VIROLOGY 21, 636-641 (1963)

The Location of the Inhibitory Action of KCN and Several

Polyamines on Bacteriophage Replication’

HARVARD REITER

U. S. Army Biological Laboratories, Fort Detrick, Frederick, Maryland

Accepted August 26, 1963’

Streptomycin, spermine, and arginine inhibit the replication of Tl, T3, T7, and PLT22 bacteriophages. Spermine and arginine inhibit adsorption as well as penetra- tion, whereas streptomycin allows adsorption but blocks the initiation of phage pene- tration. The majority of bacteria infected in the presence of streptomycin survive as viable cells. A technique based on the sensitivity of adsorbed T3 phage to antiserum has been used to define the site of action of these polyamines. This technique also showed that KCN inhibited T3 phage late in the penetration process.

INTRODUCTION

Streptomycin, spermine, and potassium cyanide have been shown to inhibit bacte- riophage replication at an early stage in the infectious cycle. Streptomycin, if present prior to or at the time of phage infection, inhibited several staphylococcal phages (Edlinger, 1949) and blocked T2r+ replica- tion in a streptomycin-resistant strain of Escherichia coli B (Bourke et al., 1952). However, according to Graham (1953), streptomycin did not alter the normal T2 infection of streptomycin-resistant E. coli B nor, according to Brock (1962)) were Tl, T2, T3, T4, T5, or T7 phages affected by streptomycin when a streptomycin-resistant mutant of E. coli 3000 was the host. Brock did show that several DNA-containing streptococcal phages as well as the RNA- containing coliphage MS2 were inhibited by streptomycin.

When present at the initiation of infec- tion, spermine inhibited the replication of PLT22 phage in Salmonella typhimurium LT2, but it allowed normal replication when added shortly after the onset of infection (Ames and Dubin, 1960).

l This investigation was part of the material submitted to the George Washington University, Department of Microbiology in partial fulfill- ment of the requirements for the Ph.D. degree.

The third inhibitor, potassium cyanide, blocks phage replication when added at any time during the infectious cycle (Anderson and Doermann, 1952). Moreover, cyanide is reportedly able to block the inject,ion of phage DNA into the host bacterium (Kel- lenberger, 1961).

The exact site of action of these com- pounds has not yet been determined, since it is difficult to differentiate among phage that are irreversibly adsorbed, phage that are in the process of penetration, or phage that have just finished introducing their DNA into the host cell. The observations of Adams and Wassermann (1956) and of Reiter (1963) that the penetration processes of adsorbed T3 or T7 phages could be slowed by specific antiphage serum form the basis for experiments that differentiate among these three stages. This paper describes the inhibitory effects of streptomycin, spermine, and several other polyamines on the infec- tion of E. coli B cells with several of the T series phages. The site of action of these in- hibitors, as well as of cyanide, is determined, and a sequence of at least three inhibitory events is est’ablished.

MATERIALS AND METHODS

Bacterial strains and phages. The strep- tomycin-resistant E. coli B was obtained

636

SITE OF INHIBITION OF PHAGE BY KCN AND POLYAMINES 637

as a spontaneous mutant of an E. coli B strain maintained in the laboratory for several years. Both the parent and mutant strains were hosts for all the T series phages. Salmonella typhimurium P22 was obtained from Dr. P. E. Hartman, as was the PLT22 phage.

Media. Nutrient broth consisted of 0.8% Difco Nutrient Broth powder and 0.5% NaCl. Nutrient agar was nutrient broth plus 1.5% agar, and soft agar was nutrient broth plus 0.7% agar. Streptomycin sulfate (Squibb) was used. Phage techniques were those described by Adams (1950)) and the probit analysis of one-step growth curves was described by Adams and Wassermann (1956).

RESULTS

The Inhibition of Bacteriophage Replication by Different PoEyamines

When either Tl, T3, or T7 bacteriophage was added to a logarithmically growing broth culture of streptomycin-resistant E. co& B cells containing, per milliter, 1 mg of streptomycin, spermine, or arginine, strong inhibition of phage growth was noted (Table 1). These polyamines also inhibited the replication of PLT22 phage on a strepto- mycin-resistant strain of Xalmonella typhi- murium LT2. However, when streptomycin was added 2 minutes after the phage, it had no inhibitory effect. The T2 and T4 phages were unaffected by strept,omycin added at any time in the infectious process (Table 1).

The Effect of the Polyamines on Free Phage and on Phage Adsorption

To determine the location of polyamine inhibition of phage replication, we examined the effects of polyamines on free phage and on phage adsorption. An experiment in which free T3 phage was exposed for 1 hour to 1 mg of either streptomycin, spermine, or arginine per milliliter showed that streptomycin inactivated less than 30% of the phage, whereas spermine or arginine had no observable effects.

