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Protein synthesis in Escherichia coli: A phage-mediated interruption of translation
PRELIMINARY NOTES
711
PN 91077
Protein synthesis in Escherichio coli: A phoge-medioted interruption of tronslation FRY AND GROS1 have reported conditions under which 9 1 % of the cells of Escherichia coli K I I 2 gave a lysogenic response after infection by phage ~. Net synthesis of DNA, RNA and protein could not be detected in these potential lysogens for approx. 12o rain following infection, fl-Galactosidase could not be induced for at least 19o min, but was inducible after the lysogens were growing at a rate comparable with uninfected cells. SIMINOV1TCHAND JACOB2 have reported that induced production of fl-galactosidase was possible during vegetative phage development following ultraviolet irradiation of ~ lysogens. Thus, a profound disturbance in the metabolism of the host is evident immediately after infection b y ~ and during the establishment of the prophage. A less serious disturbance is evident upon induction of the prophage. We are investigating the effects of a virulent m u t a n t of phage ,~(~vtr) on the metabolism of the host. The rates of synthesis of general protein and RNA are compared with those of specific protein and RNA ill an attempt to identify the event which inhibits the protein-synthesizing machinery. E. coli strain 3.000 growing with aeration at 37 ° in a medium containing (g): MgSO4 1.2, K2HPO * o.3, Na2 H P O 4 • 7 H20 0.6, (NH~)2SO 4 0.5, glycerol I.O, and vitamin B 10.5 mg per 1 was exposed to ~vir at a multiplicity of 50 phages per cell. Under these conditions less than 1 % of the bacteria survived a 3-rain exposure as indicated b y viable count. A minimum of 85 % of these non-viable cells produced infective centers after treatment with ~ antiserum. A minimum latent period of 50-60 min
25
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Fig. I. E f f e c t of p h a g e Jlvir o n p r o t e i n a n d 1RNA s y n t h e s i s . A t - - 1 5 min, for p r o t e i n L-[I-14C] leucine (0.02/*C//,g, 4 # g / m l ) ( 0 - 0 ) ; or for 1RNA, [2-z4C]uracil (o.05 pC/itg, 5#g/ml), t h y m i d i n e (5/*g/ml) ( G - C ) ) , w a s a d d e d to cells g r o w i n g a t 5" IOS cells/ml. P h a g e (5o/cell) were a d d e d a t zero t i m e . A t specified t i m e s , p r o t e i n s a m p l e s were a d d e d to a n e q u a l v o l u m e of 1o % trichloroacetic acid -- i % c a s a m i n o a c i d s solution, h e a t e d a t 90 ° for 3 ° min, t h e insoluble m a t e r i a l collected on a m e m b r a n e filter (Millipore, AA), w a s h e d w i t h 5 % trichloroacetic acid-o. 5 % eas a m i n o acids, a n d c o u n t e d in a Geiger c o u n t e r . R N A s a m p l e s were s i m i l a r l y w i t h d r a w n i n t o io % trichloroacetic acid a t o% A f t e r 30 m i n a t o °, t h e i n s o l u b l e m a t e r i a l w a s collected, w a s h e d w i t h cold 5 % trichloroacetic acid, a n d c o u n t e d as described.
Biochim. Biophys. Hcta, lO 3 (1965) 711-713
712
PRELIMINARY NOTES
and a single burst of 33 were observed. No leakage of fl-galactosidase could be detected from cells maximally induced before exposure to phage. Fig. i indicates that the rate of general protein and RNA synthesis is drastically . . . .
