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Biochimica et Biophysica Acta, 435 (1976) 184--191 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

B B A 98621

I N A C TI VATI ON OF T H E T7 COLIPHAGE BY M O N O F U N C T I O N A L ALKYLATING AGENTS ACTION ON PHAGE A D S O R P T I O N AND INJECTION OF ITS DNA B. KARSKA-WYSOCKI, L. THIBODEAU and W.G. VERLY Department of Biochemistry, University of Montreal (Canada) and Biochemistry, Faculty of Sciences, University of Liege (Belgium)

(Received December 24th, 1975)

Summary Alkylation by ethyl or m e t hyl m e t ha ne s ul fonat e to an e x t e n t t hat inactivates more than 99.5% of T7 coliphages has no effect on phage adsorption on Escherichia coli B cells, but decreases the a m o u n t o f phage DNA injected into the host cells. Depurination interferes with the injection of the phage DNA. Failure to inject the whole phage genome thus appears to be a cause of the immediate as well as of the delayed inactivation of the T7 coliphage treated by m o n o f u n c t i o n a l alkylating agents; the hypothesis that it is the only cause of inactivation, although not very likely, c a n n o t be excluded at the present time. Introduction Loveless and Stock [1] and Papirmeister [2] have studied the inactivation of t h e T2 coliphage by sulfur and nitrogen mustards. With these bifunctional alkylating agents, the adsorption of the phage o n t o the host bacteria proceeds normally, b u t there is some inhibition of the injection o f the phage DNA. Whereas the delayed inactivation of the T7 coliphage treated by m onofunctional alkylating agents is known to be due to DNA depurination [3,4], the causes o f the immediate inactivation are still partially unclear. Verly et al. [5] have shown that, after a 2-h t r e a t m e n t with ethyl methanesulfonate or m et hyl methanesulfonate, the apurinic sites and single-strand breaks in the phage DNA can explain a b o u t half of the immediate inactivation. We wanted to know whether a n o t h e r part might be due to the inability o f the phage to be adsorbed o n t o the host bacteria or to inject its DNA. It was, moreover, i m p o r t a n t to k n o w whether depurination did affect the injection of the phage DNA so t hat depurination and a DNA-injection def e c t would not be i n d e p e n d e n t causes of t h e immediate inactivation of the phage. To elucidate these problems, an investigation was undertaken of the first

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steps of the infection of Escherichia coli B cells by T7 phages labelled in their DNA and treated with ethyl- or methyl methanesulfonate. To appreciate the effect of depurination, the results of incubating the phage, after the alkylation step, on the injection of its DNA into the host bacteria were studied. Materials and Methods T7 coliphage type L [6] was added to E. coli A-1 thy- [5] grown in a medium containing 2 pg/ml of [Me-3H]thymidine (2 pCi/ml) at a multiplicity of infection of 1 phage per 20 bacteria. After a 4-h incubation at 37°C, the phages were purified from the lysate using the polyethylene/dextran sulfate m e t h o d [7]. The total number of phages was estimated from the absorbance at 260 and 280 nm of the suspension with a 10% correction for light scattering [8,9], whereas the number of viable phages was determined by the lysis plaque method [10]; about 85% of the phage particles were infectious. The phage suspension contained, per ml, 2.7 • 10 ~I particles having a radioactivity of 3.04 -106 dpm. For each experiment, a portion of the phage suspension was dialyzed at 4°C against 0.4 M sodium phosphate, pH 7.0 (phosphate buffer). To 1 ml aliquots of the dialyzed phage suspension was added either 1 ml of phosphate buffer (control) or 1 ml phosphate buffer containing alkylating agent (0.4 or 1.48 M ethyl methanesulfonate, or 0.04 M methyl methanesulfonate) (assay) before incubation at 37°C for 2 h with gentle shaking. After cooling to 4°C, the suspension was dialyzed for 15 h against 1 M NaCl, 0.01 M MgC12, 0.01 M Tris • HC1, pH 7.8, with three changes. The determination of the number of surviving phages was performed on a diluted aliquot immediately after the end of the alkylation and verified after the dialysis step (immediate inactivation). To assess the effect of depurination, T7 phages were alkylated with 0.02 M methyl methanesulfonate and, after removal of the alkylating agent by dialysis, the phage suspension was divided in two parts: one was immediately analyzed, whereas the other was incubated for 24 h at 30°C. Determinations of the number of surviving phages were performed on diluted aliquots from the two parts of the phage suspension. Comparison with titers of non-alkylated phage suspension treated in the same way (controls) yielded the immediate and delayed inactivation, respectively. The titer of non-alkylated phage did not change after an incubation. The determination of phage adsorption onto the host bacteria was carried out in the following way. To 7.2 ml E. coli B (5 • 109 or 5 • 10 ~° cells/ml) in Abelson and Thomas medium [11] was added 0.8 ml of the phage suspension ( 2 . 5 . 1 0 1 ° or 2 . 5 . 1 0 ~ particles/ml; alkylated phages or control phages) to have a ratio of one phage per two bacteria. After 8 min at 37°C, chloramphenicol was added to a final concentration of 50 pg/ml [12,13]; an aliquot was taken for an assay of radioactivity and the mixture was then cooled to 4°C and centrifuged for 15 min at 8000 rev./min in a Sorvall RC 2-B centrifuge (rotor SS-34). An aliquot of the supernatant, which contained the non-adsorbed phages, was taken for radioactivity measurement. The sediment, which contained the bacteria and the adsorbed phages, was suspended in 7 ml of 0.1 M Tris • HC1, pH 7.9; an aliquot was used to measure the a m o u n t of radioactivity pres-

