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Interactions between modified phage T4 particles and spheroplasts
VIROLOGY
63, 225435
interactions
Department
(1973)
Between
Modified
WENDY
C. BENZ’
Phage AND
T4 Particles
EDWARD
and
Spheropiasts
B. GOLDBERG
of Molecular Biology and Microbiology, Tufts University School of Medicine, 1% Harrison Avenue, Boston, Massachusetts 02111 Accepted February
9, 1973
Two types of contracted phage particles with full heads were compared. Artificially contracted T4 phage particles were produced by treatment with urea (UT+), and naturally contracted T4 phage particles lacking gene 12 product were recovered after temporary adsorption to bacteria (adslZ+). Both types can infect T4-resistant bacteria if the bacterial envelope is altered by penicillin or lysozyme-EDTA treatment. Both are inactivated by cell membrane preparations and by low concentrations of phosphatidylglycerol which cause the particles to release DNA. Both are sensitive to trypsin. Differences in the particles as to the type of spheroplasts they can infect and the degree of their sensitivity to phosphatidylglycerol and DNase are correlated with mode of contraction rather than with phenotype. The properties of these contracted phage are described in terms of the properties expected for particles at intermediate stages of adsorption. Several mutants in other baseplate genes were tested, and it was found that genes 9,11, and 12 are unnecessary for infection of spheroplasts. We conclude that baseplates, as well as long tail fibers and their corresponding receptors in the bacterial envelope, do not play a part in the infection process of these contracted particles. This suggests to LIS that these organelles are necessary only during the initial stages of infection.
richia coli B (c&12-$1) (Simon et al., 1970). Their morphological properties and interactions with bacteria and spheroplasts correspond in many respects to particles at the contracting and penetrating stages, and they appear to function as expected for all stages of adsorption after the sheath contracts.
INTRODUCTION
The interaction between T4 phage and the bacterial envelope during infection can be divided into several stages (see Fig. 1). Phage adsorption starts with landing on the cell surface and ends with the injection of DNA into the cell. Some postulated intermediate stages of this process are described in the legend to Fig. 1. In an attempt to study the details of the scheme and to identify intermediates in the adsorption process, we have examined the properties of two types of phage particles which resemble particles at inbermediate stages of adsorption: particles treated by urea (UT4) (van Arkel et al., 1961; Wais and Goldberg, 1969; Baumann et al., 1970) and particles with a baseplate mutation in gene 12 which were recovered after adsorption to Esche1 Present address: Department of Biology, The Johns Hopkins University, Baltimore, Maryland, 21205.
MATERIALS
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Media. P broth, washing fluid, LSTG, V broth, spheroplast dilution medium (SDM), and SDM top agar were described by Wais and Goldberg (1969). L Broth contained 10 g Bacto-tryptone (D&o), 5 g yeast extract (Difco), and 5 g NaCl in 1 liter distilled water and was brought to pH 7.0 by addition of 1 ml 1 N NaOH. 0.1 M Salt Solution contained 5.85 g NaCl, 3.40 g MH=J’O, , 3.55 g Na2P04 in 1 liter distilled water. All media (except SDM) were sterilized in the autoclave for 20 min at 121°C. SDM was sterilized by filtration and stored frozen. 225
Copyright All rights
AND
BENZ
AND
GOLDBERG
CONTRACTINQ
PENETRATING
FIG. 1. Stages of T4 phage adsorption. This drawing, adapted in part from Simon and Anderson (1967), represents our current view of the stages of adsorption, although much of it is still conjecture. Landing. The phage particle attaches to the LPS layer of the bacterial envelope by long tail fibers. The attachment of each fiber is reversible, and each can be repositioned as the particle moves over the surface of the bacterium once it has landed (the landing site is shown here in relation to a stationary bump drawn on the surface). Pinning. The landed particle continues to move un and down while wandering over the bacterial surface until the baseplate is in the proper position, close enough to the appropriate surface receptor, to make contact. Further movement is restricted and the particle is pinned down. Contracting. The pinned particle undergoes a series of morphological changes. The baseplate pins elongate; the baseplate itself expands and detaches from the distal end of the tail tube and is drawn away from the bacterial surface as the sheath contracts to expose the tail tube. Penetrating. The contracting particle, held taut against the bacterial envelope by elongated baseplate fibers, penet.rates the layers of the envelope with the tail tube. Unplugging. The penetrating particle reaches a tail tube receptor on the plasma membrane and is positioned for final unplugging of the tail tube. Injecting. The unplugged particle ejects DNA from the head through the tail tube. The DNA then passes into the bacterial cell.
