VIROLOGY
66, 241-252 (19751
Effect of T4 Ghosts ANGELINA
on T4 Development
QUINTERO-RUIZ’
Institut
de Biologie
AND
Physico-Chimique
Accepted
March
EDWARD N. BRODY 75005 Paris, France
7, 1975
The effect of T4 ghosts on T4 development has been examined by analysis of early RNA species and polyacrylamide gel electrophoresis of proteins. Ghosts interfere with early development from the moment of infection. The transition from immediate to delayed early RNA is slowed down by ghosts as is the appearance and disappearance of early proteins. We have concluded that the delay seen in the immediate to delayed early transition is not a direct effect of ghost alteration of a membrane site for this transition. Ghosts also have an effect on the ability of T4 bacteriophage to inhibit host protein and RNA synthesis. Host protein bands are seen as late as 10 min after simultaneous ghost-phage infection, whereas such bands are no longer visible 3 min after normal phage infection. INTRODUCTION
T4 ghost particles adsorb to and kill host bacteria even though no T4 functions are expressed in such infections (see Duckworth, 1970a for a review). The changes that take place after such infections are of two types. On the one hand, there is an inhibition of active transport of nucleosides and nucleotides, and a depletion of the intracellular pool of nucleotides; at the same time, host macromolecular syntheses are rapidly inhibited (Duckworth, 1970b). The relationship between these two types of phenomena is not known. When T4 phage infects E. coli, there is an orderly sequence of development that can be followed by examining either RNA (Salser et al., 1970) or protein (O’Farrell and Gold, 1973a) synthesis. Classes of RNA and proteins have been characterized and their control is under active investigation. T4 development is inhibited when cells are treated with phage and ghosts simultaneously (Duckworth, 1971). We have examined the effect of ghost inhibition on the various control steps of T4
development in order to determine whether ghost interaction with host membrane blocks specific control steps in T4-infected cells. MATERIALS
Bacteria
AND METHODS
and phage. E. coli B” (Su-) is
our normal host for wild-type T4 and for all experiments reported here. E. coli CR 63 (Su,+) was used to prepare stocks of am N122, a T4 amber mutant in gene 42 (dCMP hydroxymethylase). The temperature for all experiments was 30”. Media. M9 plus 1% Casamino acids is our standard medium (Bolle et al., 1968). When proteins were to be labeled with a mixture of “C-labeled amino acids, the Casamino acid concentration was reduced to 0.02%. Phage preparation. E. coli B” was grown to 2 x lO*/ml at 30” and infected with T4+ at a multiplicity of 0.01. After 2-3 hr, aliquots were shaken with several drops of chloroform. If complete clearing of the aliquot was observed, the infected cells were centrifuged 10 min at 4000g. The bacterial pellet was resuspended in l/100 the original volume in 0.01 M Tris-Cl (pH 1Present address: Universidad National Autonoma de Mexico, Facultad de Quimica, Depat. de 7.5); 0.01 M MgCl,; and 0.05 M NaCl. A Bioquimica, Mexico 20, D. f. few drops of chloroform were added and the 241 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
242
QUINTERO-RUIZ
suspension was stirred at room temperature for 5 min. Two or three crystals of pancreatic DNase I were added and the stirring continued for 30 more min. The lysate was then diluted 2-fold with the same buffer and centrifuged 5 min at 4000g. The supernatant was used as a concentrated phage stock, The same procedure was used to purify T4 am N122, except that E. coli CR 63 was the host bacterium. Purification of these phage stocks was carried out by centrifugation through a step-gradient of CsCl (densities of 1.7, 1.5, and 1.3) for 1 hr in the Spinco SW 41 rotor at 149,000g. The phage bands were dialyzed first against 0.01 M Tris-Cl (pH 7.5); O.O,l M MgCl,; and 0.5 M NaCl; then against the same buffer without NaCl. Preparation of T4 ghosts. Purified T4 am N122 were used to prepare ghosts. In following a standard procedure (Duckworth, 1970b), we occasionally found rather high levels of surviving phage particles. When this occurred, we repeated the osmotic shock treatment used to prepare ghosts. Crude ghost preparations (95-96% inactivated) were further purified by sucrose gradient centrifugation (Duckworth, 1970b). The sucrose gradient was lo-30%; centrifugation was for 1 hr at 41,000g in a Spinco SW 25 rotor. Fractions were monitored by optical density measurements at 260 and 280 nm, by infectivity and by the capacity to inhibit E. coli colony formation. Killing titers were derived from the equation S = eTm, where S is the level of bacterial survivors, and m is the multiplicity of infection. The ghost peak obtained from sucrose gradient centrifugation contained fewer than 0.5% intact phage particles. Our ghost preparations tended to lose activity during storage; their capacity to inhibit T4 development decreased as a function of time. Thus, a given ghost multiplicity had less of an effect on delaying T4 development as the ghost preparation aged. RNA preparation. Unlabeled RNA from T4-infected cells was prepared according to a published procedure (Bolle et al., 1968). CAM RNA refers to RNA extracted 5 min after infection of E. coli BE to which 200
AND BRODY
/*g/ml chloramphenicol had been added 5 min before the addition of phage. Such an RNA preparation contains, by definition, only immediate early species (Brody et al., 1970a). Labeled RNA preparations were extracted from cells infected either with phage alone, or with phage plus ghosts. We used 10 &!i (in 50 ~1) of [5-sH]uridine (C. E. A., Saclay, France) per ml of cells. Pulses were terminated by addition of an excess of unlabeled uridine and simultaneous chilling in an ethanol-Dry Ice bath. Cells were subsequently centrifuged at 2”, resuspended, and extracted with hot phenol as for the unlabeled preparations. Hybridization-competition. Method II of a previously published procedure for liquid annealing was used (Brody et al., 1970b). Hybridization was carried out in 2 SSC at 60” for 18 hr. The reaction mixture was then treated with 10 pg/ml pancreatic RNase for 15 min at 37”, diluted 15-fold with 0.5 M KCl, plus 0.01 M Tris-HCl (pH 7.5), and subsequently filtered through nitrocellulose filters. Protein labeling and polyacrylamide gel electrophoresis. In some experiments, bac-
teria were irradiated with UV light before infection (Hosoda and Levinthal, 1968) in order to suppress residual host protein synthesis. The UV treatment consisted of placing the bacteria in a Petri dish and irradiating for 3 min with a UV lamp placed 10 cm above the culture. UVirradiated bacteria were then aerated 10 min at 30” before being used for either phage or phage-ghost infection. Pulse labeling of proteins with “Clabeled amino acids and treatment of samples for electrophoresis followed a published procedure (O’Farrell, Gold and Huang, 1973). Twelve percent SDS polyacrylamide gels with stacking gels were run in slabs, also according to this procedure. Gels were fixed using 0.1% Coomassie blue stain in 50% TCA for 30 min, then decolored overnight by incubating in 7% acetic acid. These gels were placed on Whatman No. 3 paper and dried under vacuum. The gels were autoradiographed using Kodak No. 0350 W X-ray film; autoradiographs were scanned with a Joyce-Loebl mi-
T4 GHOSTS
AND T4 DEVELOPMENT
crodensitometer. Quantitative evaluation of each band was effected by cutting bands out of the microdensitometer tracings and weighing them on a Mettler high-precision balance. RESULTS
We have verified the results of Duckworth (1971) that show the inhibitory effect of ghosts on T4 development. Table 1 shows that ghosts, added simultaneously with phage, inhibit infectious center formation, and that this inhibition depends on the multiplicity of ghost infection. This decline in infectious centers is paralleled by the decline seen in total phage yield 60 min after infection. That means that the average burst size stays more or less constant as the ghost-to-phage ratio increases, but that the number of cells capable of giving an infectious center decreases as the ghost multiplicity is increased. Such an effect has been reported for other bacteriophage systems (Fabricant and Kennell, 1972). We have verified (data not shown) that immunity to ghost inhibition is complete 3 min after infection, as had been previously reported (Vallee et al., 1972). We examined the program of T4 development in those cells simultaneously inTABLE 1 INHIBITION OF INFECTIOUS CENTERS AND PHACE PRODUCTION AS A FUNCTION OF GHOST MULTIPLICITY= Ghosts (multiplicity) 1 3 6 12
Infectious centers (%)
Phage (%)
62 67 12 11 2.2 2.5 0.3 0.1
41 59 20 28 10 12 0.5 1.2
‘E. coli BE were grown in M9 at 30” to a concentration of 5 x 108/ml and then infected with three phages + r ghosts per bacterium; x being the ghost multiplicity. Infectious centers were measured 5 mm after infection, and phage yield 60 min after infection, after addition of a few drops of chloroform to each culture. The percentages listed compare the yields found in mixed phage-ghost infection to those found in an infection by T4 alone (x = 0).