186

ent in the bacterial sediment. The percentage of radioactivity in this sediment yields the percentage of phage particles which were adsorbed onto the host bacteria. The following procedure was utilized to determine the fraction of DNA from the adsorbed phages injected into the bacteria. To the Tris suspension of the bacterial sediment prepared above, were added 1.4 ml of 0.17 M EDTA and 0.7 ml of an aqueous lysozyme solution (2 mg/ml) and the mixture incubated at 25°C for 1 h to destroy the bacterial walls [14]. To the lyzed bacteria, MgSO4 was added to a final concentration of 0.02 M, and then pancreatic deoxyribonuclease to a final concentration of 100 pg/ml [15]; after a 30 min incubation at 37°C, the reaction was stopped by addition of an equal volume of 0.3 M sodium citrate/3 M NaC1 [16]. The lysate was cooled to 4°C and the bacterial debris was removed by centrifugation at 8000 rev./min for 15 min in a Sorvall RC 2-B centrifuge (rotor SS-34). The phage particles were subsequently pelleted by centrifugation at 28 000 rev./min for 5 h in the L2-65B Beckman ultracentrifuge (rotor 40). The radioactivity of the supernatant was measured. It is assumed that only the radioactive phage DNA injected into the host bacteria is fragmented by the deoxyribonuclease and that its hydrolysis products are in the supernatant, whereas the radioactive DNA which remained in phage particles is in the sediment of the high-speed centrifugation. Consequently, the percentage of radioactivity in the supernatant of the high-speed centrifugation measures the fraction of the adsorbed phage DNA injected into the host cells. For the radioactivity determinations, the labelled sample in 1 ml of aqueous solution was added to 10 ml of a mixture of 1 part of Triton X-100 and 2 parts of toluene containing 0.4% (w/v) PPO and 0.01% (w/v) dimethyl-POPOP. An TABLE I IMMEDIATE

Conc. (M)

0.20

0.74

INACTIVATION

Expt. No.

1 2 3 4 5 1 2

N a N°

80"10 95'10 95'10 75'10 75'10 4'10 3"10

-3 -3 -3 -3 -3 -3 -3

OF ETHYL METHANESULFONATE-TREATED

T7 PHAGES

A d s o r b e d p h a g e s b (%)

Injected DNA c(%)

Control

Treated

Control R°

Treated R

Reduction d (%)

