Effect of spermidine on the development of bacteriophage SP6

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 270, No. 1, April, pp. 77-83,1989

Effect of Spermidine on the Development of Bacteriophage SP6

MUKESH VERMA’

Molecular Genetics Laboram, Howard University Cancer Center, Washington, D.C. 20060

Received August 24,1988, and in revised form November 7,1988

Bacteriophage SP6 is a virulent phage of Salmonella tgphimurium which behaves differently than other phages of the same host. The effect of spermidine on SP6 infection of S. tgphimutium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infection process, the replication of the phage was inhibited but the cells did not survive. Furthermore, if spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis. 0 1999 AcademicPress,Inc.

Polyamines are ubiquitous in biological materials although the relative amounts of putrescine, spermidine, and spermine differ markedly in different organisms and in different cells (1, 2). The responses of different bacteriophages, such as T-even and T-odd phages (T2, T4, T3, T?, etc.), X, P22, 4-X-174, and others, toward these polyamines differ markedly and for some phages the interaction of polyamines starts at low concentration whereas for others it starts at higher concentrations. The tolerance of spermidine (and other polyamines) in bacteriophages varies markedly with the type of phage studied and with the polyamine composition of the host cell. The concentrations of the polyamines and their derivatives are highest in the T-even type of the Escherichia coli bacteriophages where they amount for about 40% of the phage cations (1). The role of these polyamines in phage seems to be that of a nonspecific cation for deoxyribonucleic

1 Address correspondence to the author at Depart- ment of Biochemistry, George Washington Univer- sity School of Medicine, 2300 I Street, NW, Washing- ton, DC 20037.

acid neutralization and stabilization. Low concentrations of spermidine in the medium are not inhibitory for Salmunella phages (3) and higher concentrations, like 100 mM spermidine, can only give the complete inhibitory effect of phage development.

The effect of polyamines on the development of phages has been studied in different phage host systems (4-14). For exam- ple, the growth of T-even phages is not much affected by polyamines (6,7), the rI1 mutants of bacteriophage T4, which nor- mally cannot grow in E. coli K12( X), can do so in the presence of spermidine (8,9); the development of T-odd series phages is blocked by spermidine and spermine (10, 11); the reproduction of phages like h and f2 is inhibited by polyamines (12, 13); the multiplication of b-X-174 and QR-1’76 is stimulated by a low concentration of spermidine whereas at higher concentrations, the infected cells do not lyse (13).

Bacteriophage SP6 is morphologically similar to coliphage T7 but genetically dis- tinct from the latter (14-16). It behaves more like phage P22 (3) of Salmonella typhimurium. The present report discusses the

77 0003-9861/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form resewed.

78 MUKESH VERMA

effect of spermidine on the development of SP6. Like P22, higher concentrations of spermidine, around 100 InM, were needed to see the complete inhibitory effect of spermidine on the development of bacteriophage SP6. Therefore 100 InM spermidine was used for the experiments presented in this report.

MATERIALS AND METHODS

Grwth medium for phuge and its host. S. typhimurium was grown in minimal medium, the composition of which is dihydrogen potassium phosphate, 1.5 g; disodium hydrogen phosphate, 5 g; ammonium chloride, 0.4 g; magnesium sulfate, 0.5 g; NaCl, 0.5 g; and glucose, 2.5 g per liter of distilled water. Exponen- tially growing cells (2 X lO*/ml) were infected with phage (multiplicity of infection = 10) and after 5 min of adsorption of phage to the host aeration was started. After about 90 min the cells lyse. A few drops of chloroform was added to lyse any unlysed cells. This lysate could be stored at 4°C for several months without any loss of the viability of the phage.

Determination of phage titre. Plaque-forming units (PFU)2 were determined by the agar overlay method. Infected cells were treated with chloroform before determining the phage titer as described (17). 8. tgphimurium strain LT2 was used as the plating bacteria and incubation was done at 37°C for 24 h.