243
fected with phage and ghosts. The incorporation of thymidine into DNA is shown as a function of increasing ghost-to-phage ratio (Fig. 1). Ghosts depress DNA synthesis and at high ghost multiplicities the onset of DNA synthesis is delayed: in fact, patterns of DNA synthesis with 3 phages and between 3 and 10 ghosts per bacterium resemble the DNA-synthesis kinetics seen after infection with DNA-delayed mutants of T4 (Yegian et al., 1971). Immunity to ghosts seen 3 min after phage infection is reflected in the recovery of normal DNA synthesis kinetics from the DNA delayed type of synthesis seen in simultaneous infection (data not shown). Incorporation of uridine and of amino acids into TCAinsoluble material was also depressed as a function of ghost-to-phage ratio in simultaneous infections (Quintero-Ruiz, 1974). Immunity also reverses these kinetics by 3 min after phage infection. Since the ghost effect on development is already apparent at the beginning of DNA synthesis, we examined prereplicative RNA and protein synthesis in ghost-phage simultaneous infections. The sequential appearance of early RNA is well documented (Salser et al., 1970; Brody et al., 1970a). Immediate early RNA corresponds to the species normally synthesized during the first 1.5 min after normal infection; delayed early RNA appears only after this period. RNA was pulse labeled after infection with either six phage or three phage plus three ghosts per bacterium. Surviving bacteria were the same (4 x lo-*) in the two infections. The labeled RNA preparations were used in hybridization-competition tests with unlabeled CAM RNA, normal S-min RNA, and normal 20-min RNA. Material uncompeted by CAM RNA, but competed by 5-min RNA is a measure of the delayed early species,2 and it is clear that the appearance of this set of RNA molecules is delayed in ghost-treated T4infected cells (Table 2). The delay is not completely overcome even 10 min after ‘We include here those species, called quasi-late, which are thought to be synthesized by a mode of transcription different from the bulk of the early species present at 5 min (cf. O’Farrell and Gold, 1973b).
FIG. 1. aH-thymidine incorporation in T4 infected cells as a function of ghost concentration. E. coli BE at 5 x 10’ per ml were infected with T4 wild type at a multiplicity of three phages per bacterium plus a variable multiplicity of T4 ghosts. Four minutes after infection, [‘Hlthymidine at 5 &i/ml and 10 @g/ml was added to each infected culture. At various times, lOO-~1aliquots were removed, added to 200 ~1 of a solution of unlabeled thymidine at 1 mg per ml, and
the solution brought to 5% TCA. Samples were diluted with 5% TCA, left on ice for 30 min, and filtered through GF/C Whatman glass filters. The graph shows cpm incorporated into lOO+l aliquots as a function of time after infection. 04 T4 only; 04 T4 plus one ghost per bacterium; x-x T4 plus three ghosts per bacterium; A-A T4 plus 10 ghosts per bacterium; WA 10 ghosts per bacterium without T4.
TABLE
2
EFFECT OF GHOSTS ON T4 TRANSCRIPTION AS MEASURED BY HYBRIDIZATION-COMPETITIONS
Period of pulse-label (min) (O-l)
None (cpm) CAM 5 min (cpm) (WY 5 min (cpm) (%) 20 min (cpd (%)
-G
+G
345
53
18 (5)
-G
+G
-G
+G
-G
+G
-G
+G
-G
+G
4644
528
3052
145
3214
160
1714
218
2123
186
23
984
28
834
70
2123 (100)
144 (79)
603 (13)
3 (7)
41
0 (0)
(0.9) 0 (0)
(19-20)
(l-2)
0 (0)
i,
~(9-10)
(2-3)
322 (7)
35 (5)
(3-4)
1146
(4-5)
(38)
(16)
(31)
(17)
(48)
0 (0)
0 (0)
65
15 (9)
(0) O
-
0 (0)
275 (9)
(2)
(32)
502
0 (0)
(21) 17
(8)
a Each period represents a 1-min pulse-label with 10 &i/ml of [5-3H]uridine. The infection was carried out either with six phage per bacterium (-G), or with three phage plus three ghosts per bacterium (+G). After hot phenol extraction of the RNA preparations, hybridization was carried out with excess denatured T4 DNA (9 ag/ml) in 2 SSC for 18 hr at 60”. When competitor RNA was added, it was at a final concentration of 1 mg/ml. CAM 5 min represents competition by RNA extracted 5 min after infection which had had chloramphenicol (200 &ml) added 5 min before the addition of phage. Five minutes and 20 min represent normal early and late RNA, respectively. RNase treatment and filtration of hybrids were carried out as given in Materials and Methods. bThis represents the percentage of cpm hybridized in the presence of competitor RNA compared to the cpm hybridized in the absence of competitor RNA. Please note that the pulse given in the second column was done from 2 to 3 min after infection for the control and l-2 min after infection for the phage plus ghost infection. Background counts of samples annealed without DNA (between 5 and 10 cpm for the various preparations) were subtracted in all cases. 244
245
T4 GHOSTS AND T4 DEVELOPMENT
infection. Because of the delay in the appearance of early species, there is a delay in the appearance of late species; 20 min after infection by phage there is 21% late mRNA whereas the ghost-treated T4infected cells have not yet entered the late period (all of the pulse-labeled RNA is competed by 5 min RNA). The time of appearance of late species is somewhat variable and depends on the age of the ghost preparation. Thus, the effect of ghosts can be seen as early as the time of the normal immediate to delayed early transition. In order to decide whether this is a generalized slowing down of the T4 “clock” or whether ghosts block a specific step in the immediate early period, we examined T4 proteins labeled after phage, or phage plus ghost infections. When host cells are irradiated with UV
o-1
l-2
3-4
FIG. 2. Autoradiography of T4 proteins after normal infection and simultaneous ghost-phage infection. E. coli BE at 5 x 10’ per ml were irradiated with UV light as indicated in Materials and Methods. Half of the culture was infected with T4 wild type at a multiplicity of six phages per bacterium; the other half of the culture was infected with three phages plus three ghosts per bacterium. One-minute pulses with 3.6 pCi per ml of %-labeled amino acids were terminated by addition of ice-cold 10% casamino acids. Samples were treated as indicated in Materials
light, gel patterns show no interference from E. coli proteins. The sequential appearance of T4 proteins (cf. O’Farrell and Gold, 1973a) is retarded in such irradiated cells (Fig. 2) infected by phage and ghosts and the delay is evident from the time of infection. Immediate early proteins are as retarded as the delayed early and quasilate proteins; thus, the delay seen in the RNA transition is preceded by a delay in the appearance of immediate early proteins. A retardation is seen in the appearance and disappearance of all early proteins. We have quantified the appearance and disappearance of the early bands by using microdensitometry of the autoradiographs (Fig. 3). Comparison of individual bands for normal and simultaneous phageghost infections (Table 3) show that ghost treatment does somewhat affect the quan-
4-5
9-10
19-20
and Methods. Each 1-min pulse period is represented by two gels: the left-hand one represents the normal infection, and the right-hand one the simultaneous phage-ghost infection. The pulse periods are shown underneath each pair of gels. The amino acid incorporation was reduced to about 15% of the control in all the ghost-treated samples. The infection with phage alone gave 1.8 x 1O’Ophage per ml 60 min after infection (average burst size of 34). Simultaneous phage-ghost infection gave 1.0 x 10’ phage per ml at 60 min (average burst size of 10).
