
o,'4 o'6 relative
to
dye
8 gr~
4O
02 0.4 0.6 Mobility r e l a t i v e
0.8 t o dye
Fig. I. P o l y a c r y l a m i d e gel e l e c t r o p h o r e t o g r a m s of S. lactis peptides. S, lactis (Strain N C D O 497) was g r o w n in a c o m p l e x m e d i u m ~ a n d r e s u s p e n d e d in a buffered salts s y s t e m 6 c o n t a i n i n g 0. 5 # C / ml of ?4C]threonine (208 m C / m m o l e ) or ?4C]cysteine (15 m C / m m o l e ) , T h e cells were i n c u b a t e d at 3 °° for 9o min. I - m l aliquots were h a r v e s t e d b y m e m b r a n e filtration, a n d t h e filters t r e a t e d with I m l of o.o5 M HC1 at 90 ° for to min. After c e n t r i f u g a t i o n t h e s u p e r n a t a n t c o n t a i n i n g t h e p e p t i d e s was t r e a t e d w i t h 7 vol. of acetone a t --15 °. T h e a c e t o n e p r e c i p i t a t e w a s dissolved in o.2 m l of 6o m M p o t a s s i u m f o r m a t e buffer (pH 5.3) c o n t a i n i n g 6 M u r e a plus p y r o n i n dye. T h i s solution was s e p a r a t e d b y gel electrophoresis at p H 3.3 w i t h 6 M u r e a u s i n g t h e s y s t e m of LEJSEK AND LUSENA% a n d a n a c r y l a m i d e c o n c e n t r a t i o n of IO % (w/v) plus e t h y l e n e d i a c r y l a t e a t 1 % (v/v). 1 . 2 - m m gel slices were dissolved in I ml of i M a m m o n i a solution c o n t a i n i n g I M h y a m i n e h y d r o x i d e prior to c o u n t i n g in a t o l u e n e - T r i t o n (2 : I, v/v) b a s e d scintillator. (a) A d e n s i t o m e t e r t r a c i n g of a gel s t a i n e d w i t h A m i d o black (the gel c o n t a i n e d n o n r a d i o a c t i v e p r o t e i n f r o m io m l of suspension). (b) T h e r a d i o a c t i v i t y of p e p t i d e s f r o m cells i n c u b a t e d w i t h [z4C]threonine. (c) T h e r a d i o a c t i v i t y of p e p t i d e s labelled w i t h [14C~cysteine. (d) As (c) b u t w i t h c h l o r a m p h e n i e o l (5 ° / , g / m l ) a d d e d io m i n a f t e r t h e cysteine.
Biochim. Biophys, Acta, 224 (197 o) 263-265
264
SHORT COMMUNICATIONS
antibiotic synthesis is also generally insensitive to inhibitors of protein synthesis. The only exception was the synthesis of gramicidin S, but this result has been retracted 4. However, HURSTa found that nisin synthesis was inhibited by chloramphenicol. He used a bioassay to estimate nisin and it is possible that synthesis de novo of nisin was not directly affected. In this work I used the incorporation of radioactive amino acids to confirm the report of HURST5 and also to propose a mechanism of synthesis of the lanthionine residues. A suspension of Streptococcus lactis was incubated with radioactive amino acids and the newly synthesised peptides were separated by disc gel electrophoresis as described in Fig. I. The Amido black stained gels had several bands (Fig. Ia), and one band (III) was coincident with nisin. A complex profile of radioactivity was also obtained when peptides were labelled with I14Clthreonine (Fig. ib) and with several other amino acids. The experiment was repeated with [laClcysteine which labelled Peak III preferentially (Fig. IC). This peak had the same electrophoretic mobility, relative to pyronin dye, as nisin and was shown to contain lanthionine by paper electrophoresis e of the radioactive amino acids contained in a hydrolysate of peptide eluted from the gel. More than 9 ° % of the radioactivity was associated (after hydrolysis and oxidation) with lanthionine and fl-methyllanthionine sulphone and not with cysteic acid (nisin contains no cysteine). Lanthionine peptide synthesis was measured in the