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Inactivation of infectious deoxyribonucleic acid of bacteriophage ΦX 174 by colicin E2
Biochimica et Biophysica Acta, 324 (1973) 305-308 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97835
I N A C T I V A T I O N OF I N F E C T I O U S D E O X Y R I B O N U C L E I C A C I D OF BACT E R I O P H A G E • X 174 BY C O L I C I N E2
AKIKO SETO*, TAKAO SHINOZAWA and AKIO MAEDA Department of Biophysics, Faculty of Science, Kyoto University, Kyoto (Japan)
(Received June 4th, 1973)
SUMMARY Incubation of single-stranded cPX 174 D N A with colicin E2 resulted in the impairment of the ability of the D N A to produce progeny phage particles in spheroplasts. The inactivation of ~PX 174 D N A by colicin E2 occurred even without chain scissions in DNA. The rate for the inactivation at 40 °C was some 50-fold faster than that at 30 °C. The effect of colicin E2 was reduced with the increase of ionic concentration and completely abolished by 1 M NaC1.
INTRODUCTION Colicin E2 is known to exert various kinds of effects in cells of Escherichia coli. The degradation of D N A is the most prominent phenomenon in these cells 1'2. Beppu and Arima 3 have shown that E2 causes the liberation of D N A from the complexes consisting of D N A and membrane. Ringrose 4 has further reported that E2 lowers the T m of the isolated DNA. For further analyses on the mode of interaction of E2 with DNA, other methods of approach are also helpful. An intact form of ~ X 174 D N A has been known to produce progeny phage particles in spheroplastsS'6; some kind of interaction of D N A with E2 is expected to be detected when cbX 174 D N A treated variously with E2 is put into spheroplasts. It was found that colicin E2 inactivated the infectious D N A of ~ X 174. Some features of interaction of E2 with D N A are also described. MATERIALS AND METHODS g~X 174 D N A E. coli HF4704 (hrc-, thy-)7 (lysis defective) a. Phage particles described by Y o k o y a m a et al. 9 and density gradients, tPX 174 D N A was Guthrie and Sinsheimer 1o.
was a host strain for the growth of ~PX 174 am3 labeled with [aH]thymidine were prepared as further purified by the centrifugation in sucrose extracted by the hot phenol method described by
* Present address: Institute of Life Science, Mitsubishi Kasei, Minami-Oya, Machida-shi, Tokyo (Japan).
306
A. SETO et al.
Colicin E2 Colicin E2 was extracted in 0.5 M neutralized guanidine-hydrochloride from the cells of E. coli W3110 (col E2) induced by mitomycin C, precipitated by 60 % saturation of ammonium sulfate and purified through columns of DEAE-Sephadex and CM-Sephadex according to the procedures described by Herschman and Helsinski 11. The final preparation exhibited only a single band in a disc electrophoresis at pH 4.0 (see ref. I I). The specific killing activity of this preparation was about 2 • 1014 killing units (see ref. 12 as for the definition of killing unit) per mg of protein. Assay of activity of ~ X 174 DNA A colicin E2-resistant strain of E. coli K12 1100 (end-, thi-) 13 was used for the preparation of spheroplasts. The ability of variously treated ~X 174 DNA to produce progeny particles was measured by the procedures described by Guthrie and Sinsheimer 1o. Incubation of spheroplasts was done at 30 °C instead of 35 °C. E. coli HF4714 t4 was used as an indicator strain. RESULTS
The number of phage particles produced from ~X 174 DNA was found to decrease with the increasing amounts of E2 (Fig. 1). The effects of E2 on ~X 174 DNA decreased also with the loss of its bacteriocidal activity. The relation between these two kinds of activity (i.e. the activity on DNA and activity on bacteria) in several prepara1.00 (
0.75 u
O_ I1)
.~0,50
~: 0.25
o
o
20
40 60 80 Killing units of E2 No, of ~X DNA molecules
Fig. 1. Relation between the effect o f E2 on ~ X 1 7 4 D N A a n d the killing activity o f E2. E2 (20 ~g/ml) partially inactivated by heating at 60 °C for 15 m i n ( O ) , at 70 °C for 5 m i n ( A ) o r at 70 °C for 15 m i n ( A ) was incubated with ~ X 1 7 4 D N A at 40 ° e for 40 m i n in 0.01 M T r i s - H C l buffer ( p H 8.1). O - O , results f r o m the reaction mixture containing the various a m o u n t s o f u n h e a t e d E2. T h e a m o u n t o f D N A used was calculated to be 5 • 10 t° molecules/ml f r o m the n u m b e r o f plaquef o r m i n g centers a n d the radioactivity o f original phage preparation.