a
FIG. 3. Densitometer tracings of a polyacrylamide gel shown in Fig. 2. These tracings were obtained by scanning the autoradiographs shown in Fig. 2 in a Joyce-Loebl Chromoscan. For reference we have labeled some of the T4 proteins in each tracing (cf. O’Farrell, Gold, and Huang, 1973). (a) T4 infection labeled 4-5 min after infection; (b) T4 infection labeled 9-10 min after infection; (c) T4 plus ghost infection labeled 9-10 min after infection; (d) T4 infection labeled 19-20 min after infection; (e) T4 plus ghost infection labeled 19-20 min after infection.
X 45
b Fmua~ 3b 246
T4 GHOSTS
247
AND T4 DEVELOPMENT
FIGURE
3c
i
248
QUINTERO-RUIZ
AND BRODY
FIGURE3e
titative distribution of label in T4 proteins, but that this effect is minor compared to the over-all slowing down of the T4 “clock.” (For instance, the distribution of label in proteins of cells, pulse-labeled 9-10 min after simultaneous phage-ghost infection, shows some similarities to a normal 9to lo-min labeling and some to a normal 4to 5-min labeling.) The appearance and disappearance of T4 proteins is slowed down from the time of infection, but no class of proteins (defined with respect to their mode of control) seems to be affected more than any other. This type of labeling was also done in unirradiated host cells (Fig. 4). The gels were stained prior to autoradiography in order to identify E. coli proteins. The autoradiographic pattern confirms the general delay in appearance and disappearance of T4 proteins, but also shows another effect of ghosts on T4 development. There are bands, particularly in the region of the largest proteins, which continue to be labeled up to 10 min after infection in the phage-ghost infection, but which are no
longer labeled by 3 min after a normal infection. Inspection of the stained pattern identifies these bands as E. coli proteins. Since the surviving bacteria were of the order of lo-’ in the two infections, ghosts must prevent the inhibition of host protein synthesis by T4 phage. This result is paradoxical because ghosts alone are capable of inhibiting host protein synthesis; the modes of inhibition must be different and show mutual interference. That ghost and phage inhibit host functions by different mechanisms has been suggested previously (Goldman and Lodish, 1973). It should be emphasized that the largest E. coli proteins are the last to disappear in simultaneous phage-ghost infections (cf. Discussion). DISCUSSION
T4 ghosts adsorb to E. coli and kill them without the expression of any T4 genes. Ghosts affect membrane permeability and macromolecular syntheses, but the relationship between these effects has never been clear. We have examined the specificity of the inhibition of macromolecular
T4 GHOSTS AND T4 DEVELOPMENT TABLE 3 DISTRIBUTIONOF LABELEDPROTEINSSYNTHESIZED AFTERNORMALT4 INFECTIONAND SIMULTANEOUS PHAGE-GHOSTINIWTION~ Proteins (genes) t
43
rIIA 46
39
b
0.2 6.0 0.3 4.7 5.4 23.3
;rgl (k 10)b 20Jb 3.3 7.1 3.8
2.4
1.6 5.6 1.2
0.7 4.3 2.0
-
3.9
-
3.4 21.6
4.3 23.0
3.3 26.0
3.7 3.4 5.8
19.9 16.9 6.0 9.9 0 0
10.3
19.2 6.4 0.4
45
9.0
P: ”
20.5 4.7 12.8
13.1 8.5 1.6 3.6
4.0 8.4
5.8 3.8 10.3
18.0
0
4.2
4.2
-
-
21.5
9.0 -
52
X, 63, imm, 44, 47 and 32 rIIB 2 28ooo
62 55,56,12000
Late (23)
4.9
0.9 4.9
2 +T;: (9- (191010 2010
3.3 24.5
10.1 10.8 1.7 3.2
9.0
“The percentage of proteins listed in this Table were obtained by cutting out bands from microdensitometer tracings of autoradiographs of polyacrylamide gels (those shown in Fig. 31, and by weighing each band on a high-precision balance. The percentages represent the amount of label in early protein, except for the pulse period 19-20. These percentages are given for the total early protein plus P 23, since this is the major late protein seen in simultaneous phageghost infection labeled 19-20 min after infection, although the timing of P23 appearance is variable and depends on the age of a ghost preparation. Proteins were identified according to the published description of Gold and collaborators (O’Farrell, Gold, and Huang, 19731. The numbers correspond to T4 genes; X, 2, 28000, and 12006 are proteins for which amber mutants are not known. Pi” is internal protein III (Black and Ahmand-Zadeh, 1971). In two cases proteins were grouped together because of the difficulty in quantifying overlapping bands. “The numbers in parentheses represent the time (in minutes) after infection of the pulse labeling.
syntheses by measuring the effect of ghosts in a well-characterized viral program, that of T4 development. We have found that the T4 program is slowed down by ghost treatment and that this delay starts at the time of infection. All of the indicators in this program are delayed, and we find no evidence that there is a specific interaction of a control step and a ghost-modified membrane function.
249
The appearance of both RNA and protein species is slowed down by ghost treatment of T4-infected cells, and these results parallel what had previously been found for the ghost effect on E. coli macromolecular syntheses (Duckworth, 1970b). It is impossible to say whether there is a primary direct effect of ghosts which induces secondary phenomena. Nonetheless, the rapidity with which RNA and protein synthesis are slowed down in T4-infected cells suggests that both inhibitions are direct responses to ghost treatment. Can this generalized slowdown of all early development be accounted for solely by the accelerated leakage of small molecules from the ghost-treated cells? The inhibition of protein synthesis in ghost-treated E. coli takes place at the level of the ribosome (Nugent and Kennell, 1972) and has been localized at the step of polypeptide chain release (Fukuma and Kaji, 1972). If the same mechanism operates for T4 protein synthesis, it is not immediately apparent why this step alone should be inhibited by an accelerated loss of ATP and other nucleotides from the infected cells. A recent finding that ghost treatment induces a nucleolytic cleavage of 16 S ribosomal RNA (Simon and Kennell, 1974) is intriguing since nuclease action could result in the inhibition of both RNA and protein synthesis. This slowdown in early functions leads to the delay in DNA synthesis seen in simultaneous ghost-phage infections. Although the kinetics of DNA synthesis are similar to those seen in DD (DNA delayed) mutants of T4, the mechanism of the delay must be different in the two cases, since in DD mutant infections there is no general inhibition or delay in early RNA and protein syntheses (Yegian et al., 1971). Our results show a very distinct effect of T4 ghosts on the takeover of host functions by T4 bacteriophage. Host protein synthesis is seen up to 10 min after simultaneous phage-ghost infection, whereas host protein bands disappear within 3 min after phage infection at an equivalent multiplicity. This could be an expression of the delayed appearance of a T4 protein necessary for the rapid inhibition of host protein
o-1
4-5
3-4
FIG. 4. Effect of ghosts on T4 protein synthesis in unirradiated host cells. The protocol was the same as that used for the experiment shown in Fig. 3, except that the host cells were unirradiated E. coli BE. Although the ghost multiplicity was the same as that shown in Fig. 3, the inhibition was less marked; the ghost plus phage preparations having, on the average, 45% of the amino acid incorporation found in the 250
9-10
19-20
controls. After electrophoresis of the protein samples, the gel was stained with Coomassie blue (0.1% in 50% TCA) for 30 min, then destained overnight in 7% acetic acid. Protein bands reflect total cell protein. Autoradiography was carried out as described in Materials and Methods; (a) total protein; (b) autoradiography.
