Inactivation of Escherichia coli bacteriophage T2 by poly-l -lysine. I. Nature of the inactivation process

ARCHIVES

OF

BIOCHEMISTRY

Inactivation

AND

BIOPHTSICS

of Escherichia I. Nature

CHANNA

SHALITIN,

From the Department

494-507

99,

(1962)

co/i Bacteriophage of the Inactivation

DAVID

DANON

AND

T2 by Poly-L-lysine. Process’

EPHRAIM

of Biophysics and of Polymer Research, Institute of Science, Rehovoth, Israel Received

August

KATCHALSKI The Weizmann

14, 1962

Interaction of coliphage T2 with poly-L-lysine was investigated. T2 suspended in saline loses its plaque-forming ability in the presence of polylysine. Inactivation is fast, about S&90% of phage being inactivated within 1 min., and practically independent of temperature within the range of Ck37”C. Study of the effect of pH and ionic strength on the extent of phage inactivation revealed that the interaction between phage and basic polypeptide is largely determined by the electrostatic attraction between the negatively charged virus and the positively charged polypeptide. Partial reversion of the inhibitory effect of poly-L-lysine on the growth of T2 could be carried out by incubation with trypsin or with polyaspartic acid. Populations of coliphage T2 which lost their plaque-forming ability by treatment with polylysine, were thus assumed to consist of two fractions: (a) reversibly inactivated phages, i.e., phages whose ability to form plaques is restored by tryptic digestion; and (b) irreversibly inactivated phages, i.e., phages whose ability to form plaques cannot be restored by tryptic digestion. Increase in temperature and time of incubation of T2 with polylysine caused a corresponding increase in the fraction of irreversibly inactivated phage. Electron microscopic examination has shown that inactivation of T2 by polylysine is accompanied by aggregation. Incubation of the phage aggregates with trypsin or with polyaspartic acid resulted in complete resuspension of the agglutinated particles.

seems, therefore, to represent a suitable synthetic model compound for the elucidation of the mechanism of inactivation of viruses by protamines, histones, and natural basic peptides. In the present study the interaction of coliphage T2 with poly-L-lysine was investigated in detail. The virus selected has been thoroughly investigated (10) and is easy to grow and to purify. Only few studies are available on the growth inhibition of other phages by polylysine, protamine, or histone (9, 11-15). These indicate that phage inactivation by the polycationic peptides can be partially reversed by neutralization with anionic polyelectrolytes or by proteolytic digestion. In the present paper it is shown that the ability of T2 to form plaques can be reduced

INTRODUCTION

Inactivation of tobacco mosaic virus and animal viruses by polylysine was investigated by Stahmann and his co-workers (l-6). Electrostatic attraction has been shown to play an important role in determining the interaction between the negatively charged viruses and the synthetic positively charged, basic polypeptide. In its ability to precipitate viruses and cause their inactivation, polylysine resembles basic proteins and some basic natural peptides (7-9). Polylysine 1 The research reported in this document has been sponsored by the Air Force Office of Scientific Research OAR, Grant No. AF-EOAR 61(052)391, through the European Office, Aerospace Research, U. S. Air Force, and by Grant No. A3083 of the National Institutes of Health, U. S. Public Health Service. 494

INACTIVATION

lOOO-fold on admixture with polylysine. The inactivation is rapid and practically temperature independent in the temperature range investigated. The fraction of inactivated phage is dependent on pH and ionic strength. Examination with the electron microscope revealed that inactivation is accompanied by aggregation of phage. The plaque-forming ability of the phage inactivated by polylysine is partially restored on incubation with trypsin. A population of T2 treated with polylysine thus consists of reversibly and irreversibly inactivated phage particles. The fraction of irreversibly inactivated phage was found to increase on increasing the time and temperature of preincubation of phage with polylysine. The properties of the irreversibly inactivated phage T2 are described in part II of this article. Preliminary notes on this work have appeared (16-18). EXPERIMENTAL MATERIALS Poly-n-lysine hydrobromide (19, 20) with an average degree of polymerization n = 120, polyr,-aspartic acid (n = 200) (20, 21), poly-L-glutamic acid (n = 50) (ZO), and poly-nn-alanine (n = 30) (22) were prepared according to the literature. Di-L-lysine. 2HCl was prepared according to Erlanger and Brand (23); tri-r-lysine.3HCl according to Brand et al. (24), and tetra-L-lysine. 4HCl according to Waley and Watson (25). Aqueous stock solutions of the above materials, eontaining approximately 1 mg. peptide/ml., were adjusted to pH 7.0 and kept in the refrigerator. The polyaspartic acid and polyglutamic acid were dissolved in 0.1 N NaOH and subsequently neutralized with hydrochloric acid. A sterile solution of the sodium salt of heparin was purchased from Upjohn Co., Kalamazoo, Michigan. Chondroitin sulfuric acid and calf thymus DNA were obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. Trypsin 2X crystallized (50% MgSO,) was purchased from Worthington Biochemical Corporation, Freehold, Xen Jersey. COLIPHAGE

AND HOST BACTERIA

Escherichia coli B was obtained from the Department of Bacteriology, Hebrew University, Hadassah Medical School, Jerusalem.

