J. Mol. Biol. (1970) 47, 215-229

General Inhibition of Escherichia coli Macromolecular Synthesii by High Multiplicities of Bacteriophage 4X174

ALAN B. STONE

Guinness- Lister Research Unit, Lister Institute of Preventive Medicine Chelsea Bridge Road, London X. W.1, England

(Received 17 April 1969, and in revised form 13 October 1969)

Infection by +X174 affects bacterial macromoleoular synthesis by two distinct mechanisms. At low multiplicity, host DNA synthesis stops abruptly 15 minutes after infection, as the result of a process dependent on protein synthesis, while the formation of RNA and protein continues. At higher multiplicities, all macro- molecular synthesis is suppressed; the formation of DNA, RNA and protein is inhibited by a process not prevented by chloramphenicol. Inhibition becomes more pronounced at increasing multiplicities between 4 and 40. Phages that have received numerous lethal “hits” with nitrous acid or ultraviolet radiation retain their ability to inhibit macromolecular synthesis by the second mechanism, which is distinct from “lysis from without”. The permeability of the cell is demonstrably increased after infection at high multiplicities.

Infection at low multiplicity induces partial immunity to subsequent injury by superinfection at high multiplicity. The immunity appears between 5 and 9 minutes after the primary infection. It does not develop in chloramphenicol, or if ultraviolet-killed phage are used, but once it has developed it is unaffected by chloramphenicol.

1. Introduction When Escherichia coli is infected with low multiplicities of $X174, bacterial DNA syn- thesis comes to an abrupt halt after about 15 minutes (Lindqvist & Sinsheimer, 1967). The mechanism responsible, which is blocked by CAP?, affects only two-stranded DNA; the formation of RNA, protein and phage SS DNA continues. A second 4X- induced inhibition of macromolecular synthesis is described in this report. It occurs immediately after infection, and affects not only DNA, but also the synthesis of RNA and protein. It is not blocked by CAP, and becomes more pronounced at higher multiplicities of infection.

This general inhibitory mechanism accounts for the reduced yields of progeny +X observed after infection at high multiplicities (Stone, unpublished results). It may also explain the discrepancy between the results of Lindqvist & Sinsheimer (1967), which implicate protein synthesis in the arrest of host DNA replication by +X infection, and those of Ishiwa & Tessman (1968), which do not.

Preliminary reports of this work have already appeared (Stone, 1968,1969).

t Abbreviations used: CAP, chloramphenicol; m.o.i., multiplicity of infection; p.f.u., plaque- forming units; RF, 4X replicative form DNA; SS DNA, single-stranded DNA; TNS, N-tolyl-a- naphthylamine-S-sulphonic acid.

215

216 A. B. STONE

2. Materials and Methods (a) .Bacterkx

E. coli C (BTCC 122) was used for the isotope experiments. The permissive host for +X amber mutants, E. COG CR, was used for the estimation of p.f.u. by plating; this strain is a recombinant (E. coli CR34 x E. coEi C416), described by Denhardt & Sinsheimer (1965).

(b) Phqe strains To avoid complications due to cell lysis, experiments were performed with a lysis-

defective phage strain, qbXam3, bearing an amber mutation in cistron I (Hutchison & Sinsheimer, 1966; Sinsheimer, 1968). In many experiments, the double mutant, +Xamdt&?, of Lindqvist & Sinshehner (1967) was used. The ts9 mutation, in cistron IV, results in a temperature-sensitive capsid component which indirectly precludes the synthesis of progeny SS DNA at the non-permissive temperature (Lindqvist & Sinsheimer, 1967; Sinsheimer, 1968). As the amount of RF synthesis is small compared with the amount of bacterial DNA synthesis, the use of 4Xam3ts9 in the non-suppressor host (E. coli C) at the non-permissive temperature (40°C) facilitates the prolonged measurement of bacterial DNA synthesis.

Both phage strains were kindly provided by Professor R. L. Sinsheimer, and stocks were prepared from single plaques. Small-scale lysates were made using E. coZi CR growing in 3XD medium (Fraser & Jerrel, 1953) supplemented with 10 pg thymidine/ml. Infection w&s performed at 37°C for $Xamd and at 30°C for dXam3ts9. These lysates were then used to infect large cultures of E. co.% C, the non-suppressor host. The “snakes” which resulted- elongated cells without septa, characteristic of infection with lysis-defective $X strains (Hutchison & Sinsheimer, 1966), were collected by centrifugation and lysedwithlysozyme and ETDA (Denhardt & Sinsheimer, 1965). The phage suspensions were treated with RNase, DNase and trypsin (Sinsheimer, Starman, Nagler & Guthrie, 1962), extensively dialysed against borate buffer (O-05 M, pH 9.0) and centrifuged at 150,000 g for 2 hr. The sedimented phage was resuspended, centrifuged to equilibrium in a CsCl gradient, and again extensively dialysed against borate buffer.