Determination of the burst size. Exponentially growing bacterial cells (2 X lO*/ml) were infected with phage at a multiplicity of infection of 10. After 5 min of phage adsorption, the unadsorbed phage were neutralized with antisera (neutralization constant = 2) for 5 min. Infective centers were determined by plating the antiserum-treated cell suspension on plating bacteria. Properly diluted samples of the infected cells were incubated at 37°C till the lysate formed. To determine the phage yield, the lysate was suitably diluted and plated. The burst size was determined from the number of infective centers irrespec- tive of the number of cells used for infection, as described earlier for phage MB78 and P22 of S. typhimurium (18).

Purijicution of the phage through cesium chloride gradient and preparation of %P-labeled phage particles Bacterial debris were removed from phage lysate by centrifugation at 9OOOg for 20 min at 4OC. The lysate was concentrated by a two-phase system using 6% plyethylene glycol, 0.3% dextran sulfate, and 0.25 M NaCl. The phage were finally purified in a stepwise gradient of cesium chloride (density 1.3-1.7 g/cm3). Centrifugation was done at 40,000 rpm in rotor T41 of

* Abbreviations used: PFU, plaque-forming units.

a Beckman ultracentrifuge for 90 min at 10°C. The phage band was removed by puncturing the tube from the bottom and was dialyzed overnight against 0.01 M Tris-HCl, pH 7.5 at 4°C. The preparation of labeled phage was as described (17) for phages of S. typhimu- tiU?R

Measurement of the rate of incorporation of labeled precursors into mucromolcculea The rates of incorporation of [‘Quidine and PHjthymidine into trichloroacetic acid-precipitable fraction were followed as described (19,20). [“Cjleucine incorporation was measured as described (21).

Zone sedimentatiun thruzqh sucrose gradient& the isolation of membrane DNA complex. Neutral sucrose solutions were made up containing either 5 or 10% sucrose and 10 mM Tris-HCI, 10 mM EDTA, and 0.1% sarkosyl. The final pH is 8. The sarkosyl was included to give an even drop size. Before the gradients were made, 0.25 ml of 80% (w/v) iothalamic acid and 20% (w/v) sucrose was pipetted into the bottom of the 5- ml polyallomer tubes. The gradients were then made on top of these materials. The material at the bottom facilitates the recovery of material which would oth- erwise becomes a pellet. Different samples, as described in the legend to Fig. 5, were layered on top of the gradients. These gradients were allowed to stand for 15 min before centrifugation (30,000 rpm for 45 min at 20°C) so that the phage membrane complex made an even layer on top of the gradient. Gradients are collected by puncturing the tube with a needle; the drops were collected onto numbered Whatman 3MM papers (1.5 X 1.5 cm size). These were thoroughly dried and soaked twice in cold 10% trichloroacetic acid for 30 min, washed in cold 5% trichloroacetic acid for 15 min, and finally in cold acetone for 15 min. Filters were air-dried and then counted in toulene- based scintillation fluid (22).

RESULTS AND DISCUSSION

Eflect of spermidine on the growth of S. typhimurium. Before the effect of spermidine on phage SP6 development was studied, its effect on the growth rate of the host, S. typhimurium was investigated. There was no effect on growth at low concentration (20 mM) but at higher concentrations, spermidine affected the growth of the host only transiently causing a temporary lag in growth. The lag period depended on the concentration of spermidine. The effect of 100 mM spermidine is shown in Fig. 1. In order to get the complete inhibitory effect of spermidine on the development of SP6, 100 mM spermidine was used. As shown in Table I, low concentrations of spermidine,

DEVELOPMENT OF BACTERIOPHAGE SP6 79

0.6.

0 0.4. (D

Q

0.2.