Bacteria. E. coZi B was from the collection of Dr. A. D. Hershey, E. coli 011’ (B/K hybrid) with amber suppressor II (Bsuf) from Dr. E. Stahl, E. coli B40SuI with amber suppressor I from Dr. P. Strigini, E. coli CR63 (X) with amber suppressor I from Dr. L. Simon, E. coli CR63 from Dr. R. Edgar and E. coli B/4 from Dr. H. Revel. E. coli B40suI/4 was isolated in this laboratory from E. coli B40suI. It permits growth of T2 wild type and amber mutants, but not T4 phage.
Bacteriophage strains. T4Dac41 was from the collection of Dr. A Doermann and is the wild type phage (w.t.) in this work. T4D mutants amN69 (gene 12), am N93 (gene ll), ana El7 (gene 9), and am N5.!? (gene 37) were from Dr. R. Edgar. T4DamB17amNl20rdj (which makes neither heads (gene 23-) nor tails (gene 27-) and has a deletion in rI1) was from Dr. L. Simon. The amEl’7amN52 (gene 9, gene 37) double mutant was constructed in this laboratory. Nomenclature. Phage are viable particles
ADSORPTION
OF MODIFIED
which will infect bacteria, whereas phage particles (4) denotes only the physical entities and does not imply viability. Wild type phage is abbreviated w.t., Urea-treated wild type phage is abbreviated UT& Active UT+ are those few UT+ which can infect spheroplasts. Phenotypically altered phage particles are referred to according to the number of the mutant gene and the strain of E. coli on which they were grown. For example, an 11 .B40suI phage is mutant in gene 11 and grown on E. coli B40suI. 11-d indicates phage particles grown under restrictive conditions which lack the product of gene 11 (Pll). 11-4 also lack P12 since PI1 is an obligatory precursor to P12 addition on the morphogenetic pathway (King, 1968). Phage stock Wild type and am-B&: Bacteria were aerated overnight in P broth, diluted 20-fold into P broth, and incubated at 37°C for 30 min without aeration. One overnight phage plaque per 25 ml bacterial suspension was suspended in 1 ml P broth (with a few drops of chloroform) and then added to the bacteria. The infected culture was aerated for 5 hr at 37”C, chloroform was added, and the debris sedimented at 7000 g for 5 min. The supernatant was incubated at 37°C with DNase (10 pg/ml) and RNase (100 pg/ml) for 20 min, with pancreatin (100 pg/m.l) for another 20 min, and then filtered through a O.&pm Millipore filter. The phage in the filtrate were concentrated by sedimentation at 35,000 g for 45 min and washed 3 times with washing fluid. Before the last wash, the phage were passed through a 0.45~pm Millipore filter. The phage were finally resuspended in a few ml of washing fluid. 11 .B and 12.B: Bacteria were grown to 4 X lo8 cells/ml in P broth. The culture was cooled to room temperature, and 11. B40suI or 12.B40suI phage were added t,o a m.o.i. of 3-5. The culture was aerated by bubbling for 10 min at room temperature, sedimented at 7000 g for 3-5 min at cold or room temperature, and the cells resuspended in the original volume of P broth. This procedure removed most of the unadsorbed phage. The culture was bubbled vigorously at 30°C for 145 hr, chloroform was added, and the phage particles purified by low and
T4 PARTICLES
227
high speed centrifugation as for w.t., and resuspended in a few ml of washing Auid. 9. B and 9,S7. B: This procedure was modified somewhat from preparation of 11 .B. L Broth was used instead of P broth t,o retard spontaneous inactivation. After chloroform treatment, the lysate was centrifuged at 7000 g for 3 min at room temperature, and the supernatant centrifuged at 35,000 g for 45 min. The phage were then resuspended in 0.1 M salt solution in about Woo the lysate volume and stored at room temperature. In the case of 9.B or 9, 37 .B, freshly prepared phage stocks were used for each experiment. Radioa&ve phuge stocks. 