OF COLIPHAGE

495

T2

Phage T2hrr was obtained from the Israel Institute for Biological Research in Ness Ziona (Israel). High-titer phage stocks were prepared by growing the phage on bacteria maintained in a synthetic medium according to French (2(i). The bacteriophage was purified by the method of Herriott and Barlow (27), or by differential centrifugation according to Putnam et al. (28). The purified phage was stored in the refrigerator as a suspension (1012-1013 particles/ml.) in saline-magnesium phosphate buffer, 0.15 M in NaCl, 0.001 M in MgS04 , and 0.01 M in NanHPOr , adjusted to pH F.5 with 1 M hydrochloric acid. Phage assay was carried out’ by the agar double layer method according to Adams (29). The osmotic shock-resistant mutant T4B05 (30) was kindly supplied by Dr. S. Brenner, Laboratory of Molecular Biology, Cambridge, England. Phage T4 wild type was generously donated by Dr. E. Kellenberger, Laboratory of Biophysics, University of Geneva, Geneva, Switzerland. The stock suspension of phage in saline-magnesium phosphate buffer was diluted with saline, or buffered saline, to a concentration of 104-log particles/ml. The required amount of peptide inhibitor was added, and the mixture was incubated as described in Results. The incubation mixture was then diluted to a concentration of about 103 viable particles/ml., and the active phages in 0.1 ml. were assayed by plating on an agar double layer (29). The data given in the results are average values of assays carried out on two to four agar plates. Control experiments revealed that the maximal amount of polylysine found in the phage aliquots assayed (1 pg./2.5 ml.) did not affect the growth of E. coli cells suspended in semisolid agar. ELECTRON

MICROSCOPY

Phage samples were prepared for electron microscopy by the agar filt.ration method of Kellenberger and Kellenberger (31) or by suspending the phage in volatile salt solution as described by Backus and Williams (32). An aqueous solution of 0.2% poly-nr,-alanine was used as detergent in the first method. In t.he method using volatile salt, drops of the phage suspension were deposited on electron microscopic grids carrying a Formvar film. Fixation was carried out in vapor of 40% formaline for 10 min. at room temperature. Preparations were subsequently air-dried and shadow-cast by Cr-Ni alloy at a height to shadow ratio of 1:4. RCA EMU 2A electron microscope was used. Dow latex sphere 0.340 rnr in diameter, titrated to a concentration of 8 X 10*“/ml. were used for determining phage concentrations.

496

SHALITIN,

I 0

20 (rnj4g.I

DANON

I

AND

KATCHALSKI

I

I

-+I

40 50 ml.)

I

25

I 100

( pg./ml.) Poly-L-

lysine

FIG. 1. Survival of coliphage T2 at various poly-L-lysine concentrations. The phage (lo6 particles/ml.) was incubated with polylysine in 0.14 M NaCl, pH 6.5, for 20 min. at 37”C., and the number of survivors was assayed by plating on agar double layer. RESULTS

CONCENTRATIONS OF POLYLYSINE INHIBITING GROWTH OF PHAGE T2

Preliminary studies have shown that bacteriophage T2 suspended in saline loses its plaque-forming ability in the presence of polylysine. To evaluate the minimal concentration of polylysine required to inhibit phage’ growth in unbuffered saline (initial concentration of lo6 particles/ml.), the number of survivors after incubation for 20 min. at 37°C. with the basic polypeptide at different concentrations, was determined. A lO,OOO-fold reduction in the concentration of plaque-forming particles was obtained already at a polylysine concentration of 0.01 pg./ml. (Fig. 1). Higher polylysine concentrations, up to 100 pg./ml., did not augment markedly the percentage of inactivation. In this connection it is pertinent to note that in phosphate-buffered saline, pH 6.5, relatively high concentrations of polylysine are required to inactivate the phage. Thus even at a concentration of 2 pg. polypeptide/ml. only a lOOO-fold reduction in the titer of plaqueforming particles was obtained within 20 min. (see Fig. 2, third curve from top). The effect of polylysine, in a concentration range of O-10 pg./ml., on the number of survivors using different initial concentrations

of phage in phosphate-buffered saline, is presented in Fig. 2. The data given show that in the range of phage concentration investigated (lOelOu particles/ml.) polylysine at a concentration of 2 pg./ml. suffices to reduce the infective titer lOO- to lOOO-fold. In the experiments in which a high initial phage concentration was used (1Og-1O11particles/ml.) the fraction of survivors remaining after treatment with 2.0-10.0 pg. polylysine/ml. was about ten times greater than that found in the experiments in which low initial phage titers (lOplO particles/ml.) were employed. Because of the relatively great number of survivors obtained in experiments in which a high initial phage concentration was used, appropriate dilutions in 0.14 M NaCl, pH 7.0, were made before assaying the remaining infective particles. This dilution did not cause any significant reactivation of the inactivated phage particles. EFFECT OF TIME OF INCUBATION ON INACTIVATION OF T2 BY POLY-L-LYSINE

Phage suspended in saline (lo4 particles/ ml.) was incubated with the peptide at 37”C., and the number of survivors was determined by direct plating at the time intervals specified (Fig. 3). At the three polylysine concentrations employed (0.1, 1.0, and 10.0

INACTIVATION

pg./ml.), approximately 99 % of the initial plaque-forming units were inactivated within 10 min. of incubation. The percentage of survivors decreased to a value of 0.01% on further incubation for 30 min. The susceptibility to polylysine of a progeny derived from a single surviving phage particle was tested as follows: A single plaque formed by a phage particle which survived polylysine treatment (incubation with polypeptide, 1 pg./ml. buffered saline, pH 6.5, for 20 min. at 37”C.), was subcultured on E. coli B grown for 3 hr., at 37°C. in 0.8 % Difco nutrient broth, 0.5 % in NaCl. The lysate obtained after incubation at 37°C. overnight was centrifuged at 3500 X g for 10 min. to remove cell debris. The supernatant was diluted in phosphate-buffered saline, pH 6.5, to a final concentration of lo4 infective particles/ml., and the rate of inactivation by polylysine (1 pg. or 10 pg./ml.) at 37°C. was determined. Inactivation curves similar to those given in Fig. 3 were obtained. When the newly surviving phage particles were subcultured on E. coli B as above, the progeny obtained showed a susceptibility to polylysine similar to that of the original phage stock. EFFECT OF PH AND IONIC STRENGTH ON THE INACTIVATION

OF COLIPHAGE

10’2 ,

497

T2 I

I

I 1

10’0

\

i--O-c-o

A

106 b

\

al g

B

“\,O-O-O-O

106

0) s u 0 a IO4

i

o--o-o-o-o

c

o-o-%5-+

D

102 \

L

0

I

I

I

2

6

IO

Poly -L-

lysine

( pg./ml.)