0 2 4 6 8

HOURS

FIG. 1. Growth of SPG-infected S. typhimurium in the presence of spermidine added at different times before or after infection. LT2 cells growing exponentially in minimal medium were infected with phage SP6 at a multiplicity of infection of 10. The infected

cells were divided into different batches and spermidine (100 m&Q was added to these cells at different times after infection. In one case the cells were treated with spermidine for 5 min and then infected

with the phage. (A) Control cells, no spermidine; (V) spermidine added, either no phage infection or phage

added 5 min after addition of the spermidine (identical curves for both cases); (A) spermidine added at 1, 2, or 3 min after addition of the phage (identical

curves for all three cases); (0) spermidine added 4 and 5 min following infection with phage (identical curves for both cases); (0) phage-infected cells, either no spermidine or spermidine added 10 or 20 min after

infection (identical curves).

such as 10 mnq do not affect the development of SP6, but 50 mM or 100 mM spermidine reduced the burst size to 20-30 and l- 2, respectively. To understand more about the nature of this inhibition, 100 IIIM spermidine was chosen. It is not surprising that such a high concentration of spermidine has been used because the effect of 100 mM spermidine has already been seen on the development of phage P22 of S. typhimurium (3).

Efect of spermidine on the growth of phuge-itiected S. typhimurium. The experiments with phage SP6 antiserum indicate that spermidine does not inactivate antiserum. To determine the precise limit of the time of addition of spermidine relative to the time of infection, to have the full inhibitory effect, spermidine was added at different time intervals either before or after infection (Fig. 1). The results presented in the figure clearly indicate that if sper-

midine was added before the addition of the phage the infected cells grew exactly in the same way as the uninfected cells in the presence of spermidine. A similar situation was observed when spermidine was added to the phage-infected cells within a short period (3 min). Under this condition the adsorption of the phage to the host was normal, and the number of infected cells was found to remain constant throughout the period of the study, whereas the number of viable cells increased exponentially with time (data not presented). There was abortive infection under this condition.

When spermidine was added 4 or 5 min after infection, the optical density of the cell suspension remained constant (Fig. 1). These infected cells neither multiplied nor produced phage as long as spermidine was present, but they could give rise to infective centers on plating in the absence of spermidine (at least up to 6.5 h, the period of study), indicating that the cells retain their capacity to sustain the phage development. If spermidine was added later than 10 min, the infected cells lysed (Fig. l), liberating phage particles. The yield per infected cell, however, depended on the time of addition. Thus, it does not seem surprising that the time of addition of spermidine with respect to that of the

TABLE I

EFFECT OF SPERMIDINE ON THE BURST SIZE OF PHAGE SP6

Concentration of spermidine (mM) Burst size

0 150-160 10 150-166 50 20-30

100 l-2 200 l-2

Note. Exponentially growing cells of S. tgphimu-

rium (in minimal medium, M9) were infected with phage SP6 at a multiplicity of infection of 10. Spermi-

dine was added along with the phage at different concentrations as indicated above and was present in all the dilution tubes during plating. Burst size was cal- culated on the basis of the number of infected cells obtained in each case.

80 MUKESH VERMA

TABLE II

EFFECT OF TIMING OF ADDITION OF SPERMDINE ON

THE BURST SIZE OF SP6

Time of addition of spermidine (min after infection) Burst size

Control (no spermidine) 150-160 -20 0 -10 0

-5 0 0 0 5 0

10 20-25 20 50-60 25 50-60 30 150-160 50 150-160

Note. The final concentration of spermidine was 100 mM.

phage has great influence on the course of events following phage infection.

The effect of spermidine was found to be dependent on the growth medium. When a similar experiment was carried out with cells grown in enriched medium (Luria broth- or casamino acid-supplemented minimal medium) phage development was not blocked, although it took a longer time for lysis. However, it is not known why the effect of spermidine is dependent on the composition of the medium. It may be that cells growing in enriched medium contain polyamines which are not found in cells grown in minimal medium.