T4 GHOSTS AND T4 DEVELOPMENT
synthesis, since all early functions are delayed by ghost treatment. However, it is also possible that the inhibition of the initiation of host protein synthesis is the same in phage and in phage-ghost infections, but that chain elongation is much slowed down by the ghost treatment. Thus, host bands would be seen for a longer time in the phage-ghost infection than in the phage infection, even though initiation of E. coli proteins had been inhibited with the same kinetics. We can see in Fig. 4 that the largest host bands are the last to disappear in phage-ghost infections; such an observation is consistent with the interpretation that ghosts slow down elongation of E. coli proteins more than they interfere with the inhibition of E. coli protein initiation. Another measure of host takeover by T4 indicates that ghosts slow down this process. E. coli RNA synthesis is generally inhibited by 3 min after phage infection (Adesnik and Levinthal, 1970). The annealing efficiency of pulse-labeled RNA preparations to T4 DNA is usually at a maximum by this time. In the pulselabeled RNA preparations used in the experiments shown in Table 2, we found that ghost treatment affected the annealing efficiency of T4 RNA. During normal infection the annealing efficiencies varied between 14 and 22% (after the second minute of infection). In contrast, similar preparations from simultaneous phageghost infections were between 5 and 6%. If one discounts the possibility that RNA from ghost-phage infections is more degraded (thus giving TCA-precipitable but poorly hybridizing T4 RNA) than normal T4 RNA, this is best explained by a continued labeling of host RNA in ghost-phage infections. Since surviving bacteria were the same in these infections, this would mean that ghosts prevent the passage of RNA polymerase from E. coli to T4 DNA. Here, as was the case with protein synthesis, we do not know if there is a slow appearance of a T4 protein which inhibits E. coli RNA synthesis, or whether the inhibition of the initiation of host RNA synthesis is normal in phage-ghost infection but that slow propagation across host
251
transcription units accounts for the low annealing efficiency. ACKNOWLEDGMENTS This work was supported by the Delegation Gen&ale a la Recherche Scientifique et Technique (Contract 71.7.3094) and by Centre National de la Recherche Scientifique (G.R. 18). We gratefully acknowledge the support of Dr. M. Grunberg-Manago during the course of this work. A. Q.-R. acknowledges a grant from the Division des Relations Scientifiques of the French Ministry of Foreign Affairs. This work constitutes part of a Thesis presented by A. Quintero-Ruiz to the University of Paris VII on May 4th, 1974, to obtain the degree of Docteur de 1’Universite. We thank Dr. M. Vallee for helpful discussions, and Dr. L. Gold for help with the gel electrophoreses during his sojourn in Paris. REFERENCES ADESNIK,M., and LEVINTHAL,C. (1970). RNA metabolism in T4-infected Escherichia coli. J. Mol. Biol. 48, 187-208. BLACK, L. W., and AHMAD-ZADEH,C. (1971). Internal proteins of bacteriophage T4D: their characterization and relation to head structure and assembly. J. Mol. Biol. 57, 71-92.
BOLLE, A., EPSTEIN, R. H., SALSER, W., and GEIDUSCHEK,E. P. (1968). Transcription during bacteriophage T4 development: synthesis and relative stability of early and late RNA. J. Mol. Biol. 31, 325-348.
BRODY,E., SEDEROFF, R., BOLLE,A., and EPSTEIN,R. H. (1970 a). Early transcription in T4-infected cells. Cold Spring Harbour Symp. Quad. Biol. 35, 203-211. BRODY,E. N., DIGGELMAN,H., and GEIDUSCHEK, E. P. (1970 b). Transcription of the bacteriophage T4 template: Obligate synthesis of T4 prereplicative RNA in uitro. Biochemistry 9, 1289-1299. DUCKWORTH,D. H. (1970 al. Biological activity of bacteriophage ghosts and “take-over” of host functions by bacteriophage. Bacterial. Reo. 34, 344-363. DUCKWORTH,D. H. (1970 b). The metabolism of phage-ghost infected cells. 1. Macromolecular synthesis and transport of nucleic acid and protein precursors. Virology 40, 673-684. DUCKWORTH, D. H. (1971). Inhibition of T4 bacteriophage multiplication by superinfecting ghosts and the development of tolerance after bacteriophage infection. J. Viral. 7, 8-14. FABRICANT,R., and KENNELL,D. (1972). Exclusion of bacteriophages by T2 ghosts. J. Virol. 10.872-874. FLJKUMA,I., and KAJI, A. (1972). Effect of bacteriophage ghost infection on protein synthesis in Escherichia coli. J. Viral. 10, 713-720.
252
QUINTERO-RUIZ
GOLDMAN,E., and LODISH,H. F. (1973). T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms. J. Mol. Biol. 74, 151-161. HOSODA,J., and LEVINTHAL,C. (1966). Protein synthesis in Escherichia coli infected with bacteriophage T4 D. Virology 34, 709-727. NUGENT, K., and KENNELL, D. (1972). Polypeptide synthesis by extracts from Escherichia coli treated with T2 ghosts. J. Viral. 10, 1199-1204. O’FARRELL,P., and GOLD, L. M. (1973a). Bacteriophage T4 gene expression. Evidence for two classes of prereplicative cistrons. J. Biol. Chem. 246, 5502-5511. O’FARRELL,P., and GOLD, L. M. (1973b). Transcription and translation of prereplicative bacteriophage T4 genes in vitro. J. Biol. Chem. 248, 5512-5519. O’FARRELL, P., GOLD, L. M., and HUANG, W. M. (1973). The identification of prereplicative bacteriophage T4 proteins. J. Biol. Chem. 246, 5499-5561. QIJINTERO-RUIZ, A. (1974). Etude des interactions des
AND BRODY enveloppes proteiques du phage T4 et des cellules d’E. coli infectees par ce phage. These de Doctorat de I’Universite, Universite de Paris VII. SALSER,W., BOLLE, A., and EPSTEIN,R. H. (1970). Transcription during bacteriophage T4 development: A demonstration that distinct subclasses of the “early” RNA appear at different times and that some are “turned off” at late times. J. Mol. Biol. 49, 271-295. SIMON, M., and KENNELL,D. (1974). Defective 30 S ribosomal subunits after infection of Escherichia coli by T2 ghosts. J. Viral. 14, 1316-1313. VALLBE, M., CORNETT,J. B., and BERNSTEIN,H. (1972). The action of bacteriophage T4 ghosts on Escherichia coli and the immunity to this action developed in cells preinfected with T4. Virology 48, 766-776. YEGUN, C. D., MUELLER,M., SELZER,G. Russo, V., and STAHL, F. W. (1971). Properties of the DNAdelay mutants of bacteriophage T4. Virology 46, 906919.