FIG. 2. Survival of T2 of various initial titers in the presence of polylysine. The phage was incubated with polylysine, at the concentrations specified, in phosphate-buffered saline, pH 6.5, for 20 min. at 37”C., and t’he number of survivors was assayed by plating on agar double layer. The initial phage concentrations in the experiments represented by curves A, R, C, and D were 5 X 10’1, 109, 107, and 104/ml., respectively.

The data given in Fig. 4 show that in a medium of constant ionic strength (r/2 = 0.14) the number of survivors decreases markedly on increasing the pH from 3.9 to 5.0. Under the experimental conditions used, could not, therefore, be inhibited by polyapproximately 50% of infective phage sur- lysine even at as high a concentration as 1 vived at pH 4.2 after 6 min. of incubation at mg. peptide/ml. 25°C. EFFECT OF LYSINE OLIGOPEPTIDES The effect of ionic strength (NaCl, 1’/2 = ON GROWTH OF PHAGE 0.1-0.7) on the inactivation of the phage by To clarify whether the inhibitory effect of polylysine at pH 7.4 and 25°C. is given in the upper curve of Fig. 4. The increase in polylysine described above is due to its ionic strength in this range markedly de- macromolecular, polycationic character, the creases the growth inhibitory effect of the effect of L-lysine, and of L-lysine oligopepbasic peptide. It is noteworthy that phage tides on the growth of the coliphage was (lo3 particles/ml.) suspended in 1.5 M NaCl, studied. Neither L-lysine nor di-, tri-, tetrapH 7.0, could be inactivated to an extent of or pentalysine, at a final concentration of 10 95% only, at a polylysine concentration of pg./ml., did decrease the number of viable 500 pg./ml. Polylysine precipitates in the phage (lo3 plaque-forming units/ml. of phospresence of a relatively high phosphate con- phate-buffered saline, pH 6.5) on incubation centration. Phage particles, suspended in for 20 min. at 37°C. Polylysine, under simi0.067 M (M/15) buffer. I nH \, ,&nhosnhate I I~- 7.4. -7 lar conditions, caused a complete inactiva-

498

SHALITIN,

DANON

AND

KATCHALSKI

20 0 Minutes

FIG. 3. Effect

of time of incubation on inactivation of T2 by polylysine. Coliphage T2 (lo4 particles/ml. saline, pH 6.5) was incubated at 37°C. with polylysine at a concentration of: 0.1 pg./ml. (+); 1 pg./ml. (a); or 10 pg./ml. (0). The fraction of survivors was determined by plating at the times specified. The data represented by (A) give the results recorded at 0°C. using a polylysine concentration of 10 pg./ml.

i ii

s

-:

.

. I^^ L2ll-A *t-f *2 uu

3

/

-3

0

ionic strenath .4

.5

InIn

3

4

5

6

11

.6

-74

I

7

8

PH FIG. 4. Per cent survivors

of T2 (initial concentration lo3 particles/ml.) after incubation with polylysine (1 rg./ml.) for 6 min. at 25°C. Upper figure gives per cent survivors as a function of ionic strength (NaCl), at pH 7.0. Lower figure gives per cent of survivors as a function of pH at an ionic strength (NaCl) of 0.14.

tion of phage already at a concentration 0.5 pg./ml.

of

EFFECT OF ACIDIC AND NEUTRAL POLYAMINO ACIDS ON GROWTH OF PHAGE

Poly-r,-aspartic acid (n = 200) and poly-Lglutamic acid (n = 50) at concentrations of

100 pg./ml. as well as poly-nn-alanine (n = 30) at a concentration of 2 mg./ml., did not have any growth inhibitory effect on T2 under the experimental conditions specified above (lo3 plaque-forming particles/ml. of phosphate-buffered saline, pH 6.5. In-

ISACTIVATION

cubation 37°C.).