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Fig. 2. P r o d u c t i o n of fl-galactosidase. Cells g r o w i n g at 5 " ~o8 cells/ml were i n d u c e d w i t h isopropyl-thio-fl-D-galactoside (5. lO-4 M) a t zero t i m e . A t 2 rain, t h e i n d u c e r w a s r e m o v e d b y filt r a t i o n o n a merfibrane filter (Millipore, H A ) , a n d t h e cells w a s h e d a n d r e s u s p e n d e d in i n d u c e r free m e d i u m ( O - Q ) . T h i s r e q u i r e d 1. 5 min. I n a parallel e x p e r i m e n t , p h a g e (5o/cell) were a d d e d a t 2 rain ( 0 - 0 ) . A t specified times, s a m p l e s were w i t h d r a w n a n d a s s a y e d for fl-galactosidase as described b y R E V E L , LURIA a n d ROTMAN 3. i
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Fig. 3. M e s s e n g e r R N A in i n f e c t e d a n d u n i n f e c t e d cells. Cells g r o w i n g at 5 " lO8 cells/ml were i n f e c t e d w i t h p h a g e (5o/cell) a t zero t i m e . B e t w e e n t h e io th a n d i i th m i n t h e cells were p u l s e d for 45 sec w i t h E5, 6-3H] uracil (4.o/~C//~g, 0. 5 / , g / m l ) ( O - © )- T h e i n c o r p o r a t i o n w a s stopped, carrier cells a d d e d , a n d t h e p h e n o l - e x t r a c t e d R N A p r e p a r e d as described b y O K A M O T O , SUGINO AND NOMURA4. T h e final e t h a n o l p r e c i p i t a t e w a s dissolved in a s m a l l a m o u n t of buffer, layered o n t h e 4 to 20 % s u c r o s e g r a d i e n t , a n d s e d i m e n t e d a t 39 ooo r e v . / m i n S W - 3 9 rotor, Spinco L-2 u l t r a c e n t r i f u g e , o °, for 5 to 6 h, T h e g r a d i e n t w a s collected dropwise a n d a n a l i q u o t f r o m e a c h t u b e t r a n s f e r r e d to a b l o t t i n g p a p e r disc. T h e a b s o r b a n c e a t 260 m/z w a s m e a s u r e d o n t h e d i l u t e d rem a i n d e r . T h e s e m a r k e r p e a k s are d e s i g n a t e d b y arrows. T h e cold trichloroacetic aeid-precip i t a b l e m a t e r i a l w a s p r e p a r e d o n t h e b l o t t e r discs as d e s c r i b e d b y BOLLUM 5, placed i n vials c o n t a i n i n g : t o l u e n e 8 ml, 2.5-diphenyl-oxazole 32 rag, 1,4-bis-2-(5-phenyloxzolyl)benzene o.8 rag, a n d c o u n t e d in a liquid s c i n t i l l a t i o n s p e c t r o m e t e r . U n i n f e c t e d g r o w i n g cells ( Q - O ) were p u l s e d a n d t h e R N A e x t r a c t e d , purified s e d i m e n t e d , a n d a s s a y e d as described. To correct for t h e difference in t o t a l R N A recovered f r o m t h e g r a d i e n t s , c o u n t s / r a i n are e x p r e s s e d in t e r m s of zoo % r e c o v e r y for t h e u n i n f e c t e d cells. R e c o v e r y is b a s e d on a b s o r b a n c e a t t h e 23-S m a r k e r p e a k .
Biochim. Biophys. Acta, lO 3 (1965) 711-713
PRELIMINARY NOTES
713
reduced within 3 min after phage infection. The infected rate is less than 5 % of the initial rate. Removal of the unadsorbed phage by centrifugation or treatment with 2 antiserum had no effect. The rate of decrease of fl-galactosidase production following removal of inducer (isopropyl-thio-fi-D-galactoside) by washing is compared with the rate of decrease following phage infection in Fig. 2, The dramatic "shut-off" of enzyme synthesis following phage infection is evident. The relative amounts of messenger RNA in cells growing normally and in cells IO min after infection are compared in Fig. 3. No substantial decrease in the rate of total messenger RNA synthesis is evident during the Io-min period following infection as measured between the Ioth and I l t h rain. However, as shown, the rate of general protein synthesis has been reduced to less than 5 % and the rate of synthesis of fi-galactosidase is zero. These findings are consistent with an immediate interruption of the expression (translation) of host messenger RNA in cells infected by 2vir. Further investigations are in progress. This work was supported in part by research grants CY52IO and C5175 National Institutes of Health, U.S. Public Health Service, administered by Professor B. MAGASANIK, Department of Biology, Massachusetts Institute of Technology. The author was a National Science Foundation Postdoctoral Fellow, 1962-1964.
Department o/ Biology, Massachusetts Institute o/ Technology, Cambridge, Mass. and Department o/ Biological Chemistry, College o/ Medicine, University o/Illinois, Chicago, Ill. (U.S.A.) i 2 3 4 5
WILLIAM V, HOWES
]3. A. FRY AND F. GROS,J. Gen, Microbiol., 21 (1959) 685. L. SIMINOVlTCHAND F. JACOB,Ann. Inst. Pasteur. 83 (1952) 745. H. REVEL,S. LURIAAND ]3. ROTMAN,Proc. Natl. Acad. Sci. U.S. 47 (1961) 1961. t~. OKAMOTO,Y. SUGINOANDM. NOMURA,J. Mol. Biol., 5 (1962) 527. F. J. BOLLUM,J. Biol. Chem., 234 (1959) 2733.
Received May I l t h , 1965.
