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Activity of stringent protein in ribosomes of Escherichia coli during the growth cycle
128
Biochimica et Biophysica Acta, 4 3 5 ( 1 9 7 6 ) 1 2 8 - - 1 3 1 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
BBA 98619
ACTIVITY OF S T R I N G E N T PROTEIN IN RIBOSOMES OF ESCHERICHIA COL.I D U R I N G THE GROWTH CYCLE
SUBBANAIDU
RAMAGOPAL
*
Bacterial Physiology Unit, Harvard Medical School, Boston, Mass. (U.S.A.) (Received December 17th, 1975)
Summary To investigate whether the stringent protein in Escherichia coli was lost from ribosomes at certain phase of growth as in other organisms (Bacillus subtilis, mouse embryo), cells growing at various phases of the growth cycle were harvested and ribosomes tested for activity. The results showed that in E. coli, stringent protein was associated with the ribosomes throughout the growth cycle. The peak activity for the synthesis of guanosine tetra- and pentaphosphates appeared around midlogarithmic phase.
Introduction Growth in bacteria is intimately associated with the production of ribosomes [1]. Recent evidence suggests that the stringent control mechanism operative in certain strains of bacteria regulates b o t h ribosomal R N A [2] and ribosomal protein [3] synthesis. The unusual nucleotides, ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and p p p G p p (guanosine 5'-triphosphate 3'-diphosphate), that accumulate in stringent strains during depletion of essential amino acids are implicated in the control of rRNA synthesis. These compounds are made on the ribosome by a product of the rel* gene called the stringent protein [4] which was previously shown to be associated with the 50-S subunit [5,6]. Recent evidence indicates that growth and development seem to regulate the synthesis of (p)ppGpp in some organisms. Ribosomes from sporulating cells of Bacillus subtilis made almost no (p)ppGpp compared to vegetative cells [7]. In eukaryotic system also there appears to be a definite correlation between developmental stage and stringent protein activity. For example, stringent protein activity was present in ribosomes of l l - d a y mouse embryos but none in 14-day or adult embryos [8]. * Present address: The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pa. 19111, U.S.A.
129 At present, it is not known whether such a growth-dependent regulation of stringent protein activity occurs in Escherichia coli in which the stringent system was initially discovered. Our earlier work showed that the stringent protein was a normal c o m p o n e n t of 50 S subunit and does not appear to leave the ribosome during the ribosome cycle [ 5 ] . This communication will provide evidence for the qualitative presence of stringent protein on the ribosome throughout the growth of cycle of E. coli cells. Materials and Methods
E. coli, strain CP78 (rel ÷) was grown at 37°C with aeration in Zubay's phosphate medium [ 5]. Growth was monitored by absorbance measurements of the culture in a Klett-Summerson colorimeter. The growth curve of the culture is illustrated in Fig. 1. The exponential-phase doubling time was 42 min. The cells were harvested when the culture reached early logarithmic (Klett 55), midlogarithmic (Klett 110) and stationary phases (Klett 350; 6.7 h after inoculation) of the growth cycle. Cells were washed once with cold buffer. Ribosomes were prepared by rupturing the cells with alumina and ultracentrifugation [ 5 ] , suspended in buffer (10 mM Tris • acetate, pH 7.8, 60 mM potassium acetate, 14 mM magnesium acetate and 1 mM dithiothreitol) at 6 0 - - 7 0 mg/ml (1 mg = 16 A260nm units) and stored at --76°C. The stringent protein content was assayed by incubating the various ribosome preparations in 0.05 ml reaction mixtures containing 0.05 M Tris • acetate (pH 7.8), 2 mM dithiothreitol, 12 mM Mgacetate, 28 mM a m m o n i u m acetate, 10 mM potassium acetate, 0.55 mM GTP (0.8 pCi of [a_32p] GTP, New England Nuclear) and
4.00 300
200
(D u
100
cl 0 co a~ ,<
50 40 30
20,
10 0
I 1
I 2
I 3
1 4
I 5
I 6
I 7
TIME (hours) Fig. 1. G r o w t h c u r v e f o r E . coli, s t r a i n C P 7 8 i n Z u b a y ' s p h o s p h a t e at w h i c h t h e c u l t u r e w a s h a r v e s t e d f o r r i b o s o m e
isolation.
medium.