with

polypeptide

for 20 min. at

REVERSIONOF THE INHIBITORY EFFECT OF POLYLYSINEBY POLYASPARTIC ACID OR BY TRYPSIN Poly-n-lysine is known to form multivalent salts with acidic polyelectrolytes. Furthermore, the negatively charged poly-n-aspartic acid was shown to reverse the inhibitory effect of the basic polypeptide on the growth of bacteria (33). It was, therefore, of interest to investigate whether poly-L-aspartic acid can also reverse the inhibitory effect of polylysine on the growth of coliphage. Actually when the phage (lo3 particles/ml. of Veronalbuffered saline, pH 7.2) was preincubated with polylysine (1 pg./ml.) for l-2 min., at 25°C. (a procedure shown to inactivate approximately 90 % of the phage population), and the mixture was then incubated with poly-L-aspartic acid (n = 200, final concentration 100 pg./ml.) for 60 min. at 25”C., the inhibitory effect of the basic polypeptide was completely reversed. In this connection it is pertinent to note that other acidic polyelectrolytes, such as heparin, chondroitin sulfuric acid, and calf thymus DNA, did not reverse significantly the inhibitory effect of polylysine on the growth of the coliphage when substituted for polyaspartic acid in the above experiments. Poly-n-lysine is readily digested by trypsin to yield low lysine oligopeptides (19, 34, 35). The latter are devoid of antiviral activity. A reversion of the inhibitory effect of poly-Llysine on the growth of coliphage by incubation with trypsin could thus be carried out. The phage (lo3 particles/ml. of Veronalbuffered saline, pH 7.2) was preincubated with polylysine (1 pg./ml.) for l-2 min. at 25°C. (a procedure shown to inactivate approximately 90 % of the phage population), and the mixture was incubated with trypsin (final concentration 50 pg./ml.) for 30 min. at 25’C. The inhibitory effect of the basic polypeptide was reversed, and the number of revived plaque-forming particles was found to be practically equal to that of the original phage suspension. The phage inactivation by polylysine was not affected by egg albumin (final concentration 1 mg./ml.) or by heat-denatured trypsin (36) (final concentration 1 mg./ml.).

OF COLIPHAGE

T2

499

IRREVERSIBLEINMZTI~ATIONOF T2 BY POLYLYSINE In the previous section it was shown that the inhibitory effect of polylysine can be reversed when the phage is incubated with the basic peptide for l-2 min. Prolongation of the incubation period led to only a partial restoration of phage infectivity, after treatment of the phage-polylysine mixtures with trypsin or with polyaspartic acid. A systematic study of the effect of time and temperature of incubation of T2 with polylysine on the extent of irreversible phage inactivation was thus undertaken. The data presented in Fig. 5 give the fraction of T2 survivors, after treatment with trypsin, of T2-polylysine mixtures preincubated at the temperatures and time intervals specified. The curves given for 1, 19, and 34°C. show that the fraction of the population revived at each of the temperatures tested decreased markedly with time of incubation. The fraction of survivors at 19 and 34°C. was considerably smaller than the corresponding values at 1°C. Survival curves similar to those of Fig. 5 were obtained when the tryptic digestion of the poly-L-lysine was substituted by polyelectrolyte neutralization with polyaspartic acid. In the latter case the percentage of survivors was usually somewhat lower than that in the corresponding trypsin experiments. The results described above show that a population of coliphage T2 which lost its plaque-forming ability by treatment with polylysine consists of two fractions: (a) phages whose ability to form plaques is restored by tryptic digestion under the conditions specified (see legend for Fig. 5) and (6) phages whose ability to form plaques cannot be restored by the above enzymic treatment. Phages of the first fraction will be denoted in the following as reversibly inactivated phages, while those of the second fraction as irreversibly inactivated phages. The upper curve of Fig. 6 gives the fraction of irreversibly inactivated phage as a function of initial polylysine concentration. For comparison, the survival curve in the presence of polylysine (fraction of plaque-forming particles of the T2-polylysine mixture which has not been treated with trypsin) is also included (lower curve). The percentage

500

SHALITIN,

m $ .> L 3 5

DANON

AND

KATCHALSKI

60

40

60

80

minutes FIG. 5. Survival curve of T2 (103/ml.) on incubation with polylysine (1 pg./ml.). All experiments were performed in salineMg-Verona1 buffer, pH 7.2, ionic strength 0.18. The abscissa gives the time of incubation with the peptide. After the time given, trypsin (50 rg./ml.) was added, the mixture digested for 30 min. at 25”C., and the number of survivors counted on agar plates.

of irreversibly inactivated phage was found to increase markedly under the experimental conditions used, with a corresponding increase in the concentration of polylysine. Furthermore, the amount of polylysine required to irreversibly inactivate a given percentage of phage population is markedly greater than that required to inhibit growth of the same percentage of phage in a T2polylysine mixture (lower curve). The extent of irreversible inactivation was found to be dependent also on initial phage concentration. Thus, approximately 90 % of phages were irreversibly inactivated when a mixture containing lo3 phages and 2 kg. polylysine/ml. 1% ammonium acetate solution, pH 7.4, was incubated for 60 min. at 37”C., while only 10% of phages were irreversibly inactivated when the concentration of phages in the above mixture was increased to lo9 particles/ml. SENSITIVITY TO POLYLYSINE OF FASTAND SLOW-SEDIMENTING FORMS OF T2

Cummings and Kozloff (37) reported recently that the sedimentation rates of T2, in 0.1 M NaCl, pH 6.3, at 8-19”C., and at

3240°C. were SZO,~= 1030 S and spo,W= 704-747 S, respectively. In the temperature range of 19-32°C. the presence of both the slow- and the fast-sedimenting forms was recorded. The head of the slow-sedimenting form was found to be approximately 15% longer than that of the fast-sedimenting form. Since it was observed that T2 is irreversibly inactivated at 34°C. under the conditions specified in the legend for Fig. 5, to a considerably greater extent than at l”C., it might have been assumed that the slow-sedimenting form of T2 is more susceptible to irreversible inactivation by polylysine than the fast-sedimenting form. Actually, when a slow-sedimenting form (~20,~ = 763 S) was prepared according to Cummings and Kozloff (37) (incubation of 2 X 1012phage particles/ml. 0.1 M NaCl, pH 6.3, for 1 hr. at 37”C.), the phage suspension diluted in 0.1 M NaCl, pH 6.3, to lOlo T2 particles/ml., and further incubated with polylysine (100 pg./ ml.) for 30 min. at 37”C., 90% of the phage particle were inactivated irreversibly. On the other hand, when a fast-sedimenting form (s2o.w.= 1027 S) was prepared (37) (incubation of 2 X lOI particles/ml. 0.1 M NaCl, pH 6.3, for 1 hr. at 10°C.) and incubated