32P-labeled phage stocks of w.t., am.BsuI, and 11 .B were made as above, except that lo-15 &i/ml 32P was added to the bacteria immediately before the addition of phage. In making 9.B, 12-B, and 9, 37-B, the 3eP was added after low speed centrifugation and resuspension in fresh medium. 35S-labeled w.t. stocks were made as above, except that LSTG was substituted for P broth, the bacteria were initially aerated at 37°C for 1 hr, and 100 PCi 35S was added immediately before addition of phage. Reagents. Phosphatidylglycerol (PG) was made from E. coli B/4 (Baumann et al., 1970). It was bubbled out of organic solvent into 0.1 M sodium phosphate buffer, pH 7.0, before addition to phage. Crystalline deoxyribonuclease I and lysozyme were from Worthington Biochemicals. Trypsin and trypsin inhibitor were from Sigma. Special biological preparations. Ureatreat’ed phage particles (UT4) were prepared as described by Wais and Goldberg (1969). Spheroplasts were either lysozyme-EDTA spheroplasts from E. coli B40suI/4, as described by Baumann et al. (1970), or penicillin spheroplasts of E. coli B40suI/4 as described by Wais and Goldberg (1969) for E. coli B, except that P broth was used instead of LSTG, and the cells were grown to a titer of about 8 X 10s cells/ml before adding V Broth, sucrose, and penicillin. Adsorbed i2- phage particles (adsld-4): E. coli B was grown in P broth to a titer of l-2 X lo8 cells/ml The cells were harvested and resuspended in one-tenth the original volume
228
BENZ
AND
of P broth. 12- phage were added to a m.o.i. of 3-5. The mixture was incubated at 30°C for lo-12 min and then centrifuged in the cold at 7000 g for 3 min. The supernatant was either used directly as a&12-4, or was diluted into P broth as required. Infective center assay for UT4 and Adsl2-4. UT+ and a&12-4 infectivity were tested in a spheroplast infective center assay. Lysozvme-EDTA spheroplasts of B40suI/4 were diluted 1:l with P broth after the addition of MgSOc (Baumann et al., 1970). UT+ were added to a m.o.i. of 0.1-1.0. After 10 min at 37”C, aliquots were plated directly with 3 ml SDM top agar and B40suI indicator bacteria onto plates previously spread with 3 ml SDM top agar. AdsW9 were added in similar multiplicities, but after 12 min incubation at 30°C T4D antiserum (K = 5) was added to reduce the background of about 10m5plaques/particle on B40suI. Three minutes later, aliquots were plated as for UT@. P9, Pll, and P12. Extracts containing P9, Pll, and P12 were prepared according to the method of Edgar and Lielausis (1968) as modified by Flatgaard (1969). E. coli B was grown in L Broth at 30°C to a concentration of about 4 X lo8 cells/ml. Phage amB17amNl20rclj~ CR63 were added to a m.o.i. of 5, and the culture aerated at 30°C. At 20 ‘min an equal amount of the same phage was added. At 50 min the culture was chilled in ice water and centrifuged at 7000 g for 10 min. The cells were then resuspended in >{oo the original volume of BUM buffer (Edgar and Lielausis; 1968) and frozen in small aliquots with acetone/dry ice. To test in vitro complementation, the freshly thawed extract was diluted 1: 1 with BUM buffer. One microliter of phage to be tested was then diluted into 0.20.3 ml of this extract, incubated for 2 hr at 3O”C, and titered on CR63(h). All 9-B, 9, 37*B, ll*B, and 12.B stocks used were activatible with such extracts, and this titer was used as a measure of total phage particles in the stock. DNA release from UT+ and AdslF4. TO measure DNA release from 32P-labeled phage particles after adsorption or treatment with urea of PC, samples were treated with DNase (60 pg/ml with 6 mM MgC12) for 7
GOLDBERG