gel electrophoretograms by summing the radioactivity from [t4C]cysteine present in Peak III. Fig. Id shows the profile for peptides synthesised when chloramphenicol (5o #g/ml) was added IO min after the start of the experiment. Smaller concentrations of chloramphenicol chlortetracycline and puromycin were also inhibitory (Table I). Inhibition was evident within 12 min, the smallest interval tested, with the addition of chloramphenicol (63 % inhibition with 50 #g/ml chloramphenicol). TABLE I T H E E F F E C T S OF A N T I B I O T I C S ON L A N T H I O N I N E
PEPTIDE
SYNTHESIS
S. lactis cells were incubated with [14C]cysteine for 7° min and antibiotics were added io nlin after the start of incubation. Basic peptides were extracted (as in Fig. I) and purified by electrophoresis; the figures in parentheses represent the incorporation of cysteine into hot trichloroacetic acid-precipitable material. IOO % synthesis of lanthionine peptide corresponded to t738 counts/min. A ntibiotic
[14CJCysteine incorporated into nisin (%) A ntibiot, concn. (pg/ml) :
Chloramphenicol Chlortetracycline Puromycin
o
o.3
I
IOO (IOO)
3
1o
30
60 (IO7)
37 (91)
39 (51 )
37 (73)
39 (43)
ioo
36
23
21
(lOO)
(io3)
(72)
(55)
IOO (IOO)
55 (96)
The inhibition of lanthionine peptide synthesis by chloramphenicol, chlortetracycline and puromycin argues strongly that the polypeptide chain of these peptides, which closely resemble nisin, is synthesised by a ribosomal mechanism. The Biochim. Biophys. Acts, 224 (197o) 263-265
265
SHORT COMMUNICATIONS
amino acid cysteine used for this work is a precursor of the lanthionine and fl-methyllanthionine residues of nisin. These two thioether amino acids are presumably formed after cysteine is incorporated into a polypeptide chain. Serine and threonine have already 6 been shown to be precursors of lanthionine and fl-methyllanthionine and it is proposed that serine, threonine and cysteine are first incorporated into a polypeptide chain (see Fig. 2). Serine and threonine are then dehydrated to the dehydro- - NH.CH-CO-- - - N H C H . C O - -
RJbosom(al
synthesis
from cysteine, serine ond threonine
TCH2 SH
_--
CH~C.HOH -- C H
--NH'CH'CO-- --NH'CH'CO-CH2 ~H2
I
S S ~ I I CH31CH ICH2 --CO.CH.NH -- --COCH-NH--
CH.NH
~H20 H SH i 2 CI"t -- - - C ( > C H . N H - -
--NH'~H'CO-- --NH'C'CO-(rH2 ~H 2 SH
CH:~H --CO.C-NH--
SH ~iH2 --CO.CH.NH--
Fig. 2. P r o p o s e d m e c h a n i s m for t h e s y n t h e s i s of nisin related peptides.
amino acids, which condense with cysteine residues to give lanthionine and fl-methyllanthionine. The incorporation (Fig. I) of threonine into nisin, which contains no threonine residues, is further evidence for this scheme. The antibiotic subtilin contains dehydroalanine as well as lanthionine s and may also be synthesised by this mechanism. Dehydroamino acids have also been proposed as intermediates in D-amino acid synthesis 9.
Microbiology Division, Food and Drug Directorate, Department o/National Health and Wel/are, Ottawa (Canada) I 2 3 4 5 6 7 8 9
L. INGRAM
E. GROSS AND J. L. MORELL, F E B S Letters, 2 (I968) 61. N. J. BERRIDGE, G. G. F. 1NIEWTON AND E. P. ABRAHAM, Nature, 171 (1953) 6o6. IV[. BODANSZKY AND D. PERLMAN, Nature, 2o 4 (1964) 84o. N. V. BHAGAAVAN, P. i . RAO, L. Vq. POLLARD, R. K. RAO, T. WINNICK AND J. B. HALL, Biochemistry, 5 (1966) 3844. A. HURST, J. Gen. Microbiol., 44 (1966) 2o9. L. INGRAM, Biochim. Biophys. Acta, 184 (1969) 216. K. LEJSEK AND C. V. LUSENA, Can. J. Biochem., 47 (1969) 753. E. GROSS, J. L. MORELL ANn L. C. CRAIG, Proc. Natl. Acad. Sci. U.S., 62 (1969) 952. B. W. BYCROFT, Nature, 224 (1969) 595.