INACTIVATION

OF COLIPHAGE

1

501

T2

strains to irreversible inactivation by polylysine. The data presented in Fig. 7 show clearly that the shock-resistant strain undergoes, both at 37°C. and at O”C., irreversible inactivation by polylysine considerably more readily than the nonshock-resistant wild strain. A relatively high permeability of the phage protein coat seems to facilitate irreversible inactivation by the basic polypeptide. ELECTRON MICROSCOPY

The findings of Burger and Stahmann (2) that tobacco mosaic virus is agglutinated by polylysine suggested that the inactivation of

I

0.0 -0.2

A-A-A-A--h-A

vv

-0.4

0

20 poly-L-

40 lysine

( pg./ml.

60 1

FIN. 6. T2 survivors after treatment with poly lysine at different concentrations. The phage suspensions (6.8 X lO*O particles/ml.) in salineMg-Verona1 buffer, pH 7.2, were incubated for 60 min. at 37°C. with polylysine at the concentrations specified. Trypsin was added (50 pg./ml.), the mixtures were further incubated for 30 min. at 25”C., and the number of survivors was counted on agar plates after the appropriate dilution in the above saline buffer (upper curve). For comparison the percentage of survivors was determined, as above, in the corresponding phagepolylysine mixtures which were not treated with trypsin (lower curve).

with polylysine, under the conditions specified above, only 8 % of the plaque-forming particles were inactivated ireversibly. COMPARISON OF THE IRREVERSIBLE INACTIVATION OF T4BOc WITH THAT OF T4r+

The osmotic shock-resistant mutant T4B05 , isolated by Brcnner and Barnett (30), possesses high permeability to various ions as compared with T4r+. It was, therefore, of interest to compare the susceptibility of both

ul

z .->

60

‘A

40 ij0I nl

minutes FIG. 7. Irreversible inactivation of the osmotic shock-resistant mutant T4BOs (circles) as compared with that of T4 wild type (triangles). All phage suspensions contained an initial concentration of 5 X IO3 plaque-forming particles/ml. saline, pH 7.0, and were preincubated at t,he temperature of inactivation (0” or 37°C.) for 2-3 hr. Polylysine was then added up to a final concentration of 10 pg./ml., and incubation was continued at 0°C. (T4B05 , -O-O-; T4r+, - A - A -) or at 37°C. (T4BOs, -a-@-; T4r+, -A-A-) for the time interval given. Trypsin was finally added (56 rg./ml.), and t.he mixtures were further incttbated for 30 min. at 25°C. Aliquots were withdrawn, and the number of survivors was assayed on agar double layer.

SHALITIN,

DASON

AND

KATCHALSKI

FIN. 8. (a) Coliphage T2 fixed with formalin vapors, air-dried and shadow-cast by Crfor electron microscope was made from a suspension of 2 X 10” Ni alloy. The preparation in 1’;; ammonium acet,ate, pH 7.3. Magnification X 50,000. Pa rticles/ml. (b) Phage aggregation induced by polylysinc. A T2 suspension similar to that, described (final concentration 2 pg./ml.) for 5 in t,he legend to Fig. 8n was izlcubat.ed wit,h polylysine and then prepared for electron microscopy as above. Magnificami n. at room tempcratrlre tia m X50,000.

INACTIVATION

OF COLIPHAGE

T2

503

FIG. 8--Continued (c) Nonaggregated phage obtained by redispersion of the polylysine-induced aggregates (Fig. 8b). Redispersion was achieved by incubating a suspension of the aggregates with kypsin (final concentration 50 rg./ml.) for 15 min. at 25°C. The redispersed phage particles were spun down (20,000 X y, 90 min.) and the pellet was resuspended in a 170 ammonium acet,at,e solution, pH 7.3. The resulting suspension served for electron microscopic preparations using the technique given for Figs. 8a and b. Magnification X50,000.

coliphage by the basic peptide is accompanied by aggregation. A comparison of the electron micrographs a and b of Pig. 8 demonstrates that incubation of phage (2 X log particles/ml.) with polylysine (2 pg./ml.) in 1% aqueous ammonium acetate, pH 7.3, for 5 min. at 25”, led to a pronounced agglutination. Incubation of the phage aggregates with trypsin (50 pg./ml.) rcsultcd in complete resuspension of the agglutinatrd particles (Fig. 8~). The number cf resuspended particles counted by the procedure of Kellenberger (38), using Dow latex spheres as reference, was found approximately equal to that of T2 in the original suspension. Electron micrographs similar to those given in I’ig. 8c were obtained when phagc aggregates were incubated with poly-L-aspartir acid (100 pg./ml.) for 1 hr. at room temperature. Counting of the resuspended particles by the above method was not possible in this csase,as the reference Dow latex

spheres agglutinated in the presence of polyaspartic acid. Since phage is not inactivated by polylysine at pH 3.8 (see Pig. 4), aggregation under these conditions was examined. A phage suspension containing log particles/ml. of 1% aqueous ammonium acetate adjusted to pH 3.8 with acetic acid was incubated with polylysine (final concentration 2 pg./ml.) for 5 min. at 25”C., and an aliyuot was withdrawn for electron microscopic observation. iYo agglutination of phage could be detected. In parallel experiments performed at pH 4.2, a pH at which partial inactivation occurs (see Fig. 4), aggregation of only a part of the phage particles was found in the corrcsponding electron micrographs. To determine whether the agglutination of the phage by polylysine is caused by the interaction of the peptidc with tho outer protein coat, the effect of polylysinc on phagc ghosts was studied. Ghosts of coliphage were