min at 37°C (UT+) or 30°C (a&12-4). In the case of acZ.s12-4, 1 &I of 30% bovine serum albumin (Pentex) was added per 1 ml sample. After acid precipitation in cold 3 % perchloric acid for 5 min and sedimentation at 7000 g for 5 min, radioactivity in the supernatant was measured in a low background (2-3 cpm) flow counter. RESULTS
AND
DISCUSSION
Role of the Baseplate in Attachment of UT4 to Spheroplasts UT@ infection requires neither long tail fibers nor tail fiber receptors in the lipopolysaccharide layer of the cell envelope (Wais and Goldberg, 1969; Baumann et al., 1970; van Arkel et al., 1961). Therefore, the landing stage can be bypassed. To determine whether the baseplate is required, several baseplate mutations (King and Laemmli, 1970) were examined. Phage particles, lacking only the product of gene 12 (Mason and Hazelkorn, 1972), were completed when necessary by adding P12 in vitro (Edgar and Lielausis, 1968). 9-4 and 11-4 were completed by the same method. 9-4, 11-4, and 12-4 were tested before and after urea treatment on lysozyme-EDTA spheroplasts of E. coli B40suI/4. Table 1 shows that untreated particles did not infect. The urea-treated particles, however, proved to be as infective as urea-treated wild type phage, whether grown on permissive or restrictive hosts. These findings show that the products of genes 11 and 12 (associat’ed with baseplate pins-Simon et al., 1970) and the product of gene 9 (associated with outer parts of the baseplate-Flatgaard, 1969; and J. King, personal communication) are not required in UT4 infection of spheroplasts. P9, Pll, and P12 may be atypical since, unlike other baseplate proteins, they are not required for tail morphogenesis. The product of gene 10 is required for tail morphogenesis, and it has been shown that urea-treated phage with altered, inactivated PlO are as infective as UT+ (Dawes, J., unpublished data). This further strengthens the argument that the baseplate is not required for UT4 infection of spheroplasts. Between the times of landing and DNA in-
ADSORPTION TABLE UT4
1
INFECTION OF B40suI/4 Phage particle
w.t. 11. Bsu+ 1l.B 12.B40suI 12.B 9. B40suI 9, 37.B”
OF MODIFIED
adsorbed and subsequently detached (Simon were tested on lysozyme-EDTA spheroplasts of B40suI/4 and assayed for infective centers. Table 2 shows that &12-4 are as infective as UT4 (about 0.1%). Lysozyme seemed to be the most important part of the spheroplast treatment, since treatment without lysozyme (EDTA alone) decreased infectivity of ads12-4 to 4% of that on lysozyme-EDTA spheroplasts. In comparison, infectivity of UT+ on cells treated with EDTA alone remained relatively high. This suggests that destruction of murein in the cell membrane, which is caused by lysozyme treatment, may be more necessary for ads12-4 infection than for UT4 infection. Infectivity of adsl2-4 on penicillin spheroplasts is less than 1% that of UT& Ib is possible that infection is inhibited by noncrosslinked murein or some other cell envelope component. On the other hand, such infection may require a component present on lysozyme-EDTA spheroplasts that is lacking or altered on penicillin spheroplasts. A third possibility is that ads12-9 are inactivated by a Mg*+ dependent DNase released by penicillin treatment (Balte, 1971). This is unlikely, however, because adsl2-4 do not infect penicillin spheroplasts even when excess EDTA (0.014M is present (Table 2). Since adsl2-4 infect spheroplasts of B40suI/4 (normally resistant to T4 infection but made infectible by lysozyme-EDTA treatment), they too bypass t’he landing stage. The particles lack P12, so that component of the basepate is not required either. It was not possible to test the requirement for P9 and Pll since 9-4 and II-+ would not adsorb to bacteria to produce contracted particles.