INACTIVATION OF @X 174 DNA BY COLICIN E2
307
tions o f p a r t i a l l y inactivated E2 coincided well with t h a t n o t e d for native E2. Thus, the effects with the present p r e p a r a t i o n o f E2 a p p e a r to be due to the action o f E2 molecules a n d n o t to possible c o n t a m i n a n t s . A s a m p l e o f ~ X 174 D N A inactivated to 5 ~ by E2 was analyzed in alkaline sucrose density gradients. The s e d i m e n t a t i o n p a t t e r n was a l m o s t identical with t h a t o f the c o n t r o l w i t h o u t E2. M o r e o v e r , the activity was f o u n d to be restored to 60 o f the c o n t r o l after the centrifugation. The b a c k b o n e structure o f the D N A molecules seems to be kept intact even in the situation where their activity is reduced. S h a k i n g with phenol o f E2-treated ~ X 174 D N A , however, was n o t successful in restoring the activity. The rate o f the inactivation reaction increased with the rise o f t e m p e r a t u r e f r o m 30 to 70 °C. The kinetic process o f the reaction can best be expressed as t h a t o f a first-order reaction. T h e rate at 40 °C was some 50-fold faster t h a n t h a t at 30 °C. I n c u b a t i o n o f D N A with E2 was carried o u t u n d e r a variety o f c o n d i t i o n s o f ionic c o m p o s i t i o n (Table I). The effect o f E2 on D N A was s o m e w h a t depressed in 0.05 M Tris a n d c o m p l e t e l y eliminated by 1 M NaC1 or 1 m M E D T A . The action o f E2 was inhibited a p p r e c i a b l y by 2 m M MgC12. TABLE I EFFECTS OF IONIC CONCENTRATION Reaction mixture containing 3 • 101° molecules of DNA and 1 • 1013 (Expt I and II) or 4" 1012 (Expt III) killing units of E2 in 1 ml was incubated at 30 °C for 30 rain and then diluted 50-fold with Tris-HCl buffer of the same concentration as in the incubation mixture (without including other salt) for assays with spheroplasts. Expt No.
1" II III
Composition of incubation mixture
Yields of phaye from E2-treated DN.4 relative to those of control
0.01 M Tris 0.05 M Tris 0.05 M Tris 0.05 M Tris and 1 M NaCl 0.01 M Tris 0.01 M Tris and 1 mM EDTA 0.01 M Tris and 2 mM MgCl2
0.06 0.31 0.25 0.96 0.27 0.96 0.55
* The number of phage particles from the reaction mixture without E2 in 0.05 M Tris was somewhat larger than that in 0.01 M Tris as described by Guthrie and Sinsheimer t°. DISCUSSION The results presented a b o v e indicate that colicin E2 causes a decrease o f the ability o f ~ X 174 D N A to yield p r o g e n y p h a g e particles in spheroplasts. E n d o nucleolytic scissions o f D N A are n o t required for the inactivation. T h e i n a c t i v a t i o n can be p a r t i a l l y r e m o v e d b y the centrifugation in alkaline sucrose density gradients, b u t n o t b y p h e n o l treatment. F u r t h e r experiments are required to clarify these p h e n o m e n a for u n d e r s t a n d i n g the m o d e o f interaction o f E2 with D N A . T h e i n a c t i v a t i o n o f D N A is p r o h i b i t e d at higher ionic concentrations. Singles t r a n d e d ~ X 174 D N A seems to exist in a highly extended f o r m in 0.0! M Tris (see