E#ect of spermidine on phage DNA synthesis. One of the most significant changes on infection of S. typhimurium with phage SP6 is the switch over from host DNA synthesis to phage DNA synthesis. Immedi- ately following infection there is a drop in the rate of DNA synthesis which is much greater than in the uninfected host and re- flects phage DNA synthesis. To understand the intracellular events within the host infected in the presence of spermidine the rate of DNA synthesis was measured by following pulse-labeling (using rH]- thymidine) at different times after the addition of spermidine to the S. typhimurium cells infected with SP6. The rates of DNA

synthesis in the uninfected cells in the presence and absence of spermidine were simultaneously measured. As expected, the rate of DNA synthesis increased exponentially with time in the case of uninfected cells, in both the presence and absence of spermidine (Fig. 2). If the cells were infected in the presence of spermidine, initially there was a decrease in the rate of DNA synthesis, then the rate grad- ually increased, and finally it became almost equal to that in the uninfected host. This is in agreement with the growth pat- tern and the observation that the trans-

? 0

X

I a c-l

0.2

0.1 Il. 0 IO 20 30 40

MINUTES

FIG. 2. Effect of spermidine on phage DNA synthesis. Rate of DNA synthesis was followed by pulsing 1 ml of cell suspension (2.6 X 108 cells/ml) with PHI- thymidine (4 nmol, 1O’cpm) as described under Mate- rials and Methods. Spermidine was added at different times along with the phage or 15 or 20 min after phage infection (multiplicity of infection = 10). The rate of DNA synthesis of uninfected cells (control) in the presence and absence of spermidine was also followed. Similarly the rate of DNA synthesis in phage- infected cells in the absence of spermidine was followed. (0) Uninfected cells; (0) uninfected cells in the presence of spermidine; (v) phage-infected cells in the absence of spermidine; (0) spermidine added simultaneously with the phage; (0) spermidine added 20 min after infection; (A) spermidine added 25 min after infection; (A) spermidine added 15 min after infection.

DEVELOPMENT OF BACTERIOPHAGE SP6

port undergoes a transient change in the presence of spermidine. When spermidine was added at 15 min after infection, the rate of DNA synthesis increased for a short period (5 to 10 min) and then remained constant. A more or less similar situation was observed when spermidine was added at 20 min following infection. The results clearly demonstrate that phage DNA synthesis is inhibited in the presence of spermidine.

E#ect of spermidine on phage-induced alteration of the cellular transport processes. It has been observed that transport across the membrane of S. typhimurium undergoes transient changes after infection with phage (20). To test whether spermidine in- terferes with this early event, the phage- induced alteration in the rate of incorporation of uridine into the soluble pool and into macromolecules was studied in spermidine-treated cells. Spermidine itself causes a transient change in cellular transport process(es), therefore the phage was added 20 min after the addition of spermidine (100 mM) to the cells growing exponentially in minimal medium. Within this period the cells recovered the normal rate of transport. No change in the rate of uridine incorporation was observed in SPG-infected cells (Fig. 3B). However, the untreated cells that served as control (Fig. 3A) exhibited the transient change.

As shown in Fig. 4, the first arrow (at min 5) represents the addition of spermidine in uninfected host cells and the second arrow (at min 15) represents the addition of phage to host cells (with and without spermidine). At min 5 the culture was divided into two halves, one with spermidine and other without spermidine. In the presence of spermidine the incorporation of [14C]leucine started declining immediately after the addition of spermidine and never recovered. When cells were then infected with phage in the absence of spermidine the incorporation of [‘4C]leucine first dropped and then recovered almost equal to the level of the uninfected cells. On the other hand in those cells which were infected with phage in the presence of spermidine, the incorporation of [14C]leucine dropped but did not recover. This suggests

0

81

IO 20 30 MINUTES

FIG. 3. Effect of spermidine on the phage-induced alteration of cellular transport process(es). Exponen- tially growing cells in minimal medium were divided into two batches. To one batch spermidine (100 mM) was added (B). The other batch served as control (A). Twenty minutes later portions of spermidine-treated and untreated cells were infected with phage SP6 (multiplicity of infection = lo), and the rates of incorporation of exogeneous uridine into the intracellular pool and into macromolecules were followed in all cases by pulsing with [‘%]uridine (2.2 nmol, 1.3 X lo6 cpm/ml of cell suspension) as described (19). (A) In- corporation into the soluble pool of uninfected cells; (A) incorporation into the soluble pool of infected cells; (0) incorporation into macromolecules of uninfected cells; (0) incorporation into macromolecules of infected cells.

that spermidine affects the incorporation of [14C]leucine in SPG-infected cells.