SPHEROPLASTS' et al., 1970). These adsl2-4
Relative infectivity of baseplate mutants untreated
urea-treated
SO.07 0.01 <0.002 SO.05 0.03 10.17 c
1.00 0.45 0.95 0.65 1.45 0.60 0.50
Q Infectivity was measured by the spheroplast infective center assay. T4D antiserum (K = 5) was added to untreated phage a few minutes before plating infective centers. Infectivity is expressed as the number of infective centers formed divided by the total number of phage particles. This number is about 1 X lo+ for wild type (w.t.) UT+ and is defined as a relative infectivity of 1.00 to which all the mutant preparations are compared. b A 9,37.B double mutant was used instead of a 9.B single mutant because 9.B had too high a background of phage to be useful in this system. Since 99+ lack long tail fibers and 37 is a tail fiber gene, this double mutant has the same morphology as 9- alone. c The infective phage in a 9,37.B stock were 4% as responsive to T4D antiserum as wild type T4. Therefore, not enough viable phage could be removed to make accurate measurement of spheroplast infection.
jection, phage show marked changes in appearance (Simon and Anderson, 1967). Their baseplates expand and sheaths contract to expose the tail tube. UT4 resemble phage at those intermediate stages (Daems et al., 1961). However, only 0.1% of UT4 are infective, and so electron microscopic observations of the majority may not characterize the few which are infective. Nevertheless, UTC$ fulfill the prediction that tail fibers and functional baseplates are not required for a phage infection after contraction. Infectivity
229
T4 PARTICLES
of AdslP~
To obtain more naturally contracted parCcles for comparison with UT+, 12-4 were mixed with E. coli B. Most of the particles
DNase Sensitivity
qf UT4 and Ads 12-4
The firmness and specificity of the adsorption of UT+ to spheroplasts was tested with DNase. In order to determine whether DNA from infective UT+ is exposed at any stage between pinning and final injection, UT4 and mixtures of UT+ and spheroplasts were treated with DNase. Table 3 shows that UT+ are insensitive to DNase up to 100 pg/ml and UT+spheroplast mixtures up to
230
BENZ TABLE
INFECTIVITY
OF UT+
B40suI/4
2
AND adS12-+ SPHEROPLASTP
Spheroplasts
Lysozyme + EDTA Lysozyme alone EDTA alone Penicillin Penicillin + 0.014 iM EDTA Penicillin + 0.0007 M EDTA
AND
ON E.
COk
Relative infectivity UT4
a&12-$lb
1.00 1.00 0.70 0.10 0.08 0.13
0.50 0.10 0.021 10.001 <0.0015 lO.001
a Infective center assays, lysozyme-EDTA and penicillin spheroplasts were spheroplasts, made as described in Materials and Methods. 0.1 mg/ml lysozyme and 0.7 mM EDTA were used unless noted otherwise. Relative infectivity is defined in Table 1. b The concentration of adsl2-4 was determined from the amount of 3zP in the supernatant solution after adsorption of 32P-labeled 12-4 and from the known specific activity of the particles as determined by in vitro complementation and is, therefore, a minimum estimate. Normally 25-35y0 of the 32P was found in the supernatant fluid. However, 30-40’% of this z2P was acid soluble (see also Fig. 8). Thus, the actual number of potentially active adsl2-4 is probably >$ to $6 the amount determined from the crude estimate, and the actual infectivity might be 1.5-2-fold greater than shown. Simon et al. (1970) reported that most 12- phage detached after adsorption and almost no DNA was released. However, our method of preparation differed in that our bacteria were more concentrated and a smaller fraction of the particles may have reversibly adsorbed, yielding an even higher infectivity. In any case, we chose those concentrations of phage and bacteria that gave the most infective particles on spheroplastjs.