308
A. SETO et al.
Fig. 3 of ref. 15), in which the extensive inactivation of DNA is observed. This as well as Ringrose's results 4 that E2 lowers the Tm of D N A suggests that the interaction of E2 is preferential to the coil form of DNA. A large magnitude of temperature dependence as is observed in the inactivation rate of ~X 174 D N A by E2 is also found in the reaction observed by Beppu and Arima 3 for the detachment of D N A from membrane in vitro. The present system may be useful for the detailed analyses on the mode of action of E2 on DNA. ACKNOWLEDGEMENTS
The authors wish to express their thanks to Dr T. Komano for his kind supply of strains of bacteriophages and bacteria and for his invaluable instructions through the work. Thanks are also due to Mr C. Bessyo for his invaluable discussions. The work is partly aided by a grant from the Ministry of Education of Japan. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Nomura, M. (1963) Cold Sprin# Harbor Symp. Quant. Biol. 28, 315-324 Ringrose, P. (1970) Biochim. Biophys. ,4cta 213,320-334 Beppu, T. and Arima, K. (1972) Biochim. Biophys. Acta 262, 453-462 Ringrose, P. (1972) FEBS Lett. 23,241-243 Guthrie, G. D. and Sinsheimer, R. L. (1960) J. Mol. Biol. 2, 297-305 Fiers, W. and Sinsheimer, R. L. (1962) J. Mol. Biol. 5, 424-434 Lindqvist, B. H. and Sinsheimer, R. L. (1967) J. MoL Biol. 28, 87-94 Hutchison, Ill, C. A. and Sinsheimer, R. L. (1966) J. Mol. Biol. 18,429-447 Yokoyama, Y., Komano, T. and Onodera, K. (1971) A#ric. Biol. Chem. (Tokyo) 35, 1353-1362 Guthrie, G. D. and Sinsheimer, R. L. (1963) Biochim. Biophys. Acta 72, 290-297 Herschman, 14. R. and Helinksi, D. R. (1967) J. Biol. Chem. 242, 5360-5368 Maeda, A. and Nomura, M. (1966) J. Bacteriol. 91,685-694 Diirwald, H. and Hoffmann-Berling, 14. (1968) J. MoL BioL 34, 331-346 Levine, A. J. and Sinsheimer, R. L. (1969) J. Mol. Biol. 39, 619-630 Sinsheimer, R. L. (1959) J. Mol. BioL 1, 43-53
I N A C T I V A T I O N OF I N F E C T I O U S D E O X Y R I B O N U C L E I C A C I D OF BACT E R I O P H A G E • X 174 BY C O L I C I N E2
AKIKO SETO*, TAKAO SHINOZAWA and AKIO MAEDA Department of Biophysics, Faculty of Science, Kyoto University, Kyoto (Japan)
(Received June 4th, 1973)
SUMMARY Incubation of single-stranded cPX 174 D N A with colicin E2 resulted in the impairment of the ability of the D N A to produce progeny phage particles in spheroplasts. The inactivation of ~PX 174 D N A by colicin E2 occurred even without chain scissions in DNA. The rate for the inactivation at 40 °C was some 50-fold faster than that at 30 °C. The effect of colicin E2 was reduced with the increase of ionic concentration and completely abolished by 1 M NaC1.
INTRODUCTION Colicin E2 is known to exert various kinds of effects in cells of Escherichia coli. The degradation of D N A is the most prominent phenomenon in these cells 1'2. Beppu and Arima 3 have shown that E2 causes the liberation of D N A from the complexes consisting of D N A and membrane. Ringrose 4 has further reported that E2 lowers the T m of the isolated DNA. For further analyses on the mode of interaction of E2 with DNA, other methods of approach are also helpful. An intact form of ~ X 174 D N A has been known to produce progeny phage particles in spheroplastsS'6; some kind of interaction of D N A with E2 is expected to be detected when cbX 174 D N A treated variously with E2 is put into spheroplasts. It was found that colicin E2 inactivated the infectious D N A of ~ X 174. Some features of interaction of E2 with D N A are also described. MATERIALS AND METHODS g~X 174 D N A E. coli HF4704 (hrc-, thy-)7 (lysis defective) a. Phage particles described by Y o k o y a m a et al. 9 and density gradients, tPX 174 D N A was Guthrie and Sinsheimer 1o.
was a host strain for the growth of ~PX 174 am3 labeled with [aH]thymidine were prepared as further purified by the centrifugation in sucrose extracted by the hot phenol method described by
* Present address: Institute of Life Science, Mitsubishi Kasei, Minami-Oya, Machida-shi, Tokyo (Japan).