(c) TPPQ + aa medium

This is the TPG medium of Sinsheimer et al. (1962) with KHZP04, C&l, and FeCl, increased 44-, lo- and loo-fold respectively, and supplemented with thymidine (2 pg/ml.), &dine (360 pg/ml.) and the following 18 n-amino acids: Ala, Arg, Asn, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val (each at 50 pg/ml.). The doubling-time of E. coli C in this medium was 30 min at 37°C.

When DNA synthesis was measured, [6-3H]thymidine (15 to 18 c/m-mole) was added at the time of infection to a final concentration of 5 to 10 &m1.(15 &ml. for the experi- ment illustrated in Fig. 1). The destruction of thymidine by bacterial thymidine phos- phorylitse wss prevented by the high concentration of uridine in the medium (Budman & Pardee, 1967). For the measurement of RNA synthesis, a slightly modified medium was used: the uridine concentmtion was reduced to 5 pg/ml.(2 pg/ml. for the experiment recorded in Table l(a)) and the thymidine concentration increased to 50 pg/ml. [5-3H]Uri- dine (16.3 c/m-mole) was added to a final concentration of 2 to 5 pc/ml. When protein synthesis was measured, the leucine concentration was reduced to 5 pg/ml., and [4, 5-aH]leucine (1 c/m-mole) added to a concentration of 1 to 2 pc/ml. All radiochemicals were purchased from the Radiochemical Centre, Amersham, Bucks.

(d) Growth and infection of bacteria Bacteria were grown with aeration at 37°C to a concentmtion of 4 x lo7 cells/ml.

Cells were counted in a Petroff-Hausser chamber under phase-contrast microscopy, classing a rod in the act of division as two cells if a complete septum was visible. The cultures were transferred to a water-bath at 4O”C, portions (3 ml.) were infected with 4X at the desired multiplicity in the presence of the radioactive tracer, and aeration continued. For the non-radioactive experiment recorded in Fig. 5, 20-n& portions were infected, and O.D. 450 rnp measurements were made with a Unicam SP500 spectrophotometer. In

4x174 AND MACROMOLECULAR SYNTHESIS 217

some experiments, cultures were filtered on grade HA Millipore membrane filters and resuspended in fresh warm medium before infection, but it became apparent that this step was unnecessary. CAP (Parke, Davis & Co.) was sometimes added, either before or after infection; details are given in’the legends to the Figures.

(e) Measurerned of isotope incorporation

Duplicate samples (0.05 ml.) were pipetted from the cultures at intervals and deposited on a series of filter paper discs (Whatman 3MM, diam. 2.3 cm), numbered in pencil for identihcation and resting on a Perspex tray. After allowing about 1 min for the samples to soak into the paper, the discs were transferred to a beaker containing 1 1. ice-cold 5% trichloroacetic acid, to precipitate nucleic acids and protein and to elute radioactive material of low molecular weight. An excess (200 pg/m.l.) of unlabelled thymidine, uridine or leucine was present in the acid to act as a carrier. The contents of the beaker were gently stirred for 10 to 20 min and the discs then removed and placed on a pad of filter paper to drain away excess liquid. The washing was repeated in a second beaker of acid. Subsequently, the discs were washed in ethanol, then in acetone, and allowed to dry. Numerous (100 to 200) radioactive samples, washed in batches of about 50, could be pro- cessed in a short time. Provided that the discs were not pressed tightly against each other during washing, the transfer of radioactive material from one to another was negligible. This was checked by mixing a series of blank and radioactive discs in each experiment.

Each disc was placed horizontally at the bottom of a glass counting vial containing 5 to 10 ml. of scintillation mixture consisting of 6 g butyl-PBD (Thorn Electronics, Tolworth, Surrey) in 11. toluene (analytical reagent grade). Radioactivity measurements were made with the Beckman LSZOOB liquid-scintillation system. After counting, the paper discs were removed from the counting vials; the remaining scintillation liquid could be re-used repeatedly with virtually no increase in background counts.

(f) Nitrous acid treatment of $Xam3

The phage suspension ( 1012 p.f.u. in 0.27 ml. sodium borate buffer, 0.05 M, pH 9.0) was mixed with 0.33 ml. sodium acetate buffer (3 M, pH 4.5) and 0.33 ml. NaNOz (0.05 M)

contained in a small test-tube in a water bath at 37°C. Samples (O-15 ml.) were transferred at intervals to a series of tubes, each containing 0.45 ml. Tris-HCI buffer (05 M, pH 8.0) at O”C, to stop the reaction. Phage survival was assayed by plating on E. coli CR at 37°C. Treated samples were stored in the refrigerator and used the following day in one experi- ment (Table l(a)). Their titres were unchanged when assayed again immediately after the experiment.