66 56 57 43 42

64 55 54 48 50

71 55 53 81 84 64 58

62 50 47 68 71 45 45

12 10 10 12 16 29 22

a N is t h e n u m b e r o f surviving p h a g e s in t h e s a m p l e t r e a t e d w i t h t h e a l k y l a t i n g a g e n t , a n d N ° t h e n u m b e r o f surviving p h a g e s in t h e c o n t r o l similarly t r e a t e d b u t w i t h o u t t h e drug. b T h e l a b e l e d T7 p h a g e s are i n c u b a t e d w i t h E . c o l i B ; a f t e r 8 m i n at 3 7 ° C , e h l o r a m p h e n i e o l is a d d e d to a c o n c e n t r a t i o n o f 5 0 pg/ml b e f o r e c e n t r i f u g a t i o n . T h e p e r c e n t a g e o f a d s o r b e d p h a g e s is e q u a l to 1 0 0 t i m e s t h e r a d i o a c t i v i t y carried d o w n w i t h t h e b a c t e r i a in t h e s e d i m e n t d i v i d e d b y t h e t o t a l radioactivity. c T h e b a c t e r i a o f t h e s e d i m e n t c o n t a i n i n g t h e a d s o r b e d p h a g e s are l y z e d w i t h l y s o z y m e / E D T A , and the lysate treated with pancreatic deoxyribonuclease before a low-speed followed by a high-speed c e n t r i f u g a t i o n . T h e p e r c e n t a g e i n j e c t e d D N A is e q u a l t o 1 0 0 t i m e s t h e r a d i o a c t i v i t y o f t h e h y d r o l y z e d D N A f o u n d in t h e s u p e r n a t a n t d i v i d e d b y t h e r a d i o a c t i v i t y o f t h e initial s e d i m e n t . d I f R ° is t h e p e r c e n t a g e D N A r a d i o a c t i v i t y f r o m t h e a d s o r b e d p h a g e s t h a t was i n j e c t e d i n t o t h e b a c t e r i a for t h e c o n t r o l , a n d R t h e c o r r e s p o n d i n g value for t h e d r u g - t r e a t e d , % r e d u c t i o n ~ 1 0 0 X ( R ° - - R ) / R °.

187

T A B L E II IMMEDIATE INACTIVATION Conc. (M)

0.02

Expt. No.

1 2 3 4 5

OF METHYL METHANESULFONATE-TREATED

N a N°

3-10 4-10 5"10 3"10 3-10

-3 r3 -3 -3 -3

T7 P H A G E S

Adsorbed phagesb(%)

Injected DNA c(%)

Control

Treated

Control R°

Treated R

Reduction d (%)

41 63 52 42 31

44 52 53 45 31

74 40 44 42 37

56 31 35 33 27

24 21 21 23 28

a - - d see T a b l e I,

internal [3H]hexadecane standard was used; the radioactivity, measured in a Packard Tri-Carb scintillation counter, is expressed in disintegrations per minute. Results and Discussion

It was essential to verify that the DNA in phage particles having undergone our experimental procedure was not attacked by pancreatic deoxyribonuclease and that these intact phage particles were completely sedimented by the highspeed centrifugation. A suspension (0.2 ml) of labelled T7 phages (14 200 dpm) was submitted to the incubation with lysozyme/EDTA, then to deoxyribonuclease, before the two successive centrifugations described under Materials and Methods; the final supernatant contained an insignificant amount of radioactivity ( 32 dpm). T A B L E Ill RELATIVE IMPORTANCE OF REDUCED DNA INJECTION IN THE IMMEDIATE INACTIVATION O F T7 P H A G E T R E A T E D W I T H E T H Y L A N D M E T H Y L M E T H A N E S U L F O N A T E Expt. No.

Ethyl methanesulfonate

Methyl methanesulfonate

Conc. (M)

Conc. (M)