Received April 9th, 197o Revised manuscript received July I7th, 197o Biochim. Biophys. Acta, 224 (197 o) 203-265
SHORT COMMUNICATIONS BBA 93543
A ribosomol mechonism for synthesis of peptides reloted to nisin Nisin is a large polypeptide antibiotic (molecular weight 7ooo) synthesised by Streptococcus lactis. Nisin contains two unsaturated amino acids, dehydroalanine and $-methyldehydroalanine1 and also two thioether amino acids, lanthionine and /5-methyllanthionine2. BODANSZKYAND PERLMANa suggested that all polypeptide antibiotics are made by a nonribosomal mechanism. They considered that an invariant genetic code does not permit ribosomal synthesis of antibiotics containing unusual amino acids. Peptide n
o
200, E
" " 16C
IV
L
i ~2c
Q-~-~ 0
>
L V>,
u
80
40
d2 b
&
&
E lec t r o p h o r e t i c
08
62 d4 66 08
1.0
Mobility relative to dye
mobility
.E E
E
3
o 6
8C
>
2
Z
o o o rr
d2 Mobili%y
de
o,'4 o'6 relative
to
dye
8 gr~
4O
02 0.4 0.6 Mobility r e l a t i v e
0.8 t o dye
Fig. I. P o l y a c r y l a m i d e gel e l e c t r o p h o r e t o g r a m s of S. lactis peptides. S, lactis (Strain N C D O 497) was g r o w n in a c o m p l e x m e d i u m ~ a n d r e s u s p e n d e d in a buffered salts s y s t e m 6 c o n t a i n i n g 0. 5 # C / ml of ?4C]threonine (208 m C / m m o l e ) or ?4C]cysteine (15 m C / m m o l e ) , T h e cells were i n c u b a t e d at 3 °° for 9o min. I - m l aliquots were h a r v e s t e d b y m e m b r a n e filtration, a n d t h e filters t r e a t e d with I m l of o.o5 M HC1 at 90 ° for to min. After c e n t r i f u g a t i o n t h e s u p e r n a t a n t c o n t a i n i n g t h e p e p t i d e s was t r e a t e d w i t h 7 vol. of acetone a t --15 °. T h e a c e t o n e p r e c i p i t a t e w a s dissolved in o.2 m l of 6o m M p o t a s s i u m f o r m a t e buffer (pH 5.3) c o n t a i n i n g 6 M u r e a plus p y r o n i n dye. T h i s solution was s e p a r a t e d b y gel electrophoresis at p H 3.3 w i t h 6 M u r e a u s i n g t h e s y s t e m of LEJSEK AND LUSENA% a n d a n a c r y l a m i d e c o n c e n t r a t i o n of IO % (w/v) plus e t h y l e n e d i a c r y l a t e a t 1 % (v/v). 1 . 2 - m m gel slices were dissolved in I ml of i M a m m o n i a solution c o n t a i n i n g I M h y a m i n e h y d r o x i d e prior to c o u n t i n g in a t o l u e n e - T r i t o n (2 : I, v/v) b a s e d scintillator. (a) A d e n s i t o m e t e r t r a c i n g of a gel s t a i n e d w i t h A m i d o black (the gel c o n t a i n e d n o n r a d i o a c t i v e p r o t e i n f r o m io m l of suspension). (b) T h e r a d i o a c t i v i t y of p e p t i d e s f r o m cells i n c u b a t e d w i t h [z4C]threonine. (c) T h e r a d i o a c t i v i t y of p e p t i d e s labelled w i t h [14C~cysteine. (d) As (c) b u t w i t h c h l o r a m p h e n i e o l (5 ° / , g / m l ) a d d e d io m i n a f t e r t h e cysteine.
Biochim. Biophys, Acta, 224 (197 o) 263-265
264
SHORT COMMUNICATIONS
antibiotic synthesis is also generally insensitive to inhibitors of protein synthesis. The only exception was the synthesis of gramicidin S, but this result has been retracted 4. However, HURSTa found that nisin synthesis was inhibited by chloramphenicol. He used a bioassay to estimate nisin and it is possible that synthesis de novo of nisin was not directly affected. In this work I used the incorporation of radioactive amino acids to confirm the report of HURST5 and also to propose a mechanism of synthesis of the lanthionine residues. A suspension of Streptococcus lactis was incubated with radioactive amino acids and the newly synthesised peptides were separated by disc gel electrophoresis as described in Fig. I. The Amido black stained gels had several bands (Fig. Ia), and one band (III) was coincident with nisin. A complex profile of radioactivity was also obtained when peptides were labelled with I14Clthreonine (Fig. ib) and with several other amino acids. The experiment was repeated with [laClcysteine which labelled Peak III preferentially (Fig. IC). This peak had the same electrophoretic mobility, relative to pyronin dye, as nisin and