(g) Ultraviolet irradiation of $XamStsS

Phage (2 x 1Ol1 p.f.u. in 3.2 ml. Tris-HCl buffer, 9.1 M, pH 7-O) was placed in a glass Petri dish (diam. 4.3 cm.) and irradiated with a Philips 15 w germicidal tube situated 60 cm above. The phage suspension was mixed during irradiation by means of a mechanical rooking device. Samples (0.4 ml.) were removed at intervals. Survivors were assayed by plating on E. coli CR at 30°C. Titres remained constant for several days when suspensions were stored in the refrigerator.

(h) Assessment of cell permeability

Alterations in bacterial permeability following infection with phage T5 have been investigated by measuring the penetration of TNS (Crawford, personal communication), which alone does not fluoresce but which forms a fluorescent complex with protein (Newton, 1954). The same principle was applied here in the investigation of permeability changes after +X infection.

A drop of culture was mixed on a microscope slide with a drop of stock TNS solution ( 10m3 M in 1% NaCl, adjusted with NaHCO, to pH 7.0) and a cover-glass applied. The cells were immediately examined under the microscope, using vertical fluorescence illumina- tion and direct phase-contrast illumination alternately to detect fluorescent and non- fluorescent bacteria. Slides and cover-glasses were cleaned no Haemo-Sol (Meineoke $ Co., Baltimore, Md.), then washed with distilled water and air-dried. Observations were made

15

218 A. B. STONE

with a Zeiss Standard Universal microscope fitted with a x40 Neofluar phme-contrast objective and x 10 Kpl eyepieces. Fluorescence illumination was supplied by an HBO 200-w/4 source, using exciter filter BG12 and barrier filters 53/44. TNS was a. gift from Dr D. J. R. Laurence.

(i) Formation of bacterial spheropkzds O-l-ml. portions of culture (2X lo8 cells/ml.) were added to 0.3~ml. portions of a

lysozyme mixture consisting of 1 vol. Tris-HC1 buffer (0.1 M, pH S-6), 1.6 vol. 50% w/v sucrose, 0.05 vol. 30% w/v bovine serum albumin (Armour), O-05 vol. 4% EDTA and O-1 vol. lysozyme (2 mg/ml., Armour). After 20 to 30 min at room temperature, spheroplasts were counted in a Petroff-Hausser chamber.

3. Results (a) Inhibition of macromolecular synthesis at low and high

multiplicities of infection

The arrest of host DNA synthesis 15 minutes after infection with q5Xam3ts9 at a multiplicity of 4 is shown in Figure l(a). CAP at 30 pg/ml. prevented this arrest (Fig. l(b)), in confirmation of Lindqvist & Sinsheimer (1967). The inhibition of host DNA synthesis at low m.o.i. is thus dependent on protein synthesis.

50 (a)

7 .

5 40

l------

/ .r: -5 3 s 30 x .u5

‘3 8 /

Q 20 a" J

IO

~ 0 IO 20 30 40 50 5 IO 20 30 40' 50 60

Time after infection (mid

FIG. 1. Inhibition of bacterial DNA synthesis by $XumStsS infection. (a) No CAP; (b) 30 pg CAP/ml. added at -6 min. 0, Uninfected; 0, m.o.i. = 4.

At higher m.o.i., however, inhibition of bacterial DNA synthesis occurred even in the presence of CAP, as shown in Figure 2(a) for m.o.i. of 8 and 20 in the presence of 30 pg CAP/ml. Moreover, superinfection at high multiplicity of cells previously

infected in CAP at low multiplicity abruptly arrested DNA synthesis. Pronounced inhibition of DNA synthesis followed infection at a multiplicity of 20 even if 100 pg CAP/ml. was present (Fig. 2(b)); th is inhibition occurred without significant delay

(cf. Fig. l(a)). Whereas infection at low multiplicity chiefly affects DNA synthesis, having little

effect on the formation of RNA and protein, higher multiplicities inhibit all molecular synthesis. The inhibition of RNA and protein synthesis in the absence of CAP is illustrated in Figure 3(a) and (b), respectively. In the presence of CAP, the inhibition of RNA synthesis was even more pronounced-(Fig. 4).

+X174 AND MACROMOLECULAR SYNTHESIS

, , I

3 (b)

Time after infection (mid

FIG. 2. Inhibition of bacterial DNA synthesis by $X at high m.o.i. in the presenoe of CAP. (a) $Xum3ts9, 30 pg CAP/ml. added at -7 min. 0, Uninfected; 0, m.o.i. = 4; v, m.o.i. = 8:

A, m.o.i. = 20; A, m.o.i. = 4, superinfected at 20min with m.o.i. = 20. (b) 4Xam3, 100 pg CAP/ml. added at -2 min. 0, Uninfected; A, m.o.i. = 20.