504

RHALITIN,

DANON

obtained from intact phage by osmotic shock according to Anderson (39). Adsorbed DNA was removed with deoxyribonuclease (40). A suspension of the phage ghosts (lOLo particles/ml.) in phosphate-buffered saline, pH 6.5, was incubated with polylysine (final concentration 20 pg./ml.) for 5-10 min., at room temperature. An aliquot of 0.1 ml. was then withdrawn and prepared, by the agar filtration method, for electron microscopic examination. The ghosts were aggregated in a manner similar to the aggregation of intact phage when incubated with polylysine. Phage particles devoid of tail tip fibers (41) were agglutinated by polylysine under the conditions employed in the agglutination experiments of intact phage. The loss of ability of phage to form plaques was the criterion for phage inactivation used in the present work. By means of this assay it was demonstrated that poly-L-lysine (n = 120) inactivates coliphage T2, suspended in saline (lo6 particles/ml.), at a concentration as low as 0.02 pg./ml. Inactivation is fast, about 80-90% of phage being inactivated within 1 min., and practically independent of temperature within the range of 0-37°C. The data given in Fig. 3 show that approximately 90% of the phage are inactivated by polylysine within l-2 min. Purther inactivation proceeds at a slower rate, 0.1% of survivors being left after an incubation period of lo-20 min. The course of phage inactivation thus indicates that the T2 population is heterogeneous in respect to its susceptibility to the basic polypeptide. The fraction of survivors, after lo-20 min. in phosphate-buffered saline, pH 6.5, was practically independent of initial phage concentration (lOelO” particles/ml.) or of polylysine concentration (2-100 pg./ml.) (see Figs. 1 and 2). The susceptibility to polylysine of a progeny derived from a single surviving phage particle was similar to that of the original phage stock. It might therefore be inferred that the surviving phages are phenotypic variants and not resistant mutants. A similar conclusion was drawn by Gretchmann et al. (1 I, 12) caoncerning the population heterogeneity of Tl and T5 from

AND

KATCHALSKI

their study of the susceptibility of these phages to protamine. At neutral pH, coliphage T2 bears an over-all negative electric net charge (42) ; polylysine, on the other hand, is highly positively charged (43). It is plausible to assume that the interaction between phage and basic polypeptide leading to the fast inactivation of the former, is largely determined by the electrostatic attraction between the oppositely electrically charged virus and polymer. Our findings concerning the dependence of phage inactivation on ionic strength, pH, and electric charge of the polypeptide used further support the above suggestion. An increase in ionic strength from r/2 = 0.1 to 0.7, at pH 7.0, caused a marked decrease in the fraction of phage inactivated by polylysine. This effect is to be expected since the electrostatic attraction forces between the phage and the basic peptide are markedly diminished as a result of the electrostatic shielding effect of the supporting electrolyte (44). At ionic strengths exceeding I’/2 = 0.6 no interaction between phage and polylysine seems to occur, and the phage retains full infectivity (Fig. 4). In a suspension of phage in 1.5 M sodium chloride, 90% inactivation could be attained only at a polylysine concentration of 500 pg./ml. The inactivation of coliphage T2 by polylysine has been mentioned recently by Mora and Young (45). In accord with our results, they found that the inactivation depends on ionic strength, the extent of inactivation increasing on decreasing the salt concentration of the medium. The unexpected reversal of the above trend at concentrations of sodium chloride lower than 0.12 M could not be confirmed by us. As far as we are aware, no study is available on the electrophoretic mobility of coliphage T2 as a function of pH [for the electrophoretic mobility of coliphage T6 see Putnam et al. (as)]. Phage inactivation at different pH values cannot, therefore, be correlated with phage net charge. However, as polylysine remains fully ionized in the pH range investigated (Fig. 4), (pH 3.8-7.4), it is possible that the curve describing the percentage of phage survivors as a function of pH reflects net charge variation of the virus