The arrows indicate the stages
130 2.2 mM ATP at 37°C for 30 min. The synthesis of (p)ppGpp (ppGpp + pppGpp) was determined as described before [ 5,9]. Results and Discussion Cells of E. coli were grown in a rich medium and harvested when the cultures approached early and midlogarithmic phases of growth. A nongrowing culture was also harvested a b o u t three hr after attaining the stationary phase. Ribosomes were isolated and unwashed ribosomes were first directly assayed for stringent protein activity to avoid losses ensuing further purification [5,9,10]. The level of stringent protein was estimated by the capacity of ribosomes to form (p)ppGpp in vitro. The results clearly show that stringent protein was present in ribosomes harvested at all three phases of the growth cycle (Fig. 2). Exponentially growing cultures (both early and midlogarithmic) converted 50--60% of the added GTP into (p)ppGpp (Fig. 2a, b) whereas the conversion by the stationary culture w~s only 25--30% (Fig. 2c). However, the GTPase activity of stationary-phase ribosomes was not significantly lower compared to exponential phase ribosomes (Fig. 2). Although it is likely that unwashed ribosomes retain higher amounts of stringent protein, its activity can be still masked by other inhibitors of the reaction in crude ribosome preparations [4,5]. Possibly, crude ribosomes obtained at different phases of the growth cycle contributed varying amounts of such inhibitors. In an attempt to remove those inhibitors, the crude ribosomes were partially purified by passing through a heavy sucrose cushion [5] and again tested. It is interesting to note that only a constant fraction (about 50%) of the activity observed in unwashed ribosomes was lost on purification from all three ribosome preparations (Table I, compare last column). The peak activity of stringent protein seem to occur around midlogarithmic phase which is apparent from the results obtained before or after purification of the ribosomes (Table I and Fig. 2). Partial purification of ribosomes enhanced the activity of stringent protein for p p p G p p synthesis several fold (Table I). Both ppGpp and p p p G p p synthesis were observed in various ribosome preparations, the synthesis of the former
~6o~ (o.) EARLY LOG ro~]) 12o~ GTP
0
20
40
60
(b.) MIDLOG [ GTP /
20
C I) S T A T I O N A R Y GTP
(p)ppGpp
40
60 0
20
40
60
Ribosomes (fig) Fig. 2. S t r i n g e n t p r o t e i n a c t i v i t y o f r i b o s o m e s i s o l a t e d at d i f f e r e n t P h a s e s o f t h e g r o w t h c y c l e . Cells o f s t r a i n C P 7 8 w e r e g r o w n f r o m an o v e r n i g h t i n o c u l u m to early l o g a r i t h m i c (a), m i d l o g a r i t h m i c (b) a n d s t a t i o n a r y p h a s e s (c) as d e s c r i b e d in M e t h o d s , ( p ) p p G p p d e n o t e s t h e s y n t h e s i s p p G p p p l u s p p p G p p . T h e fig. also i n d i c a t e s t h e G T P a s e a c t i v i t y o f d i f f e r e n t r i b o s o m e p r e p a r a t i o n s . G T P h y d r o l y s i s is s h o w n as t h e u n c o n v e r t e d G T P left in t h e r e a c t i o n m i x t u r e a f t e r ( p ) p p G p p s y n t h e s i s ,
131
TABLE I S Y N T H E S I S O F p p G p p A N D p p p G p p BY U N W A S H E D A N D S U C R O S E - W A S H E D R I B O S O M E S C u l t u r e s g r o w n as d e s c r i b e d in Fig. 1. U n w a s h e d and s u c r o s e - w a s h e d r i b o s o m e s w e r e isolated e x a c t l y as d e t a i l e d earlier [ 5 , 9 ] . T h e r e a c t i o n m i x t u r e s c o n t a i n e d 2 . 7 - - 3 . 1 A 260nm u n i t s o f r i b o s o m e s . U n d e r t h e assay c o n d i t i o n s t h e s y n t h e s i s o f p p G p p was linear b u t p p p G p p s y n t h e s i s was a p p r o a c h i n g t h e m a x i m u m value [ 9 ] . G r o w t h phase
E a r l y log Midlog Stationary