IO

2

0 20 40 60 Time after infection (mid

FIGI. 3. Inhibition of RNA and protein synthesis by q5Xam3ts9 at high m.o.i. (a) RNA synthesis; (b) protein synthesis. 0, Uninfected; 0, m.o.i. = 4; A, m.o.i. = 20;

0, m.0.i. = 40.

(b) Effect of high multiplicity of infection on bacterial growth

Optical density measurements following infection at a high multiplicity (Fig. 5) indi- cate that the inhibition of macromolecular synthesis was not a consequence of “lysis from without” similar to that which follows infection with high multiplicities of T-even phages and which is accompanied by visible lysis (Doermann, 1948). With 40 +X/cell, the optical density of the culture slowly increased by about SO%, then remained constant. Phase-contrast microscopy showed that whereas bacteria infected at a multiplicity of

220 A. B. STOSE

,-- “(---- l

T;me o’ter infection (m;n)

FIG. 4. Effect of CAP (30 pg/ml. added at -6 min) on RNA synthesis in cells infcctod with +JXam3ts9 at high multiplicity. 0, Uninfected; A, m.o.i. = 20.

1 4 1 I 1 ,

0 20 40 60 80 100 I20 140 160

Time after infection (mid

FIG. 5. Effect of $Xam3L99 infection on b&&al growth. 0, Uninfected; 0, m.o.i. = 4; A, m.0.i. = 10; 0, m.0.i. = 40; m, m.0.i. = 4, superinfected at 20 min with m.o.i. z 40.

(5X174 AND MACROMOLECULAR SYNTHESIS 221

4 or 10 elongated to form “snakes” (cf. Hutchison & Sinsheimer, 1966), those infected at a multiplicity of 40 did not. Figure 5 also shows that 40 p.f.u./cell inhibited less drastically when the bacteria were previously infected with 4 p.f.u./cell; these cells continued to grow and formed “snakes”. This partial immunity to superinfection produced by low multiplicities is discussed below.

(c) Development of immunity to inhibition of macromolecular synthesis

Figure 2(a) shows that 20 minutes after infection in CAP, DNA synthesis was inhibited by superinfection with 20 p.f.u./cell. The effect of omitting CAP was studied with +Xam3 (Fig. 6). After the arrest of host DNA synthesis at 15 minutes or there- abouts (cf. Fig. l(a)) in the culture infected at a multiplicity of 4, the incorporation of isotope continued, reflecting the synthesis of progeny XX DNA. It is clear that incorporation was little affected by superinfection with 20 p.f.u./cell, whereas a multiplicity of 20 without preinfection was strongly inhibitory. Substantial immunity to superinfection was apparent as early as the ninth minute after the primary infection.

Time after infection (min)

Fra. 6. Immunity to superinfection: DNA synthesis in cells infected with +Xam3. (a) 0, m.0.i. = 4; A, m.0.i. = 20; ‘I, m.o.i. = 4, superinfected at 20min with m.o.i. = 15.

(b) 0, m.o.i. = 4; v, m.o.i. = 4, superinfected at 20min with m.o.i. = 20; A, m.o.i. = 4, superinfected at 9 min. with m.o.i. = 20.

The synthesis of RNA also became partially resistant to inhibition by superin- fection at high multiplicity. RNA synthesis was measured in an experiment to study the requirement for protein synthesis in the development of immunity to super- infection, because CAP has two opposing effects on DNA synthesis in the $X-infected cell: it abolishes progeny SS DNA synthesis, yet prolongs host DNA synthesis beyond the fifteenth minute. After infection with #Xam3ts9 at a multiplicity of four, immunity did not develop when CAP was added either one minute (Pig. 7(a)) or five minutes later (Fig. 7(b)). In the former case, superinfection resulted in the degradation of some of the RNA synthesized earlier. However, if a period of protein synthesis lasting 22 minutes was allowed between the primary infection and the addition of CAP, and the cells superinfected four minutes later, in the presence of CAP, with 20 p.f.u./cell, immunity was observed (Fig. 7(c)). Hence a process requiring protein

222 A. B. STONE

synthesis takes place between five minutes (cf. Fig. 7(b)) and nine minutes (cf. Fig. 6(b)) after infection at low multiplicity, which renders nucleic acid synthesis resistant to inhibition by superinfection at high multiplicity, even if protein synthesis is then blocked.

(a)

0 20 40 60 80 '0

Time after infection (mid

FIG. 7. Effect of CAP (30 pg/ml.) on the development of immunity to superinfection. Cells infected with $Xam3ts9 at m.o.i. = 4 (0) were further treated as below. RNA synthesis wa4 measured.

(a) 0, CAP added at 1 min; A, CAP at 1 min, superinfected at 21 min with m.o.i. = 20. (b) 0, CAP added at 5 min; A, CAP at 5 min, superinfected at 9 min with m.o.i. = 20. (c) 0, CAP added at 22 min; A, CAP at 22 min, superinfected at 26 min with m.o.i. = 20.