66, 241-252 (19751
Effect of T4 Ghosts ANGELINA
on T4 Development
QUINTERO-RUIZ’
Institut
de Biologie
AND
Physico-Chimique
Accepted
March
EDWARD N. BRODY 75005 Paris, France
7, 1975
The effect of T4 ghosts on T4 development has been examined by analysis of early RNA species and polyacrylamide gel electrophoresis of proteins. Ghosts interfere with early development from the moment of infection. The transition from immediate to delayed early RNA is slowed down by ghosts as is the appearance and disappearance of early proteins. We have concluded that the delay seen in the immediate to delayed early transition is not a direct effect of ghost alteration of a membrane site for this transition. Ghosts also have an effect on the ability of T4 bacteriophage to inhibit host protein and RNA synthesis. Host protein bands are seen as late as 10 min after simultaneous ghost-phage infection, whereas such bands are no longer visible 3 min after normal phage infection. INTRODUCTION
T4 ghost particles adsorb to and kill host bacteria even though no T4 functions are expressed in such infections (see Duckworth, 1970a for a review). The changes that take place after such infections are of two types. On the one hand, there is an inhibition of active transport of nucleosides and nucleotides, and a depletion of the intracellular pool of nucleotides; at the same time, host macromolecular syntheses are rapidly inhibited (Duckworth, 1970b). The relationship between these two types of phenomena is not known. When T4 phage infects E. coli, there is an orderly sequence of development that can be followed by examining either RNA (Salser et al., 1970) or protein (O’Farrell and Gold, 1973a) synthesis. Classes of RNA and proteins have been characterized and their control is under active investigation. T4 development is inhibited when cells are treated with phage and ghosts simultaneously (Duckworth, 1971). We have examined the effect of ghost inhibition on the various control steps of T4
development in order to determine whether ghost interaction with host membrane blocks specific control steps in T4-infected cells. MATERIALS
Bacteria
AND METHODS
and phage. E. coli B” (Su-) is
our normal host for wild-type T4 and for all experiments reported here. E. coli CR 63 (Su,+) was used to prepare stocks of am N122, a T4 amber mutant in gene 42 (dCMP hydroxymethylase). The temperature for all experiments was 30”. Media. M9 plus 1% Casamino acids is our standard medium (Bolle et al., 1968). When proteins were to be labeled with a mixture of “C-labeled amino acids, the Casamino acid concentration was reduced to 0.02%. Phage preparation. E. coli B” was grown to 2 x lO*/ml at 30” and infected with T4+ at a multiplicity of 0.01. After 2-3 hr, aliquots were shaken with several drops of chloroform. If complete clearing of the aliquot was observed, the infected cells were centrifuged 10 min at 4000g. The bacterial pellet was resuspended in l/100 the original volume in 0.01 M Tris-Cl (pH 1Present address: Universidad National Autonoma de Mexico, Facultad de Quimica, Depat. de 7.5); 0.01 M MgCl,; and 0.05 M NaCl. A Bioquimica, Mexico 20, D. f. few drops of chloroform were added and the 241 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
242
QUINTERO-RUIZ
suspension was stirred at room temperature for 5 min. Two or three crystals of pancreatic DNase I were added and the stirring continued for 30 more min. The lysate was then diluted 2-fold with the same buffer and centrifuged 5 min at 4000g. The supernatant was used as a concentrated phage stock, The same procedure was used to purify T4 am N122, except that E. coli CR 63 was the host bacterium. Purification of these phage stocks was carried out by centrifugation through a step-gradient of CsCl (densities of 1.7, 1.5, and 1.3) for 1 hr in the Spinco SW 41 rotor at 149,000g. The phage bands were dialyzed first against 0.01 M Tris-Cl (pH 7.5); O.O,l M MgCl,; and 0.5 M NaCl; then against the same buffer without NaCl. Preparation of T4 ghosts. Purified T4 am N122 were used to prepare ghosts. In following a standard procedure (Duckworth, 1970b), we occasionally found rather high levels of surviving phage particles. When this occurred, we repeated the osmotic shock treatment used to prepare ghosts. Crude ghost preparations (95-96% inactivated) were further purified by sucrose gradient centrifugation (Duckworth, 1970b). The sucrose gradient was lo-30%; centrifugation was for 1 hr at 41,000g in a Spinco SW 25 rotor. Fractions were monitored by optical density measurements at 260 and 280 nm, by infectivity and by the capacity to inhibit E. coli colony formation. Killing titers were derived from the equation S = eTm, where S is the level of bacterial survivors, and m is the multiplicity of infection. The ghost peak obtained from sucrose gradient centrifugation contained fewer than 0.5% intact phage particles. Our ghost preparations tended to lose activity during storage; their capacity to inhibit T4 development decreased as a function of time. Thus, a given ghost multiplicity had less of an effect on delaying T4 development as the ghost preparation aged. RNA preparation. Unlabeled RNA from T4-infected cells was prepared according to a published procedure (Bolle et al., 1968). CAM RNA refers to RNA extracted 5 min after infection of E. coli BE to which 200
AND BRODY
/*g/ml chloramphenicol had been added 5 min before the addition of phage. Such an RNA preparation contains, by definition, only immediate early species (Brody et al., 1970a). Labeled RNA preparations were extracted from cells infected either with phage alone, or with phage plus ghosts. We used 10 &!i (in 50 ~1) of [5-sH]uridine (C. E. A., Saclay, France) per ml of cells. Pulses were terminated by addition of an excess of unlabeled uridine and simultaneous chilling in an ethanol-Dry Ice bath. Cells were subsequently centrifuged at 2”, resuspended, and extracted with hot phenol as for the unlabeled preparations. Hybridization-competition. Method II of a previously published procedure for liquid annealing was used (Brody et al., 1970b). Hybridization was carried out in 2 SSC at 60” for 18 hr. The reaction mixture was then treated with 10 pg/ml pancreatic RNase for 15 min at 37”, diluted 15-fold with 0.5 M KCl, plus 0.01 M Tris-HCl (pH 7.5), and subsequently filtered through nitrocellulose filters. Protein labeling and polyacrylamide gel electrophoresis. In some experiments, bac-
teria were irradiated with UV light before infection (Hosoda and Levinthal, 1968) in order to suppress residual host protein synthesis. The UV treatment consisted of placing the bacteria in a Petri dish and irradiating for 3 min with a UV lamp placed 10 cm above the culture. UVirradiated bacteria were then aerated 10 min at 30” before being used for either phage or phage-ghost infection. Pulse labeling of proteins with “Clabeled amino acids and treatment of samples for electrophoresis followed a published procedure (O’Farrell, Gold and Huang, 1973). Twelve percent SDS polyacrylamide gels with stacking gels were run in slabs, also according to this procedure. Gels were fixed using 0.1% Coomassie blue stain in 50% TCA for 30 min, then decolored overnight by incubating in 7% acetic acid. These gels were placed on Whatman No. 3 paper and dried under vacuum. The gels were autoradiographed using Kodak No. 0350 W X-ray film; autoradiographs were scanned with a Joyce-Loebl mi-
T4 GHOSTS
AND T4 DEVELOPMENT
crodensitometer. Quantitative evaluation of each band was effected by cutting bands out of the microdensitometer tracings and weighing them on a Mettler high-precision balance. RESULTS
We have verified the results of Duckworth (1971) that show the inhibitory effect of ghosts on T4 development. Table 1 shows that ghosts, added simultaneously with phage, inhibit infectious center formation, and that this inhibition depends on the multiplicity of ghost infection. This decline in infectious centers is paralleled by the decline seen in total phage yield 60 min after infection. That means that the average burst size stays more or less constant as the ghost-to-phage ratio increases, but that the number of cells capable of giving an infectious center decreases as the ghost multiplicity is increased. Such an effect has been reported for other bacteriophage systems (Fabricant and Kennell, 1972). We have verified (data not shown) that immunity to ghost inhibition is complete 3 min after infection, as had been previously reported (Vallee et al., 1972). We examined the program of T4 development in those cells simultaneously inTABLE 1 INHIBITION OF INFECTIOUS CENTERS AND PHACE PRODUCTION AS A FUNCTION OF GHOST MULTIPLICITY= Ghosts (multiplicity) 1 3 6 12
Infectious centers (%)
Phage (%)
62 67 12 11 2.2 2.5 0.3 0.1
41 59 20 28 10 12 0.5 1.2
‘E. coli BE were grown in M9 at 30” to a concentration of 5 x 108/ml and then infected with three phages + r ghosts per bacterium; x being the ghost multiplicity. Infectious centers were measured 5 mm after infection, and phage yield 60 min after infection, after addition of a few drops of chloroform to each culture. The percentages listed compare the yields found in mixed phage-ghost infection to those found in an infection by T4 alone (x = 0).