Ribosome preparation
unwashed sucrose-washed unwashed sucrose-washed unwashed sucrose-washed
pmol nucleoide/pmol ribosome ppGpp
pppGpp
Total
148.4 50.4 246.5 62.3 167.0 68.4
2.1 27.8 6.4 51.4 1.9 29.9
150.5 78.2 252.9 1 i 3.7 168.9 98.3
slightly increased toward stationary phase. Maximum pppGpp synthesis occurred at midlogarithmic phase; the ribosomes synthesized about equal amounts of ppGpp and pppGpp (Table I). The ribosome-associated stringent protein system responsible for ppGpp synthesis is probably less affected by the growth phase. At present, it is not clear whether the same stringent system is responsible for pppGpp synthesis in vitro as well as in vivo. In vivo studies on s p o t mutants have suggested [11] a conversion of ppGpp to pppGpp but in cell-free system, pppGpp may give rise to ppGpp in the presence of elongation factor G [12,13]. Nevertheless, the results suggest that growth phase modifies this stringent system to some extent. In conclusion, this study has shown that E. col[ ribosomes retain stringent protein activity independent of growth phase in contrast to ribosomes of Bacillus subtilis [7] or mouse embryos [8]. It was present in actively dividing and stationary phase cells. The activity differences reported for various ribosome preparations may be considered only qualitative since a thorough investigation of the components of the cell-free assay system was not undertaken. Acknowledgments I thank Dr. Bernard Davis for encouragement and support. References 1 Maaloe, O. a n d K j e l d g a a r d , N. ( 1 9 6 6 ) C o n t r o l o f M a c r o m o l e c u l a r S y n t h e s i s , W.A. B e ~ j a m i n , lnc., New York 2 L a z z a r i n i , R . A . , Cashel, M. a n d G a l l a n t , J. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 4 3 8 1 - - 4 3 8 5 3 D e n n i s , P.P. a n d N o m u r a . M. ( 1 9 7 4 ) P r o e . N a t l . A c a d . Sci. U.S. 7 1 , 3 8 1 9 - - 3 8 2 3 4 H a s e l t i n e , W.A., Block, R., G i l b e r t , W. a n d W e b e r , K. ( 1 9 7 2 ) N a t u r e 2 3 8 , 3 8 1 - - 3 8 4 5 R a m a g o p a l , S. a n d Davis, B.D. ( 1 9 7 4 ) P r o c . Natl. A c a d . Sci. U.S. 7 1 , 8 2 0 - - 8 2 4 6 R i c h t e r , D. a n d I s o n o , K. ( 1 9 7 4 ) FEBS L e t t . 44, 2 7 0 - - 2 7 3 7 R h a e s e , H.J. a n d G r o s c u r t h , R. ( 1 9 7 4 ) FEBS L e t t . 4 4 , 8 7 - - 9 3 8 Irr, J . D . , K a u l e n a s , M.S. a n d UnswoV~h, B.R. ( 1 9 7 4 ) Cell 3 , 2 4 9 - - 2 5 3 9 R a m a g o p a l , S. ( 1 9 7 4 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 5 8 , 2 6 8 - - 2 7 1 10 B l o c k , R. a n d H a s e l t i n e , W.A. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 1 2 1 2 - - 1 2 1 7 11 L a f f l e r , T. and G a n a n t , J. ( 1 9 7 4 ) Cell 1, 2 7 - - 3 0 12 C o c h r a n , J.W. a n d B y r n e , R.W. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 3 5 3 - - 3 6 0 13 H a m e l , E. a n d Cashel, M. ( 1 9 7 3 ) Proc. Natl. A c a d . Sei. U.S. 7 0 , 3 2 5 0 - - 3 2 5 4
Biochimica et Biophysica Acta, 4 3 5 ( 1 9 7 6 ) 1 2 8 - - 1 3 1 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s
BBA 98619
ACTIVITY OF S T R I N G E N T PROTEIN IN RIBOSOMES OF ESCHERICHIA COL.I D U R I N G THE GROWTH CYCLE
SUBBANAIDU
RAMAGOPAL
*
Bacterial Physiology Unit, Harvard Medical School, Boston, Mass. (U.S.A.) (Received December 17th, 1975)
Summary To investigate whether the stringent protein in Escherichia coli was lost from ribosomes at certain phase of growth as in other organisms (Bacillus subtilis, mouse embryo), cells growing at various phases of the growth cycle were harvested and ribosomes tested for activity. The results showed that in E. coli, stringent protein was associated with the ribosomes throughout the growth cycle. The peak activity for the synthesis of guanosine tetra- and pentaphosphates appeared around midlogarithmic phase.