(d) Inhibitiort by nitrous acid-treated and zcltraviolet-irradiated phuge

The role of the phage genome in the inhibition of macromolecular synthesis and in the development of resistance to inhibition was examined, using phage treated with HNO, or irradiated with ultraviolet light. The inactivation curves are presented in Figure 8. The slight deviation of the HNO, curve from an exponential curve conceiv- ably reflects multiplicity reactivation (cf. Hayes, 1968) on the plates, where, in the ease of heavily treated samples, a large excess of inactivated particles per indicator bacterium was used. If that be the case, the number of lethal “hits”/particle (8, 12, 16, 18 and approximately 22, calculated from the fractional survivals) is somewhat under- estimated, although this does not affect the interpretation of the results. In the case of the ultraviolet irradiation curve, the levelling-out below 10T6 survival may be due to the presence of a minority of aggregated phages in the suspension, or of phages masked from the ultraviolet by residual cellular debris. Tessman & Thomas (quoted in Ishiwa & Tessman, 1968) state that the ultraviolet inactivation of +X proceeds exponentially down to a survival of lo-lo. Therefore, to calculate the number of lethal “hits” sustained by the vast majority of particles in the suspension, the initial part of the curve was extrapolated, giving values of 6~5, 12, 17, 22 and 28 hits/ particle for the ultraviolet doses used.

In the following experiments with HNO,-treated and ultraviolet-irradiated phage, “multiplicity” refers to the total number of phage particles/cell; that is, live phage (p.f.u.) + killed phage.

+X174 AND MACROMOLECULAR SYNTHESIS

Ultraviolet irradiation (set)

223

Duration of HNOz treatmerit (min)

FIG. 8. Survival curves of 4X after inactivating treatments. C, Ult.raviolet irradiation of dXam3f.s9; 0, HSOs treatment of 4Xam3. ( The survival after 80 min HNOz treatment. was not measured; the value obtained by extrapolation is shown.)

Ultraviolet irradiation of +Xam3ts9 (22 hits/phage) dest.royed it.s ability to a.rrest bacterial DKA synt.hesis at a multiplicity of 4, although infection at a multiplicity of 20 was inhibitory (Fig. 9). This situation resembles that w&h live phagc in the prc- sence of CAP (cf. Pigs 1 and 2).

Time after i.&ctior. (mir)

FIG. 9. Inhibition of DSA synthesis by iufectiou with untreated or ult.raviolet-irradiated (22 hits/ phage) 4Xum31s9. 0, Uninfected; 0, untreated phagc, m.o.i. = 4; n , irradiated phage, total rn.o.i. -=- 4 (p.f.u. L killed phage); A, irradiated phage, total m.o.i. -- 20

224 A. B. STONE

For the remaining experiments, RNA synthesis was measured to avoid complica- tions resulting from the phage-induced arrest of host DNA synthesis at 15 minutes. Table 1 illustrates the effects of HNOz or ultraviolet treatment on the inhibition of RNA synthesis by a total m.o.i. of 20 particles/cell. After 8 or 12 lethal HNOs hits/

TABLE 1

RNA synthesis after infection with HNO,-treated or ultraviolet-irradiated phage at a total m.o.i. of 20 particles/cell (p.f.u. + killed phage)

Hits/ph&ge [3H]TJridine incorporated (cts/min) in 50 min

(a) HNO,-treated $Xam3: Uninfected control culture 26,000 0 hits 4800 8 hits 900

12 hits 2000 I6 hits 5700 18 hits 10,500 About 22 hits 18,400

(b) Ultraviolet-irradiated $Xam3ts9: Uninfected control culture 2320 0 hits 680 2.5 hits 350 6.5 hits 90

12 hits 80 17 hits 80 22 hits 90 28 hits 80

Phage samples prepared in the experiment shown in Fig. 8 were used.

phage, inhibition was even more pronounced than with live phage. This result is quite different from that observed in the arrest of bacterial DNA synthesis caused by low m.o.i., where eight or more HNO, hitslphage reduces inhibitory efhcacy (Stone, manuscript in preparation). The Table also shows that further inactivation with HNO, (16, 18 or about 22 hits/particle) resulted in the gradual loss of inhibitory activity. Phage irradiated with ultraviolet (65 hits/particle) was significantly more inhibitory at an m.o.i. of 20 than live phage at the same m.o.i. Phage that had received 12,17,22 or 28 hits/particle gave identical results; phage which had received 2.5 hits/particle was somewhat less inhibitory, but still more so than live phage. Protein synthesis was also inhibited more strongly by irradiated phage than by live phage.

To exclude the possibility that small-molecular toxic material was produced during irradiation of the phage suspension, a supernatant fraction was prepared from the ultraviolet-treated suspension by centrifugation for three hours at 150,000 g. This fraction neither inhibited RNA synthesis nor enhanced the inhibition produced by live +X at an m.o.i. of 20. Electron microscopic examination of the irradiated +X, kindly performed by Dr A. M. Lawn, failed to reveal any differences from normal 4X.