INACTIVATION

OF COLIPHAGE

T2

505

and agglutination under the experimental conditions used. The studied the inactivation marked increase in the percentage of sur- of coliphages of the T series by protamine. Poly-L-lysine agglutinated phage could be vivors from 1 to 100 % on decreasing the pH resuspended either by treatment with polyfrom 4.8 to 3.8, at I’/2 = 0.14 (Fig. 4), would thus suggest that functional groups with an aspartic acid or by incubation with trypsin. The macroanionic polyaspartic acid forms a apparent dissociation constant of about pK = 4.2 (possibly carboxyl groups) par- multivalent salt with the macrocationic polylysine (46) and neutralizes its inhibitory efticipate in the formation of the negative electric charge of the protein coat of the fect. Trypsin digests poly-L-lysine to low molecular weight lysine oligopeptides (19, phage, which plays a major role in the interaction between the polycationic peptide and 34,35), which under the experimental conditions used do not inactivate coliphage T2. the virus. The resuspended phage particles appeared The importance of the polycationic nature morphologically intact when observed with of polylysine in determining phage inactivathe aid of the electron microscope (Fig. 8~). tion is further emphasized by the finding that the polyanionic polyaspartic acid, as well as Biological assay revealed, however, that a the neutral poly-nn-alanine have no inhibimarked fraction of the resuspended particles tory effect. The absence of growth inhibitory lost their infectivity, the fraction of inacaction of the low lysine oligopeptides, at the tivated phage being determined by the temconcentrations tested, indicates that the perature and time of incubation of the polyelectrolytic characteristics of the lysine phage-polylysine aggregates (Fig. 5). In this peptides become apparent only at relatively connection it is pertinent to recall that high chain lengths. Rhizobium phage preparations which were The neutralization of the over-all negative aggregated and inactivated by clupein could net electric charge of the coliphage by the be resuspended and their activity partially polycationic polylysine decreases electrorestored on incubation with trypsin or with static repulsion between phage particles. The chymotrypsin (14). The plaque-forming abilphage suspension is thus rendered less stable ity of phage T5, inactivated by protamine, and aggregation sets in. The aggregates ob- could also be partially restored on incubation served in the electron microscope contain with trypsin (15). many phage particles “glued” together by The finding that part of the T2 population the multivalent polylysine molecules. No which has been inactivated by polylysine is measurements were carried out on the av- revived after digestion of the polypeptide erage size of the phage aggregates formed or with trypsin shows that the aggregates of inthe rate of aggregation. It is plausible to activated phage consist of reversibly and irassume, however, that an increase in the reversibly inactivated particles. The reinitial concentration in phage would cause a versibly inactivated phage particles are those corresponding increase in the rate of phage whose ability to form plaques is restored by agglutination. Actually, it was observed that tryptic digestion, while the irreversibly inacthe rate of inactivation of T2 in the concen- tivated phage particles are those whose abiltration range of 10”lo6 particles/ml. was ity to form plaques cannot be restored by the practically independent of the initial phage above enzymic treatment. Since the fracconcentration. It seems, therefore, that tion of irreversibly inactivated phage T2 phage inactivation is not caused by aggrega- increases with temperature and time of intion but proceeds by a different mechanism, cubation of phage with polylysine, it might which most likely precedes aggregation. be concluded that while the reversible inacThus the interaction of each of the phage tivation reaction is fast and electrostatic in particles with polylysine molecules might al- nature, the irreversible inactivation reaction ter its over-all net charge, as well as its is a more complicated reaction of still unbiological properties, such as specific adsorp- known nature. The properties of irreversibly tion to the host cell. A similar conclusion was inactivated T2 are described in Part II of reached by Gretchmann et al. (11) who this work.

506

SHALITIN,

DANON

The observation that the shock-resistant strain T4BOb is irreversibly inactivated by polylysine considerably faster than the wild strain T4r+ suggests that the process of irreversible inactivation should be correlated with the permeability characteristics of the phage protein coat. This conclusion is further supported by the observation that the slowsedimenting form of T2, assumed to possess a more permeable protein head than that of the fast-sedimenting form (37, 47), is readily inactivated irreversibly by polylysine, while the fast-sedimenting form is practically resistant to irreversible inactivation. Furthermore, preliminary ultracentrifugation studies have indicated that the fraction of reversibly inactivated phage (obtained on incubation of T2 for 60 min. at the temperature recorded in Fig. 5) corresponded to the percentage of the fast-sedimenting form in the incubation mixture. The fraction of the irreversibly inactivated phage, obtained under similar conditions, corresponded to the percentage of the slow-sedimenting form. Polylysine seems to prevent the transition of the slow-sedimenting form into the fast sedimenting one and vice versa. Such a transition has been found in the absence of the basic peptide (37, 47). If the assumption that the phage protein coat is permeable to polylysine is accepted, it is possible that the irreversible inactivation of the phage is caused by an interaction of the polycationic polylysine, which had penetrated the protein layer of the phage, with the polyanionic DNA, present in the phage head. The polylysine which had combined with phage DNA would be resistant to digestion by trypsin, since the enzyme cannot pass the protein membrane. Tryptic digestion can thus remove only polylysine attached to the outer surface of the phage but not the polypeptide molecules which have reacted with the inner components of the virus. This hypothesis might explain the nature of the inactivation process and elucidate the mode of action of trypsin on the polysine phage complex. It is important to emphasize that phage particles inactivated by polylysine and revived by incubation with trypsin are not resistant mutants but phenotypic variants. Thus, progeny derived from reversibly inac-

AND

KATCHALSKI

tivated phage could be inactivated irreversibly by polylysine, under the conditions specified in Fig. 5, similarly to the original phage stock. In view of the above hypothesis, it might be predicted that the progeny derived from each of the surviving phage particles would show a permeability distribution toward polylysine similar to that of the original phage stock. It is of interest that the interaction of phage with polylysine resembles its interaction with antiserum. A reversible and irreversible inactivation of T2 by antiserum, similar to that by polylysine, has been described recently (48). Phage neutralized with antiserum could be partially reactivated by incubation with papain (49), a procedure analogous to the reactivation of polylysineinactivated phage by incubation with trypsin. The findings reported here enabled us to obtain phage T2 preparations in which the majority of particles have been inactivated irreversibly by polylysine. Since the irreversible inactivation proceeds under mild conditions, and the inactivated particles appear morphologically intact, when observed with the aid of the electron microscope, it was of interest to investigate the properties of the above phage particles which have lost their ability to form plaques. The adsorption of coliphage T2, inactivated irreversibly by polylysine, to E. coli B, its ability to inject DNA into host cells, and its host killing power, are described in the second part of this work. REFERENCES 1. STAHMANN, M. A., GRAF, L. H., PATTERSON E.L., WALKER J. C., AND WATSON II. W., J. Biol. Chem. 189, 45 (1951). 2. BCRGER, W. C., AND STAHMANN, M. A., J. Biol. Chem. 193, 13 (1951). 3. RUBINI, J. R., RASMUSSEN, A. F., JR., AND STAHMANN, M. A., Proc. Sot. Exptl. Biol. Med. 76, 662 (1951). 4. GREEN, M., STAHMANN, M. A., AND RASMUSSEN, A. F., JR., Proc. Sot. Exptl. Biol. Med. 83, 641 (1953). 5. GREEN,M., AND STAHMANN, M. A., Proc.Soc. Exptl. Biol. Med. 83, 852 (1953). 6. GREEN, M., AND STAHMANN, M. A., Proc. SOC.Exptl. Biol. Med. 87, 507 (1954).