was shown to contain lanthionine by paper electrophoresis e of the radioactive amino acids contained in a hydrolysate of peptide eluted from the gel. More than 9 ° % of the radioactivity was associated (after hydrolysis and oxidation) with lanthionine and fl-methyllanthionine sulphone and not with cysteic acid (nisin contains no cysteine). Lanthionine peptide synthesis was measured in the gel electrophoretograms by summing the radioactivity from [t4C]cysteine present in Peak III. Fig. Id shows the profile for peptides synthesised when chloramphenicol (5o #g/ml) was added IO min after the start of the experiment. Smaller concentrations of chloramphenicol chlortetracycline and puromycin were also inhibitory (Table I). Inhibition was evident within 12 min, the smallest interval tested, with the addition of chloramphenicol (63 % inhibition with 50 #g/ml chloramphenicol). TABLE I T H E E F F E C T S OF A N T I B I O T I C S ON L A N T H I O N I N E
PEPTIDE
SYNTHESIS
S. lactis cells were incubated with [14C]cysteine for 7° min and antibiotics were added io nlin after the start of incubation. Basic peptides were extracted (as in Fig. I) and purified by electrophoresis; the figures in parentheses represent the incorporation of cysteine into hot trichloroacetic acid-precipitable material. IOO % synthesis of lanthionine peptide corresponded to t738 counts/min. A ntibiotic
[14CJCysteine incorporated into nisin (%) A ntibiot, concn. (pg/ml) :
Chloramphenicol Chlortetracycline Puromycin
o
o.3
I
IOO (IOO)
3
1o
30
60 (IO7)
37 (91)
39 (51 )
37 (73)
39 (43)
ioo
36
23
21
(lOO)
(io3)
(72)
(55)
IOO (IOO)
55 (96)
The inhibition of lanthionine peptide synthesis by chloramphenicol, chlortetracycline and puromycin argues strongly that the polypeptide chain of these peptides, which closely resemble nisin, is synthesised by a ribosomal mechanism. The Biochim. Biophys. Acts, 224 (197o) 263-265
265
SHORT COMMUNICATIONS
amino acid cysteine used for this work is a precursor of the lanthionine and fl-methyllanthionine residues of nisin. These two thioether amino acids are presumably formed after cysteine is incorporated into a polypeptide chain. Serine and threonine have already 6 been shown to be precursors of lanthionine and fl-methyllanthionine and it is proposed that serine, threonine and cysteine are first incorporated into a polypeptide chain (see Fig. 2). Serine and threonine are then dehydrated to the dehydro- - NH.CH-CO-- - - N H C H . C O - -
RJbosom(al
synthesis
from cysteine, serine ond threonine
TCH2 SH
_--
CH~C.HOH -- C H
--NH'CH'CO-- --NH'CH'CO-CH2 ~H2
I
S S ~ I I CH31CH ICH2 --CO.CH.NH -- --COCH-NH--
CH.NH
~H20 H SH i 2 CI"t -- - - C ( > C H . N H - -
--NH'~H'CO-- --NH'C'CO-(rH2 ~H 2 SH
CH:~H --CO.C-NH--
SH ~iH2 --CO.CH.NH--
Fig. 2. P r o p o s e d m e c h a n i s m for t h e s y n t h e s i s of nisin related peptides.
amino acids, which condense with cysteine residues to give lanthionine and fl-methyllanthionine. The incorporation (Fig. I) of threonine into nisin, which contains no threonine residues, is further evidence for this scheme. The antibiotic subtilin contains dehydroalanine as well as lanthionine s and may also be synthesised by this mechanism. Dehydroamino acids have also been proposed as intermediates in D-amino acid synthesis 9.
Microbiology Division, Food and Drug Directorate, Department o/National Health and Wel/are, Ottawa (Canada) I 2 3 4 5 6 7 8 9
L. INGRAM
E. GROSS AND J. L. MORELL, F E B S Letters, 2 (I968) 61. N. J. BERRIDGE, G. G. F. 1NIEWTON AND E. P. ABRAHAM, Nature, 171 (1953) 6o6. IV[. BODANSZKY AND D. PERLMAN, Nature, 2o 4 (1964) 84o. N. V. BHAGAAVAN, P. i . RAO, L. Vq. POLLARD, R. K. RAO, T. WINNICK AND J. B. HALL, Biochemistry, 5 (1966) 3844. A. HURST, J. Gen. Microbiol., 44 (1966) 2o9. L. INGRAM, Biochim. Biophys. Acta, 184 (1969) 216. K. LEJSEK AND C. V. LUSENA, Can. J. Biochem., 47 (1969) 753. E. GROSS, J. L. MORELL ANn L. C. CRAIG, Proc. Natl. Acad. Sci. U.S., 62 (1969) 952. B. W. BYCROFT, Nature, 224 (1969) 595.
Received April 9th, 197o Revised manuscript received July I7th, 197o Biochim. Biophys. Acta, 224 (197 o) 203-265