Z e

L a c k o f D N A injection z'f

%g

1 2 3 4 5

0.20 0.20 0.20 0.20 0.20

2.52 2.35 2.35 2.59 2.59

0.13 0.10 0.10 0.13 0.17

5.2 4,2 4.2 5.0 6.6

1 2

0.74 0.74

5.55 5.80

0.34 0.25

6.1 4.3

0.02 0.02 0.02 0.02 0.02

Z e

5,80 5.55 5.30 5.80 5.80

Lack of DNA injection z'f

%g

0,27 0.23 0.23 0.26 0.33

4.7 4.1 4.3 4.5 5.7

e T h e n u m b e r o f i n a c t i v a t i o n h i t s p e r p h a g e p a r t i c l e , Z, is c a l c u l a t e d w i t h t h e e q u a t i o n : N / N ° = e - Z , w h e r e N is t h e n u m b e r o f s u r v i v i n g p h a g e s in t h e s a m p l e t r e a t e d w i t h t h e a l k y l a t i n g a g e n t , a n d N ° t h e n u m b e r o f s u r v i v i n g P h a g e s in t h e c o n t r o l s i m i l a r l y t r e a t e d b u t w i t h o u t t h e d r u g . f T h e n u m b e r Z ' is d e r i v e d f r o m t h e e q u a t i o n : R / R ° = e - Z ' , w h e r e R is t h e p e r c e n t a g e D N A r a d i o a c t i v i t y i n j e c t e d b y t h e a l k y l a t e d p h a g e s a d s o r b e d on t h e b a c t e r i a , a n d R ° t h e c o r r e s p o n d i n g value for the control similarly treated but without the drug. g% = IOOZ'/Z.

188

We first analyzed the situation at the end of the phage alkylation, i.e. in the conditions in which the immediate inactivation is determined. As recorded in Table I, labelled T7 phages were treated for 2 h at 37°C by 0.20 and 0.74 M ethyl methanesulfonate. The surviving fractions were measured after the dialysis step. With 0.20 M ethyl methanesulfonate, the average inactivation was 92%. Five different experiments were carried out and in each of them the adsorbed phage fractions, for et hyl m e t ha ne s ul f ona t e treated and control, were compared. There was no significant difference, the averages were respectively 54 ± 3 and 53 +- 5%. In contrast, the fraction of DNA from the adsorbed phages injected into bacterial cells was systematically lower with the treated phage than with the control; the percentage reduction has been calculated in each case; it has an average value o f 12%. When t he ethyl methanesulfonate c o n c e n t r a t i o n was raised to 0.74 M, which brought the inactivated fraction to 99.7%, the average reduction of the injected phage DNA increased to 26%. T7 phages were also t re a ted for 2 h at 37°C by 0.02 M m et hyl methanesulfonate (Table II). The average inactivation was 99.6%. There was no effect on phage adsorption, but the t r e a t m e n t p r o duced an average reduction of 23% in the a m o u n t of DNA injected by th e adsorbed phages. This value is quite similar to that f o u n d with 0.74 M ethyl methanesulfonate, which gave approxi m at el y the same level of inactivation. The immediate inactivation of the T7 phage is a one-hit process [4,17]; the average n u m b e r Z of inactivation hits per phage particle due to the action of the m o n o f u n c t i o n a l alkylating agent can be calculated from the surviving fraction: N / N ° = e - Z where N is the n u m b e r of infectious phages immediately after the t r e a t m e n t with the drug, and N ° the num ber of infectious phages in the c o n tr o l similarly treated but w i t h o u t the drug. The results are shown in Table III. It was interesting to check whether the DNA injection defect was also a TABLE IV COMPARISON OF PHAGE ADSORPTION AND DNA INJECTION IMMEDIATELY AFTER METHYL METHANESULFONATE TREATMENT AND FOLLOWING A POST-ALKYLATION INCUBATION Labelled T7 phages are treated for 2 h at 37°C with 0.02 M methyl methanesulfonate and the alkylating agent is removed by dialysis. Aliquots from the same phage suspension are used to determine the surviving fraction and the percentages of phage adsorption and DNA injection either after the alkylation (immediate) or following a 24-h incubation at 30GC (delayed); the bacteria concentration is 4.5. 109 in (A)and 4 . 5 • 1 0 l0 c e l l s ] m l i n ( B ) (1 p h a g e f o r 2 b a c t e r i a ) . F o r ( a ) , ( b ) , ( c ) , a n d ( d ) , s e e T a b l e I ; f o r ( g ) a n d ( f ) , see Table III. Inactivation Conditions

N N°

a

Adsorbed (%)

phages b

Injected DNA c

Control

Treated

Control R ° (%)

69 70

68 69

54 .

14.80

69 63

81 70

47 53

23 20

52 62

0.73 0.97

Z e

6.65

Treated R (%) 43

Reductiond (%)

Z' f

20

0.22

Immediate (A)

1.30" 10 -3

Delayed (A)

3.64" 10 -7

Immediate (B)

3.30"

10 -3

5.71

79 73

72 70

80 87

67 72

16 17

0.17 0.19

Delayed (B)

6.27.