INACTIVATION 7. BAWDEN, F. C., AND PIRIE, N. W., Proc. Roy. Sot. (London) B123, 274 (1937). 8. KLECZKOWSKI, A., Biochem. J. 40, 677 (1946). 9. WATSON, D. W., AND BLOOM, W. L., Proc. Sot. Exptl. Biol. Med. 81, 29 (1952). 10. ADAMS, M. H., “Bacteriophages.” Interscience Publ., New York, 1959. 11. GRETCHMANN, G., BRANDIS, H., FISHER, H., AND LIPPERT, W., Schweiz. 2. allgem. Pathol. u. Bakteriol. 20, 322 (1957). 12. GRETCHMANN, G., AND KAPLAN, R. W., Arch. Mikrobiol. 30, 363 (1958). 13. FISHER, H., AND BRANDIS, H., Naturwissenschaften 41, 533 (1954). 14. KLECZKOWSKI, J., AND KLECZKOWSKI, A., J. Gen. Microbial. 14, 449 (1956). 38, 223 15. GRETCHMANN, G., Arch. Mikrobiol. (1961). 16. SHALITIN, CH., AND KATCHALSKI, E., Bull. Research Council Israel Sect. A 6, 314 (1957). 17. SHALITIN, CH., AND KATCHALSKI, E., Bull. Research Council Israel Sect. E 8, No. l-2 (1959). 18. SHALITIN, CH., AND KATCHALSKI, E. Bull. Research Council Israel Sect. A 11, 64 (1962). 19. KATCHALSKI, E., GROSSFELD, S., AND FRANKEL, M., J. 4m. Chem. Sot. 70, 2094 (1948). 20. KATCHALSKI, E., AND BERGER, A., in “Meth ods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, pp. 540-g. Academic Press, New York, 1957. 21. BERGER, A., AND KATCHALSKI, E., J. Am. Chem. Sot. 73, 4084 (1951). 22. ASTBURY, W. T., DALGLIESH, C. E., DARMON, S. E., AND SUTHERLAND, G. B. B. M., Nature 162, 596 (1948). 23. ERLANGER, B. F., AND BRAND, E., J. Am. Chem. Sot. 73, 4025 (1951). 24. BRAND, E., ERLANGER, B. F., POLATNJCK, J., SACKS, H., AND KIRSCHENBAUM, D., J. Am. Chem. Sot. 73, 4027 (1951). 25. WALEY, S. G., AND WATSON, J., J. Chem. Sot. 1953, 475. 26. FRENCH, R. C., J. Bacterial. 67, 45 (1954). 27. HERRIOTT, R. M., AND BARLOW, J. L., J. Gen. Physiol. 36, 17 (1952).

OF COLIPHAGE

T2

507

28. PUTNAM, F. W., KOZLOFF, L. M., AND NEIL, J. C., J. Biol. Chem. 179, 303 (1949). 29. ADAMS, M. H., Methods in Med. Research 2, l-73 (1950). 30. BRENNER, S., AND BARNETT, L., Brookhaven Symposia in Biol. No. 12, 86 (1959). 31. KELLENBERGER, E., AND KELLENBERGER, G., Proc. Intern. Conf. Electron Microscopy, London, 1.954, p. 265. 32. BACKUS, R. C., AND WILLIAMS, R. C., J. Appl. Phys. 21, 11 (1950). 33. KATCHALSKI, E., BICHOWSKI-SLOMNITZKI, L., AND VOLCANI, B. E., Biochem. J. 55, 671 (1953). 34. WALEY, S. G., AND WATSON, J. J., Biochem. J. 55, 328 (1953). 35. LEVIN, Y., BERGER, A., AND KATCHALSKI, E., Biochem. J. 63, 308 (1956). 36. NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, Enzymes” 2nd ed. R. M., “Crystalline Columbia Univ. Press, New York, 1948. 37. CUMMINGS, D. J., AND KOZLOFF, L. M., Biochim. et Biophys. Acta 74, 445 (1960). 38. KELLENBERGER, E., Virology 3, 245 (1957). 39. ANDERSON, T. F., J. Appl. Phys. 21.70 (1950). 40. BONIFAS, V., AND KELLENBERGER, E., Biochim. et Biophys. Acta 16, 330 (1955). 41. KELLENBERGER, E., AND ARBER, W., 2. Katurforsch. lob, 698 (1955). 42. PUCK, T. T., AND SAGIK, B. P., J. Exptl. Med. 97, 807 (1953). 43. KATCHALSKI, A., SHAVIT, N., AND EISENBERG, H., J. Polymer Sci. 13, 69 (1954). 44. DEBYE, P., AND H~~CKEL, E., Phys. Z. 24, 185 (1923). 45. MORA, P. T., AND YOUNG, B. G., J. Biol. Chem. 237, 1870 (1962). 46. SELA, M., AND KATCHALSKI, E., Advances in Protein Chem. 15, 391 (1959). of 47. CUMMINGS, D. J., Ph.D. Thesis. Univ. Chicago, Chicago, 1959. L. O., Acta Pathol. Microbial. 48. KALLINGS, Stand. 52. 82 (1961). 49. KALMANSON, G. M., AND BRONFENBRENNER. J., J. Immunol. 47, 387 (1943).