10 Hg/ml (or 100 pg/ml DNase, P. Arscott, unpublished experiments). This insensitivity to DNase and the rapid and specific adsorption of active UT+ to spheroplasts supports the idea of a firm and specific union leading to a direct, protected transfer of DNA from particle to host. These findings also rule out transfection as the mechanism of DNA transfer of UT& Table 3 also shows that udsl2-4 are sensitive to 0.1 fig/ml DNase under conditions in which UT+ and 12-4 are unaffected
GOLDBERG
by lo4 times that concentration. In order to determine if DNase attacks DNA while still in the phage particle or during injection, a&12-4 were incubated with 0.1 pg/ml Dn’ase and diluted into spheroplasts at various times. Figure 2 shows that inhibiTABLE INHIBITION
OF a&12-+
DNase concn Gww
0 0.01 0.1 1.0 10.0 100.0
Fraction
3 BY DNASE~
INFECTIVITY
of infectivity
remaining
UT4
&12-4
12-4
1.00 0.93 1.00 0.93 1.00 1.00
1.00 1.00 0.37 0.10 0.04 0.01
1.00
0.92 0.97
a UT+, adsl2-4, and 12-+ were incubated with various concentrations of DNase and 0.006 M MgClz at 30’ C for 7 min. Infectivity of adsl2-4 and of UT4 was then assayed by the spheroplast infective center assay, in which the DNase concentration was diluted lo-fold. Infectivity of the (unadsorbed) 124 was assayed by the ability to be activated in the in vitro complementation assay.
-
PREINCUBATION
TIME
(MIN)
FIG. 2. Effect of preincubation with DNase on infectivity of adslr+. Samples of adslic4 were incubated with 0.1 fig/ml DNase in 0.006 E4 MgCls at 30’ C for the times indicated and then assayed by the spheroplast infective center assay. The ads124 particles are somewhat unstable at 30” C; therefore the experiment was controlled with parallel incubations of the ads124 at 30” C with MgC12 only (-DNase).
ADSORPTION
OF MODIFIED
tion of infective center formation is dependent on the time of preincubation with DNase. Therefore DNase inactivated a&12-4 before any contact with spheroplasts. We do not know why the value (extrapolated from Fig. 2) for no preineubation is 25% inhibition of infective centers. It is possible that 0.01 pg DNase, under these conditions, may also interfere with transfer of DNA from UT4 after contact with spheroplasts. It is unlikely, however, that a DNA molecule as large as that of T4 could remain intact in a solution of 0.1 pg/ml DNase or even 0.01 pg/ml DNase. Yet, Table 3 shows that UT+ and 12-4 remain fully infective and lo-40 % of a&12-I#J remain infective under those conditions. Such high frequencies of transfection (> 10-4) have not yet been demonstrated (Baltz, 1971; Benzinger et ai., 1971). As further confirmation that UT4 and a&12-4 do not transfer their DNA by transfection, the particles were treated with
CTRYPSINI
pa/ml
FIG. 3. Sensitivity of UT+ and adsl2-+ to trypsin. The phage were incubated with various concentrations of trypsin for 7 min at 30” C and assayed by the spheroplast infective cent.er assay. Unadsorbed 12+ were assayed for reactivation by in vitro complementation. Wild type phage were assayed by titering on CR63. Since the various phage preparations had to be kept in different media (0.1 M PO1 , pH 7.0, 2% BSA for UT+; P broth for adsl2-4; BUM buffer for 12-4), sensitivity of UT+ to trypsin was tested in each of the media and found not to vary (unpublished data).
gg z f z
231
T4 PARTICLES
2
3 PG CONTENT
4
5
6
X IO5 M
FIG. 4. Inhibition of UT+ by different bacterial membrane layers. The four membrane fractions from S. typhimurium were separately incubated with UT+ preparations for 10 min at 37’C at the concentrations indicated. The UT+ were then assayed by the spheroplast infective center assay. The PG concentrations of the membrane fractions were calculated from their relative protein and PG content. The rmoles PG/mg protein (determined by J. Carson) are: (A) 0.052, (B) 0.032, (C) 0.031, (D) 0.029. The fractions A-D represent successive layers of the cell envelope from inner plasma membrane to outer lipopolysaccharide membrane (Osborn et al., 1972).
trypsin at various concentrations. Figure 3 shows that both are sensitive to concentrations as low as 0.5 pg/ml. The addition of trypsin inhibitor immediately after trypsin treatment and before assaying for infective centers did not alter these results. This suggests that trypsin, like DNase, acts directly on the contracted phage particles themselves. The sensitivity of UT# infectivity to low concentrations of trypsin and theinsensitivity to high concentrations of DNase prove that DNA transfer during UT4 infection is not by transfection. For a&12-4, however, it is still possible that release of DNA into the medium is triggered by a specific interaction between the particle and the spheroplast which is inhibited by trypsin action. Such a mode of infection should be somewhat sensitive to DNase, and this might also help to explain the residual inhibition of infective centers at zero time in Fig. 2.