10 -7

14.30

48 47

50 60

85 87

51 53

40 39

0.51 0.49

.

.

.

189

one-hit process and, consequently, a constant part of the immediate inactivation. For that, we used an equation similar to the preceeding one: R / R ° = e -Z', where R is the percentage DNA radioactivity injected by the alkylated phages adsorbed onto bacteria, and R o the corresponding value for the control similarly treated but without the drug. Table III indicates that the ratio Z ' / Z is very constant: 5.1% with 0.20 M ethyl methanesulfonate, 5.2% with 0.74 M ethyl methanesulfonate and 4.7% with 0.02 M methyl methanesulfonate. Using a T7 amber m u t a n t in gene 11, it has been shown (Karska-Wysocki, B., Mamet-Bratley, M.D. and Verly, W.G., unpublished) that alkylation with ethyl- or methyl methanesulfonate decreased the recombination frequency, measured immediately at the end of the drug treatment, with another T7 amber m u t a n t much more when the mutation was in gene 19 than when it was in gene 2 or 4. This result confirmed the conclusion of Pao and Speyer [18] that the T7 genome is injected in the host bacteria beginning with gene 1 and that gene 19 is the last to penetrate. It also meant that, after alkylation, some phages injected only a part of their DNA. The ratio Z'/Z, calculated above, would indicate the fraction of inactivation hits due to the lack of DNA injection if the phage DNA were injected completely or not at all; but, because partial injection of the DNA is also an inactivation hit, we conclude that the part of the immediate inactivation of the T7 phage by ethyl- or methyl methanesulfonate due to the failure of injecting the whole phage genome is greater than 5%. To see whether apurinic sites in the T7 phage DNA might prevent its injection into the host bacteria, T7 phages labelled in their DNA were alkylated for 2 h at 37°C with 0.02 M methyl methanesulfonate, and after removal of the alkylating agent, were incubated for 24 h at 30°C so that the DNA could lose alkylated purines. Table IV shows that the post-treatment incubation significantly reduced the a m o u n t of phage DNA injected into the bacterial cells. The lethal effect of depurination is thus at least partially due to a defect in DNA injection; the aldehyde function of the apurinic sites might lead to crosslinks between the DNA and the coat proteins of the phage particle. We wanted to know what was the importance of the phage genome injection defect in the total inactivating action of the DNA depurination. The delayed inactivation of the T7 phage is also a one-hit process [4,17]; in Table IV, we have calculated the average numbers, Z, of inactivation hits per phage particle at the end of the alkylation (immediate) and after the 24-h post-treatment incubation at 30°C (delayed) from the corresponding surviving fractions N / N °. From the radioactivities R and R o of the DNA injected in the host bacteria, we have also calculated Z' values for the two situations (immediate and delayed). The differences, AZ and AZ~ between what is observed at the end of the alkylation and after the post-treatment incubation, pertain to the delayed inactivation. The average ratio A Z ' / A Z is around 6% *; this ratio would represent the part of the delayed inactivation hits due to a defect in the injection of the phage genome if the phage were always injecting its DNA completely or not at all. We know that it is not true for the phage immediate inactivation and there is no reason to think that it might be different for the delayed inactivation. Be* T h i s v a l u e is q u i t e n e a r t h e m e a n v a l u e ( a r o u n d 5%) c a l c u l a t e d f r o m t h e i m m e d i a t e i n a c t i v a t i o n data (Table liD, following the same reasoning.