306
A. SETO et al.
Colicin E2 Colicin E2 was extracted in 0.5 M neutralized guanidine-hydrochloride from the cells of E. coli W3110 (col E2) induced by mitomycin C, precipitated by 60 % saturation of ammonium sulfate and purified through columns of DEAE-Sephadex and CM-Sephadex according to the procedures described by Herschman and Helsinski 11. The final preparation exhibited only a single band in a disc electrophoresis at pH 4.0 (see ref. I I). The specific killing activity of this preparation was about 2 • 1014 killing units (see ref. 12 as for the definition of killing unit) per mg of protein. Assay of activity of ~ X 174 DNA A colicin E2-resistant strain of E. coli K12 1100 (end-, thi-) 13 was used for the preparation of spheroplasts. The ability of variously treated ~X 174 DNA to produce progeny particles was measured by the procedures described by Guthrie and Sinsheimer 1o. Incubation of spheroplasts was done at 30 °C instead of 35 °C. E. coli HF4714 t4 was used as an indicator strain. RESULTS
The number of phage particles produced from ~X 174 DNA was found to decrease with the increasing amounts of E2 (Fig. 1). The effects of E2 on ~X 174 DNA decreased also with the loss of its bacteriocidal activity. The relation between these two kinds of activity (i.e. the activity on DNA and activity on bacteria) in several prepara1.00 (
0.75 u
O_ I1)
.~0,50
~: 0.25
o
o
20
40 60 80 Killing units of E2 No, of ~X DNA molecules
Fig. 1. Relation between the effect o f E2 on ~ X 1 7 4 D N A a n d the killing activity o f E2. E2 (20 ~g/ml) partially inactivated by heating at 60 °C for 15 m i n ( O ) , at 70 °C for 5 m i n ( A ) o r at 70 °C for 15 m i n ( A ) was incubated with ~ X 1 7 4 D N A at 40 ° e for 40 m i n in 0.01 M T r i s - H C l buffer ( p H 8.1). O - O , results f r o m the reaction mixture containing the various a m o u n t s o f u n h e a t e d E2. T h e a m o u n t o f D N A used was calculated to be 5 • 10 t° molecules/ml f r o m the n u m b e r o f plaquef o r m i n g centers a n d the radioactivity o f original phage preparation.
INACTIVATION OF @X 174 DNA BY COLICIN E2
307
tions o f p a r t i a l l y inactivated E2 coincided well with t h a t n o t e d for native E2. Thus, the effects with the present p r e p a r a t i o n o f E2 a p p e a r to be due to the action o f E2 molecules a n d n o t to possible c o n t a m i n a n t s . A s a m p l e o f ~ X 174 D N A inactivated to 5 ~ by E2 was analyzed in alkaline sucrose density gradients. The s e d i m e n t a t i o n p a t t e r n was a l m o s t identical with t h a t o f the c o n t r o l w i t h o u t E2. M o r e o v e r , the activity was f o u n d to be restored to 60 o f the c o n t r o l after the centrifugation. The b a c k b o n e structure o f the D N A molecules seems to be kept intact even in the situation where their activity is reduced. S h a k i n g with phenol o f E2-treated ~ X 174 D N A , however, was n o t successful in restoring the activity. The rate o f the inactivation reaction increased with the rise o f t e m p e r a t u r e f r o m 30 to 70 °C. The kinetic process o f the reaction can best be expressed as t h a t o f a first-order reaction. T h e rate at 40 °C was some 50-fold faster t h a n t h a t at 30 °C. I n c u b a t i o n o f D N A with E2 was carried o u t u n d e r a variety o f c o n d i t i o n s o f ionic c o m p o s i t i o n (Table I). The effect o f E2 on D N A was s o m e w h a t depressed in 0.05 M Tris a n d c o m p l e t e l y eliminated by 1 M NaC1 or 1 m M E D T A . The action o f E2 was inhibited a p p r e c i a b l y by 2 m M MgC12. TABLE I EFFECTS OF IONIC CONCENTRATION Reaction mixture containing 3 • 101° molecules of DNA and 1 • 1013 (Expt I and II) or 4" 1012 (Expt III) killing units of E2 in 1 ml was incubated at 30 °C for 30 rain and then diluted 50-fold with Tris-HCl buffer of the same concentration as in the incubation mixture (without including other salt) for assays with spheroplasts. Expt No.