Introduction Growth in bacteria is intimately associated with the production of ribosomes [1]. Recent evidence suggests that the stringent control mechanism operative in certain strains of bacteria regulates b o t h ribosomal R N A [2] and ribosomal protein [3] synthesis. The unusual nucleotides, ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and p p p G p p (guanosine 5'-triphosphate 3'-diphosphate), that accumulate in stringent strains during depletion of essential amino acids are implicated in the control of rRNA synthesis. These compounds are made on the ribosome by a product of the rel* gene called the stringent protein [4] which was previously shown to be associated with the 50-S subunit [5,6]. Recent evidence indicates that growth and development seem to regulate the synthesis of (p)ppGpp in some organisms. Ribosomes from sporulating cells of Bacillus subtilis made almost no (p)ppGpp compared to vegetative cells [7]. In eukaryotic system also there appears to be a definite correlation between developmental stage and stringent protein activity. For example, stringent protein activity was present in ribosomes of l l - d a y mouse embryos but none in 14-day or adult embryos [8]. * Present address: The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pa. 19111, U.S.A.
129 At present, it is not known whether such a growth-dependent regulation of stringent protein activity occurs in Escherichia coli in which the stringent system was initially discovered. Our earlier work showed that the stringent protein was a normal c o m p o n e n t of 50 S subunit and does not appear to leave the ribosome during the ribosome cycle [ 5 ] . This communication will provide evidence for the qualitative presence of stringent protein on the ribosome throughout the growth of cycle of E. coli cells. Materials and Methods
E. coli, strain CP78 (rel ÷) was grown at 37°C with aeration in Zubay's phosphate medium [ 5]. Growth was monitored by absorbance measurements of the culture in a Klett-Summerson colorimeter. The growth curve of the culture is illustrated in Fig. 1. The exponential-phase doubling time was 42 min. The cells were harvested when the culture reached early logarithmic (Klett 55), midlogarithmic (Klett 110) and stationary phases (Klett 350; 6.7 h after inoculation) of the growth cycle. Cells were washed once with cold buffer. Ribosomes were prepared by rupturing the cells with alumina and ultracentrifugation [ 5 ] , suspended in buffer (10 mM Tris • acetate, pH 7.8, 60 mM potassium acetate, 14 mM magnesium acetate and 1 mM dithiothreitol) at 6 0 - - 7 0 mg/ml (1 mg = 16 A260nm units) and stored at --76°C. The stringent protein content was assayed by incubating the various ribosome preparations in 0.05 ml reaction mixtures containing 0.05 M Tris • acetate (pH 7.8), 2 mM dithiothreitol, 12 mM Mgacetate, 28 mM a m m o n i u m acetate, 10 mM potassium acetate, 0.55 mM GTP (0.8 pCi of [a_32p] GTP, New England Nuclear) and
4.00 300
200
(D u
100
cl 0 co a~ ,<
50 40 30
20,
10 0
I 1
I 2
I 3
1 4
I 5
I 6
I 7
TIME (hours) Fig. 1. G r o w t h c u r v e f o r E . coli, s t r a i n C P 7 8 i n Z u b a y ' s p h o s p h a t e at w h i c h t h e c u l t u r e w a s h a r v e s t e d f o r r i b o s o m e
isolation.
medium.