Although infection with live phage at a multiplicity of four induced appreciable resistance to inhibition by subsequent superinfection at a high multiplicity with either live phage or ultraviolet-irradiated phage, primary infection with irradiated phage bad no suoh effect. This was demonstrable for both RNA (Table 2) and prot.ein

d,Xl74 AND MACROMOLECULAR SYSTHESIS 225

TABLE 2

RNA synthesis after hz$h multiplicity infection of ceuS pre-injected 16 minutes earlier with live or ultraviolet-irradiated (22 hit.s/phuge) +XamStsS.

Pre-infection High multiplicity infection

---- ___-- ___~-_--_._--

- -

live +X at m.0.i. 4 live +X at m.0.i. 4 live $X at m.0.i. 4

live 4X at m.0.i. 40 irradiated +X at m.0.i. 20 live +X at m.0.i. 40 irradiated +X at m.0.i. 20

-

1300 1100 3800 4700 7100

irradiatod +X at, m.o.i. 4 live +X at m.0.i. 40 800 irradiated +X at m.o.i. 4 - 10,800

[3R]Uridine incorporated (cts/min) in 50 min.

synthesis. In its inability to induce immunity, therefore, the ultraviolet-treat,cd +X behaved like live +X in the presence of CAP (cf. Fig. 7).

It is clear t,hat the presence of CAP, or ultraviolet treatment, of the phage, enhances the inhibitory effect of 4X on RNA synthesis (cf. Fig. 4 and Table 1). The relationship between live $X in the presence of CAP a.nd ultraviolet-treated +X is exemplified further by the experiment illustrated in Figure 10, which compares the degree of inhibition produced by live and by ultraviolet-treated 4X, both in the presence of CAP. An m.o.i. of 10 was used, so that. in neither case was inhibition at a maximum. The data show that, in the presence of CAP, live $X and ult.raviolet-treated $X were equa.lly inhibitory.

Time after infection (min)

Fro. 10. Inhibition of RSA synthesis by infection with untreated or ultraviolet-irradiated (22 hitslphago) +Xam3&9 in the presence of CAP. ,4 suspension of ba&eria was divided into three portions 6 min after the addition of CAP (30 pg/ml.). 0, Uninfected; 3, untreated phage, m.o.i. : 10: A, irradiated phage, t.otal m.o.i. :-G 10 (p.f.u. + killed phage).

226 A. B. STONE

(e) Chungm in cell permeability after infection

Uninfected E. coli C were impermeable to TNS: fluorescence did not develop even after exposure to the compound for 20 to 30 minutes. Indeed, the addition of an equal volume of stock TNS solution to a growing culture had no effect on the bacterial growth rate.

On the other hand, cells became slightly but distinctly permeable 5 minutes after infection with +Xam3ts9 (live or ultraviolet-killed) at a multiplicity of 4. A faint, pale green iluorescence appeared during the first three minutes of exposure to TNS and remained constant thereafter. Cells infected with live phage for periods up to 60 minutes, which formed %nakes”, retained this slight permeability. After two to three hours the “snakes” became more permeable to TNS, and exhibited an intense, pale green fluorescence.

Infection with 40 live or ultraviolet-killed particles/cell resulted in an immediate increase in permeability to TNS. Some fluorescence was detectable one minute after infection, and was relatively intense within five minutes of infection. That this fluorescence reflected the actual penetration of the TNS into the cell, rather than its conjugation with $X capsid protein adsorbed to the outside of the cell, is indicated by the fact that the bacterial nucleoids were clearly visible as dark areas in a fluorescent cytoplasm.

When bacteria infected at a multiplicity of 4 were superinfected 25 minutes later with live +X at a multiplicity of 40, or with ultraviolet-killed (6X at a multiplicity of 20, no further increase in permeability was demonstrable in the majority of cells. Before superinfection, all cells fluoresced faintly when mixed with TNS. When TNS was added 5, 15 or 30 minutes after superinfection, only 10 to 20% of the cells fluoresced more intensely; these more permeable cells were those which had not developed into “snakes”. When the primary infection at a multiplicity of 4 was per- formed in CAP, or if ultraviolet-killed $X were used, then, after superinfection, all cells fluoresced more intensely when mixed with TNS. These findings are summarized in Table 3.

The increased permeability of E. coli after infection with +X at high multiplicity does not result from a gross disintegration of the cell membrane. Between 80 and 100% of bacteria infected for up to two hours with 4 or 35 p.f.u./cell still yielded intact spheroplasts when treated with lysozyme and EDTA in a stabilizing medium.