Phage adsorption was measured by add- ing phage to a culture of bacteria in nutrient broth. After a period of incubation the cul-

TABLE 1

THE EFFECT OF VARIOUS POLYAMINES ON PHAGE REPLICATION

Per cent increase in infectious centers

In the presence of

Phr.ge In nutrient

brotha

polyamines

Added Added before after phageb phagec

Tl T2 T3 T4 T7 P22

Tl T3 T4 P22

3000 500

4200 2300 2200 2000

- -

T3 -

Tl -

T3 -

T7 -

T3 -

Tl -

T3 -

Tl -

T3 -

Streptomycin, 1 mg/ml -30 3800 1300 700 -18 5600 4700 3100 -45 2200 -17 2100

Spermine, 1 mg/ml 24 2800

461 3300 3000 2900

10 2200 Spermidine, 1 mg/ml

170 -

Arginine, 1 mg/mL

7 3500 -15 5400

7 2300 Glutamine, 1 mg/ml

1072 4500 Acrijtavine, 10 pgjml

-58 -46 -70 -52

ProjZavine, 10 pg/ml -74 -55 -68 -48

a Phage was added to a 37” nutrient broth suspension of logarithmically growing streptomy- cin-resistant Escherichia coli B cells. The host for P22 phage was a streptomycin-resistant strain of Salmonella typhimurium LT2. The data shown are the percentage increase in the number of phage present after one cycle of growth had been com- pleted at 40 minutes as compared with the number of phage present 5 minutes after the start of in- fection.

b The amine was added to a nutrient broth sus- pension of cells 2 minutes before the addition of phage. Samples were diluted in nutrient broth and assayed for phage at 5 and 40 minutes after in- fection.

c The amine was added 2 minutes after the addi-

tion of phage.

638 REITER

ture was diluted 102-fold and centrifuged to remove the cells and adsorbed phage. This phage, not eluted from the bact,cria by dilution, was considered to have been ad- sorbed irreversibly. Experiments showed that irreversible adsorption was not sig- nificantly altered by 1 mg of streptomycin

O-1, ANTISERUM AT 4 MINUTES

AT 1 MINUTE

10 30 50 70 90 MINUTES

FIG. 1. The effects of streptomycin, T3 anti- serum, and streptomycin plus T3 antiserum on the one-step growth curve of T3 phage. Curve A: The normal one-step growth of T3 phage at 37°C. Curve B: The skewed one-step curve due to inhibition of phage penetration when T3 anti- serum is added 1 minute after the addition of phage. Curve C: The normal one-step growth curve that results when the addition of T3 anti- serum is delayed until 4 minutes after the onset of infection. Curve D : The normal one-step growth curve that starts immediately after the removal of streptomycin. Two hundred micrograms of streptomycin per milliliter had been added to the cells 2 minutes before the addition of T3 phage. The culture WM incubated at 37” for 10 minutes after the addition of phage, then diluted lo-fold with nutrient broth, incubated 4 minutes, then further diluted 104-fold with nutrient broth. Sam- ples of this were then plated for infectious centers at intervals. Curve E: The skewed one-step growth curve resulting when cells infected in the presence of streptomycin were diluted lo-fold with T3 antiserum. This was incubated 4 minutes, then diluted 104-fold with nutrient broth. Samples of this were then plated for infectious centers at intervals.

per milliliter. When streptomycin was present in nutrient broth at 37”, 52% of the phage were adsorbed in 5 minutes; in the absence of streptomycin, 56% of the phage were adsorbed. In contrast, no measurable adsorption occurred in the presence of 1 mg of either spermine or arginine per milli- liter. When the concentration of spermine or arginine was decreased to 400 rg/ml, 2% of the phage were adsorbed. Thus the strep- tomycin inhibition of phage replication ap- parently occurred after irreversible adsorp- tion, whereas spermine and arginine blocked irreversible adsorption.