243
fected with phage and ghosts. The incorporation of thymidine into DNA is shown as a function of increasing ghost-to-phage ratio (Fig. 1). Ghosts depress DNA synthesis and at high ghost multiplicities the onset of DNA synthesis is delayed: in fact, patterns of DNA synthesis with 3 phages and between 3 and 10 ghosts per bacterium resemble the DNA-synthesis kinetics seen after infection with DNA-delayed mutants of T4 (Yegian et al., 1971). Immunity to ghosts seen 3 min after phage infection is reflected in the recovery of normal DNA synthesis kinetics from the DNA delayed type of synthesis seen in simultaneous infection (data not shown). Incorporation of uridine and of amino acids into TCAinsoluble material was also depressed as a function of ghost-to-phage ratio in simultaneous infections (Quintero-Ruiz, 1974). Immunity also reverses these kinetics by 3 min after phage infection. Since the ghost effect on development is already apparent at the beginning of DNA synthesis, we examined prereplicative RNA and protein synthesis in ghost-phage simultaneous infections. The sequential appearance of early RNA is well documented (Salser et al., 1970; Brody et al., 1970a). Immediate early RNA corresponds to the species normally synthesized during the first 1.5 min after normal infection; delayed early RNA appears only after this period. RNA was pulse labeled after infection with either six phage or three phage plus three ghosts per bacterium. Surviving bacteria were the same (4 x lo-*) in the two infections. The labeled RNA preparations were used in hybridization-competition tests with unlabeled CAM RNA, normal S-min RNA, and normal 20-min RNA. Material uncompeted by CAM RNA, but competed by 5-min RNA is a measure of the delayed early species,2 and it is clear that the appearance of this set of RNA molecules is delayed in ghost-treated T4infected cells (Table 2). The delay is not completely overcome even 10 min after ‘We include here those species, called quasi-late, which are thought to be synthesized by a mode of transcription different from the bulk of the early species present at 5 min (cf. O’Farrell and Gold, 1973b).
FIG. 1. aH-thymidine incorporation in T4 infected cells as a function of ghost concentration. E. coli BE at 5 x 10’ per ml were infected with T4 wild type at a multiplicity of three phages per bacterium plus a variable multiplicity of T4 ghosts. Four minutes after infection, [‘Hlthymidine at 5 &i/ml and 10 @g/ml was added to each infected culture. At various times, lOO-~1aliquots were removed, added to 200 ~1 of a solution of unlabeled thymidine at 1 mg per ml, and
the solution brought to 5% TCA. Samples were diluted with 5% TCA, left on ice for 30 min, and filtered through GF/C Whatman glass filters. The graph shows cpm incorporated into lOO+l aliquots as a function of time after infection. 04 T4 only; 04 T4 plus one ghost per bacterium; x-x T4 plus three ghosts per bacterium; A-A T4 plus 10 ghosts per bacterium; WA 10 ghosts per bacterium without T4.
TABLE
2
EFFECT OF GHOSTS ON T4 TRANSCRIPTION AS MEASURED BY HYBRIDIZATION-COMPETITIONS
Period of pulse-label (min) (O-l)
None (cpm) CAM 5 min (cpm) (WY 5 min (cpm) (%) 20 min (cpd (%)
-G
+G
345
53
18 (5)
-G
+G
-G
+G
-G
+G
-G
+G
-G
+G
4644
528
3052
145
3214
160
1714
218
2123
186
23
984
28
834
70
2123 (100)
144 (79)
603 (13)
3 (7)
41
0 (0)
(0.9) 0 (0)
(19-20)
(l-2)
0 (0)
i,
~(9-10)
(2-3)
322 (7)
35 (5)
(3-4)
1146
(4-5)
(38)
(16)
(31)
(17)
(48)
0 (0)
0 (0)
65
15 (9)
(0) O
-
0 (0)
275 (9)
(2)
(32)
502
0 (0)
(21) 17
(8)
a Each period represents a 1-min pulse-label with 10 &i/ml of [5-3H]uridine. The infection was carried out either with six phage per bacterium (-G), or with three phage plus three ghosts per bacterium (+G). After hot phenol extraction of the RNA preparations, hybridization was carried out with excess denatured T4 DNA (9 ag/ml) in 2 SSC for 18 hr at 60”. When competitor RNA was added, it was at a final concentration of 1 mg/ml. CAM 5 min represents competition by RNA extracted 5 min after infection which had had chloramphenicol (200 &ml) added 5 min before the addition of phage. Five minutes and 20 min represent normal early and late RNA, respectively. RNase treatment and filtration of hybrids were carried out as given in Materials and Methods. bThis represents the percentage of cpm hybridized in the presence of competitor RNA compared to the cpm hybridized in the absence of competitor RNA. Please note that the pulse given in the second column was done from 2 to 3 min after infection for the control and l-2 min after infection for the phage plus ghost infection. Background counts of samples annealed without DNA (between 5 and 10 cpm for the various preparations) were subtracted in all cases. 244
245
T4 GHOSTS AND T4 DEVELOPMENT
infection. Because of the delay in the appearance of early species, there is a delay in the appearance of late species; 20 min after infection by phage there is 21% late mRNA whereas the ghost-treated T4infected cells have not yet entered the late period (all of the pulse-labeled RNA is competed by 5 min RNA). The time of appearance of late species is somewhat variable and depends on the age of the ghost preparation. Thus, the effect of ghosts can be seen as early as the time of the normal immediate to delayed early transition. In order to decide whether this is a generalized slowing down of the T4 “clock” or whether ghosts block a specific step in the immediate early period, we examined T4 proteins labeled after phage, or phage plus ghost infections. When host cells are irradiated with UV
o-1
l-2
3-4
FIG. 2. Autoradiography of T4 proteins after normal infection and simultaneous ghost-phage infection. E. coli BE at 5 x 10’ per ml were irradiated with UV light as indicated in Materials and Methods. Half of the culture was infected with T4 wild type at a multiplicity of six phages per bacterium; the other half of the culture was infected with three phages plus three ghosts per bacterium. One-minute pulses with 3.6 pCi per ml of %-labeled amino acids were terminated by addition of ice-cold 10% casamino acids. Samples were treated as indicated in Materials
light, gel patterns show no interference from E. coli proteins. The sequential appearance of T4 proteins (cf. O’Farrell and Gold, 1973a) is retarded in such irradiated cells (Fig. 2) infected by phage and ghosts and the delay is evident from the time of infection. Immediate early proteins are as retarded as the delayed early and quasilate proteins; thus, the delay seen in the RNA transition is preceded by a delay in the appearance of immediate early proteins. A retardation is seen in the appearance and disappearance of all early proteins. We have quantified the appearance and disappearance of the early bands by using microdensitometry of the autoradiographs (Fig. 3). Comparison of individual bands for normal and simultaneous phageghost infections (Table 3) show that ghost treatment does somewhat affect the quan-
4-5
9-10
19-20
and Methods. Each 1-min pulse period is represented by two gels: the left-hand one represents the normal infection, and the right-hand one the simultaneous phage-ghost infection. The pulse periods are shown underneath each pair of gels. The amino acid incorporation was reduced to about 15% of the control in all the ghost-treated samples. The infection with phage alone gave 1.8 x 1O’Ophage per ml 60 min after infection (average burst size of 34). Simultaneous phage-ghost infection gave 1.0 x 10’ phage per ml at 60 min (average burst size of 10).