232
BENZ
PG CONTENT
AND
X IO’ M
FIG. 5. Inhibition of UT+ and a&12-+ by membrane vesicles. S. typhimurium membrane vesicles (Kaback and Stadtmann, 1966) were incubated for 10 min at 30°C with either UT+ or a&12-6. Membrane vesicles, diluted to the appropriate concentration in the buffer used for the final stage of their preparation, were mixed with an equal volume of phage particles for incubation. Control preparations, used for determination of 100% infectivity, lacked membrane vesicles in the buffer. The PG concentration of the membrane vesicles was calculated from the protein content according to the ratio 0.09 rmoles PG/mg protein (Kaback and Dittmer, 1969).
E$ect of Membrane Components on the Injectivity of UT4 and AdslFqi The differences between UT+ and a&12-9 in sensitivity to DNase and ability to infect penicillin spheroplasts suggest’a difference in their mode of infection. Therefore, we investigated the effects of various components of the spheroplast membrane. LysozymeEDTA spheroplasts of Salmonella typhimurium were sonicated and the debris separated on a sucrose gradient containing EDTA (Osborn et al., 1972). The four fractions (a gift from J. Carson) correspond to layers of the cell envelope. Figure 4 shows that all fractions inactivate UT& although the inner layer seems to be a little less effective. Membrane vesicles of S. typhimurium (Kaback and Stadtman, 1966) were also tested, and Fig. 5 shows that they inactivate UT4 and a&12-+ to an equal degree at low concentrations. At higher concentrations, 75 % of UT+ are inactivated while 100% of a&12-4 are inactivated. The fraction of
GOLDBERG
UT+ which was not inactivated is not yet explained. Baumann et al. (1970) showed that phosphatidylglycerol (PG), alone of all components of the gram-negative cell membrane, inactivates UT+ Since all the membrane fractions and membrane vesicles tested contained PG, UT+ and a&12-9 were tested for sensitivity with purified PG. Figure 6 shows that’ a&12-4 are 100-1000 times more sensitive than UT4. To determine whether this might be due to the absence of P12 or to the method of contraction, UT12-$3 were compared with UT+. Figure 7 shows that UT+ and UT12-4 are almost equally sensitive to PG. The same was true with UT9-+ and UTll-4. Thus, these baseplate components are not involved in the inactivation of active UT+ by PG. If PG does play a role in phage infection, a&12-I$ appear to be at a stage for eflicient interaction with PG. The ektreme sensitivity of these naturally contracted particles suggests that similar interactions occur in vivo.
z $ loo- o--oodl12-q /y--/p oI--Q XX I-x UT+ ii ,I ; 80: 2 ,’ x iiy 601’ 1’ w ,’ > F ,’ 0 40lk! ,/‘O x1 z N k 20/’ 0,’ : , F , 5 I .’ /’ XIJ i O 10-7 10-G IO 10-5 10-g 10-8 z LPGI
hi
FIG. 6. Inhibition of UT+ and a&12-4 infection by phosphatidylglycerol. UT+ were incubated with various PG concentrations at 37” C for 8 min and adslZ+ at 0” for 7 min. The lower temperature was used for ads12+ because it has been shown (unpublished data) that these particles are inactivated by PG faster and as completely at 0” C as at higher temperatures and that higher temperatures tend to inactivate adsW+ even in the absence of PG. Infectivity was then assayed by the spheroplast infective center assay.