Biochim. Biophys. Acta, lO3 (1965) 711-713
711
PN 91077
Protein synthesis in Escherichio coli: A phoge-medioted interruption of tronslation FRY AND GROS1 have reported conditions under which 9 1 % of the cells of Escherichia coli K I I 2 gave a lysogenic response after infection by phage ~. Net synthesis of DNA, RNA and protein could not be detected in these potential lysogens for approx. 12o rain following infection, fl-Galactosidase could not be induced for at least 19o min, but was inducible after the lysogens were growing at a rate comparable with uninfected cells. SIMINOV1TCHAND JACOB2 have reported that induced production of fl-galactosidase was possible during vegetative phage development following ultraviolet irradiation of ~ lysogens. Thus, a profound disturbance in the metabolism of the host is evident immediately after infection b y ~ and during the establishment of the prophage. A less serious disturbance is evident upon induction of the prophage. We are investigating the effects of a virulent m u t a n t of phage ,~(~vtr) on the metabolism of the host. The rates of synthesis of general protein and RNA are compared with those of specific protein and RNA ill an attempt to identify the event which inhibits the protein-synthesizing machinery. E. coli strain 3.000 growing with aeration at 37 ° in a medium containing (g): MgSO4 1.2, K2HPO * o.3, Na2 H P O 4 • 7 H20 0.6, (NH~)2SO 4 0.5, glycerol I.O, and vitamin B 10.5 mg per 1 was exposed to ~vir at a multiplicity of 50 phages per cell. Under these conditions less than 1 % of the bacteria survived a 3-rain exposure as indicated b y viable count. A minimum of 85 % of these non-viable cells produced infective centers after treatment with ~ antiserum. A minimum latent period of 50-60 min
25
P H AG E
I
2O
o x
o
n
0
! 20
15
z
OI
-15
,
-IO
k
~
,
,
,
"5
0
5
'0
15
TIME
(MINi
Fig. I. E f f e c t of p h a g e Jlvir o n p r o t e i n a n d 1RNA s y n t h e s i s . A t - - 1 5 min, for p r o t e i n L-[I-14C] leucine (0.02/*C//,g, 4 # g / m l ) ( 0 - 0 ) ; or for 1RNA, [2-z4C]uracil (o.05 pC/itg, 5#g/ml), t h y m i d i n e (5/*g/ml) ( G - C ) ) , w a s a d d e d to cells g r o w i n g a t 5" IOS cells/ml. P h a g e (5o/cell) were a d d e d a t zero t i m e . A t specified t i m e s , p r o t e i n s a m p l e s were a d d e d to a n e q u a l v o l u m e of 1o % trichloroacetic acid -- i % c a s a m i n o a c i d s solution, h e a t e d a t 90 ° for 3 ° min, t h e insoluble m a t e r i a l collected on a m e m b r a n e filter (Millipore, AA), w a s h e d w i t h 5 % trichloroacetic acid-o. 5 % eas a m i n o acids, a n d c o u n t e d in a Geiger c o u n t e r . R N A s a m p l e s were s i m i l a r l y w i t h d r a w n i n t o io % trichloroacetic acid a t o% A f t e r 30 m i n a t o °, t h e i n s o l u b l e m a t e r i a l w a s collected, w a s h e d w i t h cold 5 % trichloroacetic acid, a n d c o u n t e d as described.
Biochim. Biophys. Hcta, lO 3 (1965) 711-713
712
PRELIMINARY NOTES
and a single burst of 33 were observed. No leakage of fl-galactosidase could be detected from cells maximally induced before exposure to phage. Fig. i indicates that the rate of general protein and RNA synthesis is drastically . . . .