Location of the Streptomycin Block of TS Infection

Since the penetration of adsorbed T3 phage could be inhibited by phage-specific antiserum, it was possible to determine pre- cisely the site of streptomycin action. Con- trol experiments showed that the normal T3 one-step growth curve was markedly altered if T3 antiserum was added to the system 1 minute after the onset of infection. When the antiserum was added 4 minutes after the addition of T3 phage, penetration was not affected and a normal one-step curve resulted (Fig. 1). Therefore, if phage that had been adsorbed and incubated in the presence of streptomycin for more than 4 minutes was still susceptible to antiserum inhibition, one could conclude that the streptomycin acted prior to, or during the penetration process. Figure 1 shows the re- sults of an experiment in which T3 phage was added to cells in the presence of 200 pg of streptomycin per milliliter, incubated for 10 minutes, and then freed of strepto- mycin by dilution with nutrient broth. The resulting delay in the onset of lysis corres- ponded to the time of exposure to strepto- mycin. The straight-line probit shows that once lysis had begun it proceeded normally. When the phage-cell-streptomycin system was diluted with nutrient broth plus phage- specific antiserum, incubated for 4 more minutes and further diluted with nutrient broth, the resulting one-step growth curve resembled the curve for the experiment in which antiserum was added to T3 cells 1 minute after adsorption. Thus it appears

SITE OF INHIBITION OF PHAGE BY KCN AND POLYAMINES 639

that streptomycin holds the phage at a point where it is susceptible to antiserum inhibition.

The Effect of Streptomycin on the Infec- tious Process after the Initiation of Penetration

An experiment was performed to deter- mine the effect of streptomycin on the infec- tious process after the initiation of pene- t,ration. Phage T3 was added to the host bacteria at 12” and held for 10 minutes so that reversible adsorption, but no penetra- tion, occurred. The culture was then incu- bated at 37” for 2 minutes. By the end of this time penetration had presumably begun and the process was either still underway or was completed. The culture was then chilled to 12” and tested for sensitivity to strepto- mycin or to antiserum. Figure 2 shows that the normal replication of the phage was not affected by streptomycin. On the other hand, approximately 50% of the phage population was inhibited by antiserum. Presumably these phages were still in the process of penetration. It appears reason- able to conclude that streptomycin blocked only the initiation of the penetration process. Once T3 penetration had begun, however, streptomycin had no adverse effect on any subsequent step in the infectious process.

The Rescue of Bacteria and Phage by Streptomycin

Another experiment was performed to see whether the host bacteria were killed by phage that had been adsorbed irreversibly in the presence of streptomycin. Cells were infected with T3 at a multiplicity of 10 in the presence of 1 mg of streptomycin per milliliter. One sample of these cells was di- luted in nutrient broth and plated on nutri- ent agar. A second sample was diluted in nu- trient broth containing streptomycin (1 mg/ml) and plated on nutrient agar contain- ing streptomycin (1 mg,/ml) . Approximately 85% of the cells plated on the nonstrepto- mycin medium were killed; only 15% of the cells plated on streptomycin medium were killed. Thus, streptomycin rescued approxi- mately 72% of the infected cells. Conversely,

EPTOMYCIN AT 2 MINUTES 7

AT 2 MINUTES 6

VI k

85 %

4

3 10 30 50 70 90

MINUTES FIG. 2. The effects of streptomycin and of T3

antiserum on the one-step growth curve of T3 phage after the initiation of penetration. T3 phage was added to cells in nutrient broth at 12”. Ten minutes later this culture was warmed to 37” for 2 minutes, again brought to 12”, and divided among three tubes. The first control tube was warmed to 37”, diluted with nutrient broth and assayed at intervals for infectious centers. One milligram of streptomycin per milliliter was added to the second tube before it was warmed to 37”, diluted with nutrient broth containing 1 mg streptomycin per milliliter, and assayed at inter- vals for infectious centers. T3 antiserum was added to the third tube before it was warmed to 37” for 4 minutes, diluted with nutrient broth, and assayed at intervals for infectious centers.

it was found that when bacteria that had been infected in the presence of streptomy- cin were killed with chloroform, approxi- mately 90-96% of the adsorbed phage sur- vived as infectious units. It appears that the phage, although irreversibly adsorbed in the presence of streptomycin, had not been al- tered structurally so as to become nonin- fectious.

The Location of KCN Inhibition

Streptomycin inhibits the initiation of penetration, and antiserum inhibits the penetration process itself. We utilized this information to determine the point of action of KCN on the penetration process. We found that phage replication was inhibited by 10-3M KCN and that this inhibition was reversed immediately when the KCN

640 REITER

7

6 I f x= KCN + STREPTOMYCIN

O= KCN

r

$5

A= KCN t T3 ANTISERUM

w L

4

3L 10 30 50 70 90

MINUTES FIG. 3. The effects of KCN, KCN plus strepto-

mycin and KCN plus T3 antiserum on the one- step growth curve of T3 phage. T3 phage was added to cells in the presence of lo-’ 44 KCN. After 10 minutes of incubation at 37”, one sample of the culture was diluted lo*-fold with nutrient broth, a second sample with nutrient broth con- taining 1 mg of streptomycin per milliliter, and a third with nutrient broth containing T3 antiserum. After 6 minutes of incubation, each sample was further diluted 103-fold with nutrient broth and assayed at intervals for infectious centers.

was diluted with nutrient broth (Fig. 3). If the KCN inhibited replication after the point of streptomycin sensitivity, the presence of streptomycin in the nutrient broth diluent would not effect the immedi- ate resumption of phage replication. If the point of KCN inhibition occurred before the point of streptomycin sensitivity, the lytic cycle would be arrested even after dilution of the KCN with nutrient broth and strep- tomycin. Figure 3 shows that the lytic cycle was not altered when streptomycin was present in the diluent; thus, the KCN block occurred after the streptomycin block.