TABLET

Increase in cell permeability to TNS after infection

Primary infection Secondary infection Fluorescence after TNS

Uninfected control live #X at m.0.i. 4 irradiated +X at m.o.i. 4 live (bX at m.0.i. 40 irradiated +X at m.o.i. 20 live +X at m.0.i. 4 live 4X at m.0.i. 4 CAP, live 4X at m.o.i. 4 irradiated $X at m.o.i. 4

- - - - -

live $X at m.0.i. 40 irradiated 4X at m.o.i. 20 live $X at m.0.i. 40 live $X at m.0.i. 40

none slight slight intense intense slight slight intense intense

4x174 AND MACROMOLECULAR SYNTHESIS 227

4. Discussion These observations suggest that the inhibitory effects of high multiplicities may

result from peripheral damage to the cell. Inhibition occurs immediately after infection. It is not restricted to one class of macromolecule, but affects the synthesis of DNA, RNA and protein. The infected cells barely grow and do not form “snakes”. The inhibition of the synthesis of all three types of molecule by high m.o.i. is counteracted by a process which develops after low m.o.i. Furthermore, nucleic acid synthesis is inhibited even under conditions which prohibit protein synthesis, and killing the phage with HNO, or ultraviolet light does not destroy its inhibitory e%icacy. Lastly, although infection at a high multiplicity does not induce “lysis from without”, it increases the permeability of the cell to TNS, an effect that can be prevented by a previous infection with live phage at a low multiplicity.

This situation may be compared with the inhibitory effects of low multiplicities. With live +X, host DNA synthesis is arrested after 15 minutes by a process which depends on protein synthesis; the formation of RNA, protein and SS DNA continues, and the infected cells elongate without septum formation, producing “snakes”. With ultraviolet-killed +X at low m.o.i., DNA, RNA and protein synthesis completely recover after a slight initial depression, and the cells continue to grow and divide at the normal rate. Irradiated #X may fail to inject functional DNA and may therefore be unable to express the information required for the arrest of host DNA synthesis and septation.

Infection with certain other phages is known to increase cell permeability and to inhibit macromolecular synthesis. For example, T2 at multiplicities below 5, when “Iysis from without” does not occur, increases permeability and leakage of aoid- soluble substance from the cells (Puck & Lee, 1954,1955). Some five to ten minutes after infection, the normal state appears to be restored (Puck & Lee, 1955) reflecting, perhaps, a general strengthening of the cells which may account for the finding of Visconti (1953) that secondary infection with T2 at a high multiplicity fails to cause “lysis from without”. Crawford (personal communication) has shown that cell permeability to TNS is increased when CAP is added som.e 20 minutes after infection with T5 at a multiplicity of five to ten; phage DNA synthesis comes to a halt about five minutes later (Pfefferkorn & Amos, 1958; Crawford, 1959). Presumably, the integrity of the cell membrane after T5 infection is dependent on a continuous repair process, requiring protein synthesis.

Nomura, Witten, Mantei & Eohols (1966) and Terzi (1967) have demonstrated a multiplicity-dependent, CAP-insensitive, general inhibition of macromolecular synthesis after infection with phage T4. A related situation is reported for DNA and protein synthesis after phage X infection (Terzi & Levinthal, 1967); here, inhibition requires the presence within the cell of the intact phage DNA, for it is not caused by empty phage capsids or host-modified phage, the DNA of which is degraded after penetration (Dussoix & Arber, 1962).

Substantial immunity to the inhibitory effects of high multiplicities of 4X develops between five and nine minutes after infection at low multiplicity. It does not develop in CAP, nor with primary phages killed with ultraviolet light; but once immunity has developed, nucleic acid synthesis is unaffected by high m.o.i. even in the presence of CAP. These findings suggest that a protein, the synthesis of which is induced by the primary phage, is responsible for the immunity. The enhancement by CAP of the inhibition by high m.o.i. may be accounted for in the following manner. Individual

228 A.B.STONE

phages adsorbed to the bacterial surface penetrate the cell asynchronously, so that in the absence of CAP, the “immunity” protein synthesized by the phages that penetrate early is able to prevent serious damage caused by the penetration of further phages. Hence, when protein synthesis is prevented by CAP, both early and late penetrations lead to damage, and inhibition is greater. In a similar way, phages treated with HNO, or ultraviolet light, and which as a result may be unable to synthesize “immunity” protein, are more inhibitory than live phages. This idea is supported by the finding that when protein synthesis is prevented by CAP, live $X is as inhibitory as ultraviolet- irradiated 4X.