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Fig. 2. P r o d u c t i o n of fl-galactosidase. Cells g r o w i n g at 5 " ~o8 cells/ml were i n d u c e d w i t h isopropyl-thio-fl-D-galactoside (5. lO-4 M) a t zero t i m e . A t 2 rain, t h e i n d u c e r w a s r e m o v e d b y filt r a t i o n o n a merfibrane filter (Millipore, H A ) , a n d t h e cells w a s h e d a n d r e s u s p e n d e d in i n d u c e r free m e d i u m ( O - Q ) . T h i s r e q u i r e d 1. 5 min. I n a parallel e x p e r i m e n t , p h a g e (5o/cell) were a d d e d a t 2 rain ( 0 - 0 ) . A t specified times, s a m p l e s were w i t h d r a w n a n d a s s a y e d for fl-galactosidase as described b y R E V E L , LURIA a n d ROTMAN 3. i
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Fig. 3. M e s s e n g e r R N A in i n f e c t e d a n d u n i n f e c t e d cells. Cells g r o w i n g at 5 " lO8 cells/ml were i n f e c t e d w i t h p h a g e (5o/cell) a t zero t i m e . B e t w e e n t h e io th a n d i i th m i n t h e cells were p u l s e d for 45 sec w i t h E5, 6-3H] uracil (4.o/~C//~g, 0. 5 / , g / m l ) ( O - © )- T h e i n c o r p o r a t i o n w a s stopped, carrier cells a d d e d , a n d t h e p h e n o l - e x t r a c t e d R N A p r e p a r e d as described b y O K A M O T O , SUGINO AND NOMURA4. T h e final e t h a n o l p r e c i p i t a t e w a s dissolved in a s m a l l a m o u n t of buffer, layered o n t h e 4 to 20 % s u c r o s e g r a d i e n t , a n d s e d i m e n t e d a t 39 ooo r e v . / m i n S W - 3 9 rotor, Spinco L-2 u l t r a c e n t r i f u g e , o °, for 5 to 6 h, T h e g r a d i e n t w a s collected dropwise a n d a n a l i q u o t f r o m e a c h t u b e t r a n s f e r r e d to a b l o t t i n g p a p e r disc. T h e a b s o r b a n c e a t 260 m/z w a s m e a s u r e d o n t h e d i l u t e d rem a i n d e r . T h e s e m a r k e r p e a k s are d e s i g n a t e d b y arrows. T h e cold trichloroacetic aeid-precip i t a b l e m a t e r i a l w a s p r e p a r e d o n t h e b l o t t e r discs as d e s c r i b e d b y BOLLUM 5, placed i n vials c o n t a i n i n g : t o l u e n e 8 ml, 2.5-diphenyl-oxazole 32 rag, 1,4-bis-2-(5-phenyloxzolyl)benzene o.8 rag, a n d c o u n t e d in a liquid s c i n t i l l a t i o n s p e c t r o m e t e r . U n i n f e c t e d g r o w i n g cells ( Q - O ) were p u l s e d a n d t h e R N A e x t r a c t e d , purified s e d i m e n t e d , a n d a s s a y e d as described. To correct for t h e difference in t o t a l R N A recovered f r o m t h e g r a d i e n t s , c o u n t s / r a i n are e x p r e s s e d in t e r m s of zoo % r e c o v e r y for t h e u n i n f e c t e d cells. R e c o v e r y is b a s e d on a b s o r b a n c e a t t h e 23-S m a r k e r p e a k .
Biochim. Biophys. Acta, lO 3 (1965) 711-713
PRELIMINARY NOTES
713
reduced within 3 min after phage infection. The infected rate is less than 5 % of the initial rate. Removal of the unadsorbed phage by centrifugation or treatment with 2 antiserum had no effect. The rate of decrease of fl-galactosidase production following removal of inducer (isopropyl-thio-fi-D-galactoside) by washing is compared with the rate of decrease following phage infection in Fig. 2, The dramatic "shut-off" of enzyme synthesis following phage infection is evident. The relative amounts of messenger RNA in cells growing normally and in cells IO min after infection are compared in Fig. 3. No substantial decrease in the rate of total messenger RNA synthesis is evident during the Io-min period following infection as measured between the Ioth and I l t h rain. However, as shown, the rate of general protein synthesis has been reduced to less than 5 % and the rate of synthesis of fi-galactosidase is zero. These findings are consistent with an immediate interruption of the expression (translation) of host messenger RNA in cells infected by 2vir. Further investigations are in progress. This work was supported in part by research grants CY52IO and C5175 National Institutes of Health, U.S. Public Health Service, administered by Professor B. MAGASANIK, Department of Biology, Massachusetts Institute of Technology. The author was a National Science Foundation Postdoctoral Fellow, 1962-1964.
Department o/ Biology, Massachusetts Institute o/ Technology, Cambridge, Mass. and Department o/ Biological Chemistry, College o/ Medicine, University o/Illinois, Chicago, Ill. (U.S.A.) i 2 3 4 5
WILLIAM V, HOWES
]3. A. FRY AND F. GROS,J. Gen, Microbiol., 21 (1959) 685. L. SIMINOVlTCHAND F. JACOB,Ann. Inst. Pasteur. 83 (1952) 745. H. REVEL,S. LURIAAND ]3. ROTMAN,Proc. Natl. Acad. Sci. U.S. 47 (1961) 1961. t~. OKAMOTO,Y. SUGINOANDM. NOMURA,J. Mol. Biol., 5 (1962) 527. F. J. BOLLUM,J. Biol. Chem., 234 (1959) 2733.
Received May I l t h , 1965.
Biochim. Biophys. Acta, lO3 (1965) 711-713