a
FIG. 3. Densitometer tracings of a polyacrylamide gel shown in Fig. 2. These tracings were obtained by scanning the autoradiographs shown in Fig. 2 in a Joyce-Loebl Chromoscan. For reference we have labeled some of the T4 proteins in each tracing (cf. O’Farrell, Gold, and Huang, 1973). (a) T4 infection labeled 4-5 min after infection; (b) T4 infection labeled 9-10 min after infection; (c) T4 plus ghost infection labeled 9-10 min after infection; (d) T4 infection labeled 19-20 min after infection; (e) T4 plus ghost infection labeled 19-20 min after infection.
X 45
b Fmua~ 3b 246
T4 GHOSTS
247
AND T4 DEVELOPMENT
FIGURE
3c
i
248
QUINTERO-RUIZ
AND BRODY
FIGURE3e
titative distribution of label in T4 proteins, but that this effect is minor compared to the over-all slowing down of the T4 “clock.” (For instance, the distribution of label in proteins of cells, pulse-labeled 9-10 min after simultaneous phage-ghost infection, shows some similarities to a normal 9to lo-min labeling and some to a normal 4to 5-min labeling.) The appearance and disappearance of T4 proteins is slowed down from the time of infection, but no class of proteins (defined with respect to their mode of control) seems to be affected more than any other. This type of labeling was also done in unirradiated host cells (Fig. 4). The gels were stained prior to autoradiography in order to identify E. coli proteins. The autoradiographic pattern confirms the general delay in appearance and disappearance of T4 proteins, but also shows another effect of ghosts on T4 development. There are bands, particularly in the region of the largest proteins, which continue to be labeled up to 10 min after infection in the phage-ghost infection, but which are no
longer labeled by 3 min after a normal infection. Inspection of the stained pattern identifies these bands as E. coli proteins. Since the surviving bacteria were of the order of lo-’ in the two infections, ghosts must prevent the inhibition of host protein synthesis by T4 phage. This result is paradoxical because ghosts alone are capable of inhibiting host protein synthesis; the modes of inhibition must be different and show mutual interference. That ghost and phage inhibit host functions by different mechanisms has been suggested previously (Goldman and Lodish, 1973). It should be emphasized that the largest E. coli proteins are the last to disappear in simultaneous phage-ghost infections (cf. Discussion). DISCUSSION
T4 ghosts adsorb to E. coli and kill them without the expression of any T4 genes. Ghosts affect membrane permeability and macromolecular syntheses, but the relationship between these effects has never been clear. We have examined the specificity of the inhibition of macromolecular
T4 GHOSTS AND T4 DEVELOPMENT TABLE 3 DISTRIBUTIONOF LABELEDPROTEINSSYNTHESIZED AFTERNORMALT4 INFECTIONAND SIMULTANEOUS PHAGE-GHOSTINIWTION~ Proteins (genes) t
43
rIIA 46
39
b
0.2 6.0 0.3 4.7 5.4 23.3
;rgl (k 10)b 20Jb 3.3 7.1 3.8
2.4
1.6 5.6 1.2
0.7 4.3 2.0
-
3.9
-
3.4 21.6
4.3 23.0
3.3 26.0
3.7 3.4 5.8
19.9 16.9 6.0 9.9 0 0
10.3
19.2 6.4 0.4
45
9.0
P: ”
20.5 4.7 12.8
13.1 8.5 1.6 3.6
4.0 8.4
5.8 3.8 10.3
18.0
0
4.2
4.2
-
-
21.5
9.0 -
52
X, 63, imm, 44, 47 and 32 rIIB 2 28ooo
62 55,56,12000
Late (23)
4.9
0.9 4.9
2 +T;: (9- (191010 2010
3.3 24.5
10.1 10.8 1.7 3.2
9.0
“The percentage of proteins listed in this Table were obtained by cutting out bands from microdensitometer tracings of autoradiographs of polyacrylamide gels (those shown in Fig. 31, and by weighing each band on a high-precision balance. The percentages represent the amount of label in early protein, except for the pulse period 19-20. These percentages are given for the total early protein plus P 23, since this is the major late protein seen in simultaneous phageghost infection labeled 19-20 min after infection, although the timing of P23 appearance is variable and depends on the age of a ghost preparation. Proteins were identified according to the published description of Gold and collaborators (O’Farrell, Gold, and Huang, 19731. The numbers correspond to T4 genes; X, 2, 28000, and 12006 are proteins for which amber mutants are not known. Pi” is internal protein III (Black and Ahmand-Zadeh, 1971). In two cases proteins were grouped together because of the difficulty in quantifying overlapping bands. “The numbers in parentheses represent the time (in minutes) after infection of the pulse labeling.
syntheses by measuring the effect of ghosts in a well-characterized viral program, that of T4 development. We have found that the T4 program is slowed down by ghost treatment and that this delay starts at the time of infection. All of the indicators in this program are delayed, and we find no evidence that there is a specific interaction of a control step and a ghost-modified membrane function.
249
The appearance of both RNA and protein species is slowed down by ghost treatment of T4-infected cells, and these results parallel what had previously been found for the ghost effect on E. coli macromolecular syntheses (Duckworth, 1970b). It is impossible to say whether there is a primary direct effect of ghosts which induces secondary phenomena. Nonetheless, the rapidity with which RNA and protein synthesis are slowed down in T4-infected cells suggests that both inhibitions are direct responses to ghost treatment. Can this generalized slowdown of all early development be accounted for solely by the accelerated leakage of small molecules from the ghost-treated cells? The inhibition of protein synthesis in ghost-treated E. coli takes place at the level of the ribosome (Nugent and Kennell, 1972) and has been localized at the step of polypeptide chain release (Fukuma and Kaji, 1972). If the same mechanism operates for T4 protein synthesis, it is not immediately apparent why this step alone should be inhibited by an accelerated loss of ATP and other nucleotides from the infected cells. A recent finding that ghost treatment induces a nucleolytic cleavage of 16 S ribosomal RNA (Simon and Kennell, 1974) is intriguing since nuclease action could result in the inhibition of both RNA and protein synthesis. This slowdown in early functions leads to the delay in DNA synthesis seen in simultaneous ghost-phage infections. Although the kinetics of DNA synthesis are similar to those seen in DD (DNA delayed) mutants of T4, the mechanism of the delay must be different in the two cases, since in DD mutant infections there is no general inhibition or delay in early RNA and protein syntheses (Yegian et al., 1971). Our results show a very distinct effect of T4 ghosts on the takeover of host functions by T4 bacteriophage. Host protein synthesis is seen up to 10 min after simultaneous phage-ghost infection, whereas host protein bands disappear within 3 min after phage infection at an equivalent multiplicity. This could be an expression of the delayed appearance of a T4 protein necessary for the rapid inhibition of host protein
o-1
4-5
3-4
FIG. 4. Effect of ghosts on T4 protein synthesis in unirradiated host cells. The protocol was the same as that used for the experiment shown in Fig. 3, except that the host cells were unirradiated E. coli BE. Although the ghost multiplicity was the same as that shown in Fig. 3, the inhibition was less marked; the ghost plus phage preparations having, on the average, 45% of the amino acid incorporation found in the 250
9-10
19-20
controls. After electrophoresis of the protein samples, the gel was stained with Coomassie blue (0.1% in 50% TCA) for 30 min, then destained overnight in 7% acetic acid. Protein bands reflect total cell protein. Autoradiography was carried out as described in Materials and Methods; (a) total protein; (b) autoradiography.