The apparent failure of 4X which had received eight lethal HN& “hits” to induce immunity suggests that the protein responsible may be phage-coded. In this regard, there is some evidence that the product of the rI1 region of T4 is responsible for main- tenance of the cell membrane (Buller & Astrachan, 1968). In the case of +X, the timing of the development of immunity suggests that the gene product responsible may be one of the capsid proteins. A greater amount of capsid protein is manufactured by the +X-infected cell than is required for the assembly of progeny virions; a 13-fold excess has been observed by Krane (quoted in Knippers & Sinsheimer, 1968). Its synthesis begins some 5 to 8 minutes after infection; that is, at about the time immunity is first demonstrable. This protein has a high affinity for +X SS DNA, and attaches to progeny strands simultaneously with their release from the RF @nippers & Sinsheimer, 1968). It may similarly attach to the SS DNA of superinfecting +X as soon as penetration begins, preventing its further entry. This concept has previously been proposed as an explanation of “temporal exclusion”, the failure of superinfecting 4X DNA to form RF or to express its genetic potential (Hutchison & Sinsheimer, 1966). To explain immunity to cell damage, the assumption is required that injury to the cells is dependent on penetration of the phage DNA.

Alternatively, the failure of superinfecting phage to injure the cell may be a consequence of an alteration of the cell surface caused by the primary infection, Such an alteration would have to be more subtle than a general repairing and strengthening of the outer layers of the cell, for the slight permeability to TNSinduced by four +X/cell persists even at times when immunity to superinfection is demonstrable.

The specific, delayed arrest of host DNA synthesis after infection at low multi- plicity depends on protein synthesis at some time during the period preceding arrest (Lindqvist & Sinsheimer, 1967; see also Fig. 1). However, Ishiwa & Tessman (1968) have reached the opposite conclusion, but this may reflect the phenomenon described here. At the m.o.i. they used (10 p.f.u./cell), inhibition of DNA and RNA synthesis is pronounced even in the presence of CAP (see Figs 2 and 10). This non-specific effect would account for their observation that the inhibition of DNA synthesis occurred with little delay and was insensitive to CAP. It would also be compatible with their finding that a lysis-defective 513 strain, bearing a mutation only in cistron V (analo- gous to 4X cistron I), failed to synthesize a significant amount of SS DNA (cf. Fig. 6 of the present report).

The possible consequences of peripheral damage to the cells must evidently be borne in mind when high multiplicities of +X are used. However, in a study of RF replication at various multiplicities, from which it was concluded that replication is limited to one parental RF molecule/cell in slowly growing bacteria (Stone, 1967), many of the experiments were performed at multiplicities low enough to avoid these complications.

+X174 AND MACROMOLECULAR SYNTHESIS 229

I thank Professor R. L. Sinsheimer for supplying the phage mutants and the bacterial strains, and Dr D. J. R. Laurence for his generous gift of TNS. Part of this work was supported by the Medical Research Council.

REFERENCES Budman, D. R. & Pardee, A. B. (1967). J. Bact. 94, 1546. Buller, C. S. & Astrachan, L. (1968). J. VGoZ. 2, 298. Crawford, L. V. (1959). Virology, 7, 359. Denhardt, D. T. & Sinsheimer, R. L. (1965). J. Mol. Biol. 12, 647. Doermann, A. H. (1948). J. Bad. 55, 257. Dussoix, D. & Arber, W. (1962). J. Mol. Biol. 5, 37. Fraser, D. & Jerrel, E. A. (1953). J. BioZ. Chem. 205, 291. Hayes, W. (1968). The Genetics of Bacteria mnd their Viruses, p. 528. Oxford and Edinburgh:

Blackwell Scientific Publons. Hutchison, C. A., III & Sinsheimer, R. L. (1966). J. Mol. BioZ. 18, 429. Ishiwa, H. & Tessman, I. R. (1968). J. Mol. BioZ. 37, 467. Knippers, R. & Sinsheimer, R. L. (1968). J. Mol. BioZ. 35, 591. Lindqvist, B. H. & Sinsheimer, R. L. (1967). J. Mol. BioZ. 28, 87. Newton, B. A. (1954). J. Cien. Microbial. 10, 491. Nomura, M., Witten, C., Mantei, N. & Echols, H. (1966). J. Mol. BioZ. 17, 273. Pfefferkorn, E. & Amos, H. (1958). Virology, 6, 299. Puck, T. T. & Lee, H. H. (1954). J. Exp. Med. 99, 481. Puck, T. T. & Lee, H. H. (1955). J. Exp. Med. 101, 151. Sinsheimer, R. L. (1968). Prog. iVuc. Acid Res. and Mol. Biol. 8, 115. Sinsheimer, R. L., Starman, B., Nagler, C. & Guthrie, S. (1962). J. Mol. BioZ. 4, 142. Stone, A. B. (1967). Biochem. Biophys. Res. Comm. 26, 247. Stone, A. B. (1968). Abstr. European Phuge Mtg., Internatl. Lab. Genetics and Biophysics,

Naples. Stone, A. B. (1969). Biochem. J. 111, 39P. Terzi, M. (1967). J. Mol. BioZ. 28, 37. Terzi, M. & Levinthal, C. (1967). J. Mol. BioZ. 26, 525. Visconti, N. (1953). J. Bat. 66, 247.