The arrows indicate the stages
130 2.2 mM ATP at 37°C for 30 min. The synthesis of (p)ppGpp (ppGpp + pppGpp) was determined as described before [ 5,9]. Results and Discussion Cells of E. coli were grown in a rich medium and harvested when the cultures approached early and midlogarithmic phases of growth. A nongrowing culture was also harvested a b o u t three hr after attaining the stationary phase. Ribosomes were isolated and unwashed ribosomes were first directly assayed for stringent protein activity to avoid losses ensuing further purification [5,9,10]. The level of stringent protein was estimated by the capacity of ribosomes to form (p)ppGpp in vitro. The results clearly show that stringent protein was present in ribosomes harvested at all three phases of the growth cycle (Fig. 2). Exponentially growing cultures (both early and midlogarithmic) converted 50--60% of the added GTP into (p)ppGpp (Fig. 2a, b) whereas the conversion by the stationary culture w~s only 25--30% (Fig. 2c). However, the GTPase activity of stationary-phase ribosomes was not significantly lower compared to exponential phase ribosomes (Fig. 2). Although it is likely that unwashed ribosomes retain higher amounts of stringent protein, its activity can be still masked by other inhibitors of the reaction in crude ribosome preparations [4,5]. Possibly, crude ribosomes obtained at different phases of the growth cycle contributed varying amounts of such inhibitors. In an attempt to remove those inhibitors, the crude ribosomes were partially purified by passing through a heavy sucrose cushion [5] and again tested. It is interesting to note that only a constant fraction (about 50%) of the activity observed in unwashed ribosomes was lost on purification from all three ribosome preparations (Table I, compare last column). The peak activity of stringent protein seem to occur around midlogarithmic phase which is apparent from the results obtained before or after purification of the ribosomes (Table I and Fig. 2). Partial purification of ribosomes enhanced the activity of stringent protein for p p p G p p synthesis several fold (Table I). Both ppGpp and p p p G p p synthesis were observed in various ribosome preparations, the synthesis of the former
~6o~ (o.) EARLY LOG ro~]) 12o~ GTP
0
20
40
60
(b.) MIDLOG [ GTP /
20
C I) S T A T I O N A R Y GTP
(p)ppGpp
40
60 0
20
40
60
Ribosomes (fig) Fig. 2. S t r i n g e n t p r o t e i n a c t i v i t y o f r i b o s o m e s i s o l a t e d at d i f f e r e n t P h a s e s o f t h e g r o w t h c y c l e . Cells o f s t r a i n C P 7 8 w e r e g r o w n f r o m an o v e r n i g h t i n o c u l u m to early l o g a r i t h m i c (a), m i d l o g a r i t h m i c (b) a n d s t a t i o n a r y p h a s e s (c) as d e s c r i b e d in M e t h o d s , ( p ) p p G p p d e n o t e s t h e s y n t h e s i s p p G p p p l u s p p p G p p . T h e fig. also i n d i c a t e s t h e G T P a s e a c t i v i t y o f d i f f e r e n t r i b o s o m e p r e p a r a t i o n s . G T P h y d r o l y s i s is s h o w n as t h e u n c o n v e r t e d G T P left in t h e r e a c t i o n m i x t u r e a f t e r ( p ) p p G p p s y n t h e s i s ,
131