T4 GHOSTS AND T4 DEVELOPMENT
synthesis, since all early functions are delayed by ghost treatment. However, it is also possible that the inhibition of the initiation of host protein synthesis is the same in phage and in phage-ghost infections, but that chain elongation is much slowed down by the ghost treatment. Thus, host bands would be seen for a longer time in the phage-ghost infection than in the phage infection, even though initiation of E. coli proteins had been inhibited with the same kinetics. We can see in Fig. 4 that the largest host bands are the last to disappear in phage-ghost infections; such an observation is consistent with the interpretation that ghosts slow down elongation of E. coli proteins more than they interfere with the inhibition of E. coli protein initiation. Another measure of host takeover by T4 indicates that ghosts slow down this process. E. coli RNA synthesis is generally inhibited by 3 min after phage infection (Adesnik and Levinthal, 1970). The annealing efficiency of pulse-labeled RNA preparations to T4 DNA is usually at a maximum by this time. In the pulselabeled RNA preparations used in the experiments shown in Table 2, we found that ghost treatment affected the annealing efficiency of T4 RNA. During normal infection the annealing efficiencies varied between 14 and 22% (after the second minute of infection). In contrast, similar preparations from simultaneous phageghost infections were between 5 and 6%. If one discounts the possibility that RNA from ghost-phage infections is more degraded (thus giving TCA-precipitable but poorly hybridizing T4 RNA) than normal T4 RNA, this is best explained by a continued labeling of host RNA in ghost-phage infections. Since surviving bacteria were the same in these infections, this would mean that ghosts prevent the passage of RNA polymerase from E. coli to T4 DNA. Here, as was the case with protein synthesis, we do not know if there is a slow appearance of a T4 protein which inhibits E. coli RNA synthesis, or whether the inhibition of the initiation of host RNA synthesis is normal in phage-ghost infection but that slow propagation across host
251
transcription units accounts for the low annealing efficiency. ACKNOWLEDGMENTS This work was supported by the Delegation Gen&ale a la Recherche Scientifique et Technique (Contract 71.7.3094) and by Centre National de la Recherche Scientifique (G.R. 18). We gratefully acknowledge the support of Dr. M. Grunberg-Manago during the course of this work. A. Q.-R. acknowledges a grant from the Division des Relations Scientifiques of the French Ministry of Foreign Affairs. This work constitutes part of a Thesis presented by A. Quintero-Ruiz to the University of Paris VII on May 4th, 1974, to obtain the degree of Docteur de 1’Universite. We thank Dr. M. Vallee for helpful discussions, and Dr. L. Gold for help with the gel electrophoreses during his sojourn in Paris. REFERENCES ADESNIK,M., and LEVINTHAL,C. (1970). RNA metabolism in T4-infected Escherichia coli. J. Mol. Biol. 48, 187-208. BLACK, L. W., and AHMAD-ZADEH,C. (1971). Internal proteins of bacteriophage T4D: their characterization and relation to head structure and assembly. J. Mol. Biol. 57, 71-92.
BOLLE, A., EPSTEIN, R. H., SALSER, W., and GEIDUSCHEK,E. P. (1968). Transcription during bacteriophage T4 development: synthesis and relative stability of early and late RNA. J. Mol. Biol. 31, 325-348.
BRODY,E., SEDEROFF, R., BOLLE,A., and EPSTEIN,R. H. (1970 a). Early transcription in T4-infected cells. Cold Spring Harbour Symp. Quad. Biol. 35, 203-211. BRODY,E. N., DIGGELMAN,H., and GEIDUSCHEK, E. P. (1970 b). Transcription of the bacteriophage T4 template: Obligate synthesis of T4 prereplicative RNA in uitro. Biochemistry 9, 1289-1299. DUCKWORTH,D. H. (1970 al. Biological activity of bacteriophage ghosts and “take-over” of host functions by bacteriophage. Bacterial. Reo. 34, 344-363. DUCKWORTH,D. H. (1970 b). The metabolism of phage-ghost infected cells. 1. Macromolecular synthesis and transport of nucleic acid and protein precursors. Virology 40, 673-684. DUCKWORTH, D. H. (1971). Inhibition of T4 bacteriophage multiplication by superinfecting ghosts and the development of tolerance after bacteriophage infection. J. Viral. 7, 8-14. FABRICANT,R., and KENNELL,D. (1972). Exclusion of bacteriophages by T2 ghosts. J. Virol. 10.872-874. FLJKUMA,I., and KAJI, A. (1972). Effect of bacteriophage ghost infection on protein synthesis in Escherichia coli. J. Viral. 10, 713-720.
252
QUINTERO-RUIZ
GOLDMAN,E., and LODISH,H. F. (1973). T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms. J. Mol. Biol. 74, 151-161. HOSODA,J., and LEVINTHAL,C. (1966). Protein synthesis in Escherichia coli infected with bacteriophage T4 D. Virology 34, 709-727. NUGENT, K., and KENNELL, D. (1972). Polypeptide synthesis by extracts from Escherichia coli treated with T2 ghosts. J. Viral. 10, 1199-1204. O’FARRELL,P., and GOLD, L. M. (1973a). Bacteriophage T4 gene expression. Evidence for two classes of prereplicative cistrons. J. Biol. Chem. 246, 5502-5511. O’FARRELL,P., and GOLD, L. M. (1973b). Transcription and translation of prereplicative bacteriophage T4 genes in vitro. J. Biol. Chem. 248, 5512-5519. O’FARRELL, P., GOLD, L. M., and HUANG, W. M. (1973). The identification of prereplicative bacteriophage T4 proteins. J. Biol. Chem. 246, 5499-5561. QIJINTERO-RUIZ, A. (1974). Etude des interactions des
AND BRODY enveloppes proteiques du phage T4 et des cellules d’E. coli infectees par ce phage. These de Doctorat de I’Universite, Universite de Paris VII. SALSER,W., BOLLE, A., and EPSTEIN,R. H. (1970). Transcription during bacteriophage T4 development: A demonstration that distinct subclasses of the “early” RNA appear at different times and that some are “turned off” at late times. J. Mol. Biol. 49, 271-295. SIMON, M., and KENNELL,D. (1974). Defective 30 S ribosomal subunits after infection of Escherichia coli by T2 ghosts. J. Viral. 14, 1316-1313. VALLBE, M., CORNETT,J. B., and BERNSTEIN,H. (1972). The action of bacteriophage T4 ghosts on Escherichia coli and the immunity to this action developed in cells preinfected with T4. Virology 48, 766-776. YEGUN, C. D., MUELLER,M., SELZER,G. Russo, V., and STAHL, F. W. (1971). Properties of the DNAdelay mutants of bacteriophage T4. Virology 46, 906919.