190

cause depurination is the only cause of delayed inactivation [ 3,4 ], we conclude that the part of the inactivating action of depurination due to a DNA-injection defect is greater than 6% and, in the absence of pertinent data, one can even not exclude the possibility that depurination inactivates the T7 phage only by stopping the injection of its genome into the host bacteria. A correct estimation of the importance of the DNA-injection defect for the inactivation, either immediate or delayed, of the T7 phage treated by monofunctional alkylating agents, awaits for an experimental determination of the fraction of the treated phages which do not inject their last gene (gene 19). The possibility cannot be discarded that the immediate as well as the delayed inactivations might be uniquely due to the failure of injecting the complete phage genome; this hypothesis has the obvious merit of explaining why no host-cell reactivation was observed with the T7 phage, the inactivation being strictly first order [ 17 ]. Our results clearly show that, in the conditions of immediate inactivation, a dose of ethyl- or methyl methanesulfonate, which inactivates more than 99.5% of the T7 phages, has no influence on their adsorption on E. coli B cells, while it does affect the injection of the phage DNA. Similar results were reported with bifunctional alkylating agents and T2 phages [ 1,2]. There is thus a clear frontier between phage adsorption, which is not affected by alkylating agents, and phage DNA injection which is decreased by the same drugs. Several suggestions can be made to explain why alkylating agents interfere with the penetration of the phage genome into the host bacteria: alkylation of a coat protein might prevent the perforation of the bacterial wall or the contraction of the phage capsid needed for the injection of the DNA; alkylation of a terminal phosphate might be a hinderance to the injection of the DNA; the alkylating agent might induce the formation of covalent linkages between DNA and coat proteins as was shown to occur with nitrous acid [7]. The latter inactivating mechanism is easily understood with bifunctional alkylating agents, and it might depend on the formation of apurinic sites in the case of the monofunctional alkylating agents. Acknowledgements We thank Mrs. Louise Gu~vremont for her skillful assistance. This work was supported by grants from the Medical Research Council of Canada and from the Research Office of the Montreal University. References 1 2 3 4 5 6 7 8 9 10

Loveless, A. a n d S t o c k , J . C . ( 1 9 5 9 ) P r o c . R. S o c . L o n d . Ser. B, 1 5 0 , 4 8 6 - - 4 9 6 P a p i r m e i s t e r , B. ( 1 9 6 1 ) C R D L , S p e c . P u b l . U.S. A r m y C h e m i c a l C e n t e r , M d . , U . S . A . , N o . 2 - 4 5 , 51 L a w l e y , P . D . , L e t h b r i d g e , J . H . , E d w a r d s , P.A. a n d S h o o t e r , K . V . ( 1 9 6 9 ) J. Mol. Biol. 3 9 , 1 8 1 - - 1 9 8 B r a k i e r , L. a n d V e r l y , W.G. ( 1 9 7 0 ) B i o c h i m . B i o p h y s . A c t a 2 1 3 , 2 9 6 - - 3 1 1 V e r l y , W . G . , C r i n e , P., B a n n o n , P. a n d F o r g e t , A. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 9 , 2 0 4 - - 2 1 3 S t u d i e r , W.F. ( 1 9 6 9 ) V i r o l o g y 3 9 , 5 6 2 - - 5 7 4 D u s s a u l t , P., B o u r g a u l t , J . M . a n d V e r l y , W.G. ( 1 9 7 0 ) B i o c h i m . B i o p h y s . A c t a 2 1 3 , 3 1 2 - - 3 1 9 D a v i s o n , P. a n d F r e i f e l d e r , D. ( 1 9 6 2 ) J. Mol. Biol. 5, 6 3 5 - - 6 4 2 D a v i s o n , P. a n d F r e i f e l d e r , D. ( 1 9 6 2 ) J. Mol. Biol. 5, 6 4 3 - - 6 4 9 A d a m s , M.H. ( 1 9 5 9 ) B a c t e r i o p h a g e s , I n t e r s i c e n c e P u b l . I n c . , N e w Y o r k

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11 12 13 14 15

Abelson, J. and Thomas, C.A. (1966) J. Mol. Biol. 18, 262--291 Hausman, R. and Larue, K. (1969) J. Virol. 3, 278--281 Schlegel, R.A. and Thomas, J.R. (1972) J, Mol. Biol. 68, 319--345 Fraser, D. an d Mahler, H,R. (1957) Arch. Biochem. Biophys. 6 9 , 1 6 6 - - 1 7 7 Kaback, H.R. (1971) in Methods in E n z y m o l o g y (Colowick, S.P, and Kaplan, N.O., eds.), Vol, 22, p. 107, Academic Press, New Y o r k 16 Roades, M. and Roades, A. (1972) J. Mol. Biol. 69, 187--200 17 Verly, W.G. and Brakier, L. (1969) Biochim. Biophys. Acta 174, 674--685 18 Pao. C.C. and Speyer, J.F. (1973) J. Virol. 11, 1024--1026