TABLE I S Y N T H E S I S O F p p G p p A N D p p p G p p BY U N W A S H E D A N D S U C R O S E - W A S H E D R I B O S O M E S C u l t u r e s g r o w n as d e s c r i b e d in Fig. 1. U n w a s h e d and s u c r o s e - w a s h e d r i b o s o m e s w e r e isolated e x a c t l y as d e t a i l e d earlier [ 5 , 9 ] . T h e r e a c t i o n m i x t u r e s c o n t a i n e d 2 . 7 - - 3 . 1 A 260nm u n i t s o f r i b o s o m e s . U n d e r t h e assay c o n d i t i o n s t h e s y n t h e s i s o f p p G p p was linear b u t p p p G p p s y n t h e s i s was a p p r o a c h i n g t h e m a x i m u m value [ 9 ] . G r o w t h phase
E a r l y log Midlog Stationary
Ribosome preparation
unwashed sucrose-washed unwashed sucrose-washed unwashed sucrose-washed
pmol nucleoide/pmol ribosome ppGpp
pppGpp
Total
148.4 50.4 246.5 62.3 167.0 68.4
2.1 27.8 6.4 51.4 1.9 29.9
150.5 78.2 252.9 1 i 3.7 168.9 98.3
slightly increased toward stationary phase. Maximum pppGpp synthesis occurred at midlogarithmic phase; the ribosomes synthesized about equal amounts of ppGpp and pppGpp (Table I). The ribosome-associated stringent protein system responsible for ppGpp synthesis is probably less affected by the growth phase. At present, it is not clear whether the same stringent system is responsible for pppGpp synthesis in vitro as well as in vivo. In vivo studies on s p o t mutants have suggested [11] a conversion of ppGpp to pppGpp but in cell-free system, pppGpp may give rise to ppGpp in the presence of elongation factor G [12,13]. Nevertheless, the results suggest that growth phase modifies this stringent system to some extent. In conclusion, this study has shown that E. col[ ribosomes retain stringent protein activity independent of growth phase in contrast to ribosomes of Bacillus subtilis [7] or mouse embryos [8]. It was present in actively dividing and stationary phase cells. The activity differences reported for various ribosome preparations may be considered only qualitative since a thorough investigation of the components of the cell-free assay system was not undertaken. Acknowledgments I thank Dr. Bernard Davis for encouragement and support. References 1 Maaloe, O. a n d K j e l d g a a r d , N. ( 1 9 6 6 ) C o n t r o l o f M a c r o m o l e c u l a r S y n t h e s i s , W.A. B e ~ j a m i n , lnc., New York 2 L a z z a r i n i , R . A . , Cashel, M. a n d G a l l a n t , J. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 4 3 8 1 - - 4 3 8 5 3 D e n n i s , P.P. a n d N o m u r a . M. ( 1 9 7 4 ) P r o e . N a t l . A c a d . Sci. U.S. 7 1 , 3 8 1 9 - - 3 8 2 3 4 H a s e l t i n e , W.A., Block, R., G i l b e r t , W. a n d W e b e r , K. ( 1 9 7 2 ) N a t u r e 2 3 8 , 3 8 1 - - 3 8 4 5 R a m a g o p a l , S. a n d Davis, B.D. ( 1 9 7 4 ) P r o c . Natl. A c a d . Sci. U.S. 7 1 , 8 2 0 - - 8 2 4 6 R i c h t e r , D. a n d I s o n o , K. ( 1 9 7 4 ) FEBS L e t t . 44, 2 7 0 - - 2 7 3 7 R h a e s e , H.J. a n d G r o s c u r t h , R. ( 1 9 7 4 ) FEBS L e t t . 4 4 , 8 7 - - 9 3 8 Irr, J . D . , K a u l e n a s , M.S. a n d UnswoV~h, B.R. ( 1 9 7 4 ) Cell 3 , 2 4 9 - - 2 5 3 9 R a m a g o p a l , S. ( 1 9 7 4 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 5 8 , 2 6 8 - - 2 7 1 10 B l o c k , R. a n d H a s e l t i n e , W.A. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 1 2 1 2 - - 1 2 1 7 11 L a f f l e r , T. and G a n a n t , J. ( 1 9 7 4 ) Cell 1, 2 7 - - 3 0 12 C o c h r a n , J.W. a n d B y r n e , R.W. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 3 5 3 - - 3 6 0 13 H a m e l , E. a n d Cashel, M. ( 1 9 7 3 ) Proc. Natl. A c a d . Sei. U.S. 7 0 , 3 2 5 0 - - 3 2 5 4