Association of phage SPS DNA with cell membrane of the host in the presence of spermidine. It appears from the results presented above that the addition of spermidine before infection blocks phage development at a stage between adsorption of the phage and the early phage function responsible for the alteration of the cellular transport process. In order to understand whether phage DNA is injected at all under this condition the association of the phage DNA with the membrane of the host

82 MUKESH VERMA

1 0 10 20 30

hi INUTES

FIG. 4. Effect of spermidine on the phage SPG-induced efflux of intracellular leucine. Exponentially growing cells were harvested, washed, and suspended in mineral base medium, then starved for 90 min and treated with chloramphenicol (50 mM) for 15 min. L-

[‘4C]leucine was added to the cells (10 nmol, 2.2 X 10” cpm/ml of cell suspension) and amino acid incorporation was followed as described (20). At min 5 when the extent of incorporation of leucine became maxi- mal, the cells were divided into two batches and spermidine (50 mM) was added to one batch. At min 15, when the intracellular pool of the spermidine-treated cells attained new equilibrium, both spermidine- treated and untreated cells were infected with phage SP6 (multiplicity of infection = 10) as indicated in the figure. (A) Uninfected cells in the absence of spermidine; (A) uninfected cells in the presence of spermidine; (0) infected cells in the absence of spermidine; (0) infected cells in the presence of spermidine (spermidine added before infection).

was studied as described (22). Following infection the invading SP6, DNA was found associated with the membrane, which may be the site for phage DNA synthesis. A complex having a sedimentation coefficient of 9000 S has been isolated by density gradient centrifugation of the cell lysate. Injection of DNA into the cell inte- rior is a prerequisite for this complex formation. The association of the labeled invading phage SP6 DNA with the membrane to form the high sedimentation value complex at different times following infection of the spermidine-treated and untreated cells is presented in Fig. 5. The maximum association of the parental DNA with the membrane takes place in the untreated cells around 30 min.

In the case of spermidine-treated cells, a huge excess of input DNA (about ‘70%) became associated with the membrane complex from the very beginning and remained associated as long as spermidine was present. If spermidine was added even 5 min after infection a similar situation was observed. However, as soon as spermidine was removed, DNA left the fast sedi- menting fraction but started reappearing in the same fraction after 20 minor so (Fig. 5). The subsequent steps were just as described for normal intermediate synthesis.

From the above description it is clear that spermidine affects the development of SP6 differently than has been reported for T-phages, X, and f2 phages (9, 12, 13) and it is concluded that the polyamines act in

MINUTES AFTER INFECTION

FIG. 5. Kinetics of membrane complex formation in the presence of spermidine. A culture of exponentially growing cells was divided into two batches; to one batch spermidine (100 mM) was added 10 min before infection. After infection (with ?-labeled SP6 phage) of both batches, each batch was divided into two batches again. Spermidine was added 5 min after infection to the half of the infected cells that was not treated earlier with spermidine. Spermidine was removed from one-half of the spermidine-treated and phage-infected cell suspension 5 min after infection by quick filtration and washing through a membrane filter. These cells were then suspended in fresh medium without any spermidine. The cells were lysed at the times indicated in the figure, and the high value membrane complex formation was followed as described (22). (A) Cells infected with phage in the absence of spermidine; (0) spermidine (100 mM) added 10 min before infection; (0) spermidine added 5 min after infection; (A) spermidine added 10 min before infection but removed 5 min after infection.

DEVELOPMENT OF BACTERIOPHAGE SP6 83

different ways in different host-virus systems.

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Effect of spermidine on the development of bacteriophage SP6