The location of the KCN block with reference to the antiserum-sensitive period was determined in a similar experiment. The inhibiting KCN was diluted with nutrient broth containing T3 antiserum. The re- sulting one-step growth curves were ex- amined to determine whether the lytic cycle was altered by the antiserum in the diluent. Figure 3 shows that the lytic cycle

was not altered by t,he antiserum. The KCS block therefore occurred after the antiserum block. J1Te conclude that phage that have proceeded in the infectious process as far as the point of KCX inhibit’ion are not sensi- tive to inhibition by either streptomycin or antiserum.

DISCUSSION The data show t’hat the inhibitors tested

act at different points in the penetration process. Arginine and spermine block ir- reversible adsorpt,ion, streptomycin allows adsorption but blocks initiation of penetra- tion, and cyanide blocks phage replication after the antiserum-sensitive stage of pene- tration has passed. The observation that streptomycin acts externally to the bacteria is consistent with the observation that strep- tomycin-resistant bacteria are not easily permeable to streptomycin (Anand et al., 1960). Indeed, since the streptomycin- inhibited phages are almost totally recover- able after elimination of the host, it appears that the irreversible adsorption was not concomitant with any irreversible altera- tion in phage structure.

Cyanide inhibits phage penetration (Kellenberger, 1961) ; furthermore, accord- ing to our findings, this inhibition occurs relatively late in the penetration process, after the antiserum-sensitive period of penetration has passed. Since cyanide in- hibits primarily oxidative phosphorylation and since, at present, there are no known complex enzyme systems in phage, it is possible that the cyanide inhibits the activ- ity of the host bacterium. The host hacte- rium, therefore, may normally participate actively in the penetration process. It is noteworthy that the uptake of transforming DNA has also been shown to require the 2,4-dinitrophenol-sensitive (Stuy, 1962)) KCN-sensitive (Herriott, private communi- cation), active participation of the host cell. The later stages of phage penetration may be similar to the processes by which transforming DNA penetrates into the cell.

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terial viruses. Methods Med. Res. 2, l-73. ADAMS, M. H., and WASSERMANN, F. E. (1956).

SITE OF INHIBITION OF PHAGE BY KCN AND POLYAMINES 641

Frequency distribution of phage release in the one-step growth experiment. Virology 2, 96108.

AMES, B. N., and DUBIN, D. T. (1960). The role of polyamines in the neutralization of bac- teriophage deoxyribonucleic acid. J. Biol. Chem. 235, 769-775.

ANAND, N., DAVIS, B. D., and ARMITAGE, A. K. (1960). Uptake of streptomycin by Escherichia coli. Nature 185, 23-24.

ANDERSON, T. F., and DOERMANN, A. H. (1952). The intracellular growth of bacteriophages. II. The growth of T3 studied by sonic disintegration and by T6-cyanide lysis of infected cells. J. Gen. Microbial. 35, 657-667.

BOURKE, A. R., ROBBINS, M. L., and SMITH, P. K. (1952). Studies on the chemical inhibition of TZr+ bacteriophage. J. Immunol. 69, 75-88.

BROCK, T. D. (1962). The inhibition of an RNA

bacteriophage by streptomycin, using host bac- teria resistant to the antibiotic. Biochem. Bio- phys. Res. Commun. 9, 184-187.

EDLINGER, E. (1949). Antibiotiques et lyse bac- tkriophagique. I. Protection des colonies de- veloppees & la peripherie de la zone d’action de la streptomycine contre l’action du bacterio- phage. Ann. Inst. Pasteur 76, 396-405.

GRAHAM, A. F. (1953). The fate of the infecting phage particle. Ann. inst. Pasteur 84, 96-98.

KELLENBERGER, E. (1961). Vegetative bacterio- phage and the maturation of the virus particles. Advan. Virus Res. 8, l-61.

REITER, H. (1963). The effects of antiserum on adsorbed phage. J. Bacterial., in press.

STUY, J. H. (1962). Transformability of Hae- mophilus influenzae. J. Gen. Micro&ok 29, 537- 549.