1" II III
Composition of incubation mixture
Yields of phaye from E2-treated DN.4 relative to those of control
0.01 M Tris 0.05 M Tris 0.05 M Tris 0.05 M Tris and 1 M NaCl 0.01 M Tris 0.01 M Tris and 1 mM EDTA 0.01 M Tris and 2 mM MgCl2
0.06 0.31 0.25 0.96 0.27 0.96 0.55
* The number of phage particles from the reaction mixture without E2 in 0.05 M Tris was somewhat larger than that in 0.01 M Tris as described by Guthrie and Sinsheimer t°. DISCUSSION The results presented a b o v e indicate that colicin E2 causes a decrease o f the ability o f ~ X 174 D N A to yield p r o g e n y p h a g e particles in spheroplasts. E n d o nucleolytic scissions o f D N A are n o t required for the inactivation. T h e i n a c t i v a t i o n can be p a r t i a l l y r e m o v e d b y the centrifugation in alkaline sucrose density gradients, b u t n o t b y p h e n o l treatment. F u r t h e r experiments are required to clarify these p h e n o m e n a for u n d e r s t a n d i n g the m o d e o f interaction o f E2 with D N A . T h e i n a c t i v a t i o n o f D N A is p r o h i b i t e d at higher ionic concentrations. Singles t r a n d e d ~ X 174 D N A seems to exist in a highly extended f o r m in 0.0! M Tris (see
308
A. SETO et al.
Fig. 3 of ref. 15), in which the extensive inactivation of DNA is observed. This as well as Ringrose's results 4 that E2 lowers the Tm of D N A suggests that the interaction of E2 is preferential to the coil form of DNA. A large magnitude of temperature dependence as is observed in the inactivation rate of ~X 174 D N A by E2 is also found in the reaction observed by Beppu and Arima 3 for the detachment of D N A from membrane in vitro. The present system may be useful for the detailed analyses on the mode of action of E2 on DNA. ACKNOWLEDGEMENTS
The authors wish to express their thanks to Dr T. Komano for his kind supply of strains of bacteriophages and bacteria and for his invaluable instructions through the work. Thanks are also due to Mr C. Bessyo for his invaluable discussions. The work is partly aided by a grant from the Ministry of Education of Japan. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Nomura, M. (1963) Cold Sprin# Harbor Symp. Quant. Biol. 28, 315-324 Ringrose, P. (1970) Biochim. Biophys. ,4cta 213,320-334 Beppu, T. and Arima, K. (1972) Biochim. Biophys. Acta 262, 453-462 Ringrose, P. (1972) FEBS Lett. 23,241-243 Guthrie, G. D. and Sinsheimer, R. L. (1960) J. Mol. Biol. 2, 297-305 Fiers, W. and Sinsheimer, R. L. (1962) J. Mol. Biol. 5, 424-434 Lindqvist, B. H. and Sinsheimer, R. L. (1967) J. MoL Biol. 28, 87-94 Hutchison, Ill, C. A. and Sinsheimer, R. L. (1966) J. Mol. Biol. 18,429-447 Yokoyama, Y., Komano, T. and Onodera, K. (1971) A#ric. Biol. Chem. (Tokyo) 35, 1353-1362 Guthrie, G. D. and Sinsheimer, R. L. (1963) Biochim. Biophys. Acta 72, 290-297 Herschman, 14. R. and Helinksi, D. R. (1967) J. Biol. Chem. 242, 5360-5368 Maeda, A. and Nomura, M. (1966) J. Bacteriol. 91,685-694 Diirwald, H. and Hoffmann-Berling, 14. (1968) J. MoL BioL 34, 331-346 Levine, A. J. and Sinsheimer, R. L. (1969) J. Mol. Biol. 39, 619-630 Sinsheimer, R. L. (1959) J. Mol. BioL 1, 43-53