ADSORPTION
DNA
Ejection from Mediated by PG
UT+
OF MODIFIED
and Ads 12-4
mechanism of inhibition of infection, since DNA was released most easily from noninfective particles. Figure 8 shows that UT9-4, UTll-r$, and UTl2-C$ release their DNA as easily as UT& However, since a smaller fraction of DNA is released by the action of PG on UTll-C#I and UTl2-9, Pll and P12 may be involved to some extent in the release of DNA from UT# by PG. A comparison of Figs. 6 and 8 shows that maximal DNA release by UT4 is complete with lo+’ M PG. This confirms the results of Baumann et al. (1970) that DNA release in UT+ is more sensitive to the action of PG than is infectivity. The existence of different classes of UT+ reinforces the possibility t’hat infective UT+ may differ structurally from the majority. Urea treatment of 9- phage particles and reversible adsorption of 12- phage particles both lead to loss of over 40 % of the DNA even before addition of PG. A&12-4 release the maximum amount of DNA (60 %) at 5 X lo+ f&l PC (see Fig. S), which is about the same concentrat,ion of PG needed for maximal loss of infectivity (100 %) (see Fig. 6). The uniformity of a&12-$ with respect to inhibition of infectivity and release of DNA by PG makes it less likely that in-
Baumann et al. (1970) reported a second action of PC on UT+: the release of DNA from about one half of all UT& They were unable to relate this observation to the
CPGI X Id%
7. Inhibition of infection by phosphatidylglycerol for UT+ of various genotypes. Ureatreated phage preparations were incubated with various concentrations of PG for 8 min at 37” C, and their subsequent infectivity assayed by the spheroplast infective center assay. The same 32P-labeled phage preparations were also used for the experiments shown in Fig. 8. FIG.
I I
I 2
233
T4 PARTICLES
I 3
I 4
I 5
I 6
I 7
CPGI X IO6 M FIG. 8. DNA release by phosphatidylglycerol from UT+ of various genotypes. The same 32P-labeled adslZ$~ as in Figs. 6 and 7 were incubated with PG as described in the legend to Fig. 6, and DNA release was determined. The DNA released in controls without PG varied from about 15% to 45% and depended upon the genotype of the phage to be treated. In addition, the amount of free DNA before PG addition for UT+, UTll-4, and UT124 varied in different preparations from 5% to 26% but had no effect on the maximum amount of DNA released with PG (unpublished data).
234
BENZ
AND
fective particles differ grossly from the maj ority. CONCLUDING
REMARKS
We have altered phage by two completely different treatments: with the general denaturing agent, urea, and by interruption of the natural adsorption of phage by alteration of the baseplate protein, P12. The resulting particles have many of the properties postulated for phage at intermediate stages of adsorption. It is interesting that both methods yield such similar particles, although the second method is more natural. Morphologically, both have contracted tails and full heads (Daems et al., 1962; Flatgaard. 1969; Baumann et al., 1970; Simon et al., 1970). Both will infect spheroplasts and can bypass the landing stage of adsorption. In both cases, only 10e3 of the particles are infective. The differences in host range and sensitivity to PG and DNase may reflect only minor differences in the particle, which may in turn belie finer distinction in the exact stage of adsorption at which the particle must commence infection of the spheroplast,. It is possible that a&12-4 are at a more advanced stage than UT+. The overall similarity of the more physiological adsl2-4 to UT#, however, lends support to the idea that UT+ also are adsorption intermediates. The existence of infective particles with contracted sheaths and full heads shows that the main purpose of contraction is probably to bring the tip of the tail tube to the inner membrane surface (Simon and Anderson, 1967) for the final joining of the tail tube with the plasma membrane. Only then, aft,er sheath contraction is complete, is DNA ejected from the phage and taken up by the bacterium. This process may well involve PG in the bacterial membrane as illustrated with UT4 and especially with adsl2-4. Not all contracted particles with full heads, however, are infective on spheroplasts. Spontaneously contracted 9-4 (Flatgaard, 1969) will not infect spheroplast,s (W.C.B., unpublished results). In fact, the means of sheath contraction is crucial to infection, since phenotypically identical particles contracted by different means show different infection properties on sphero-
GOLDBERG
plasts. Thus, UT9-4 are infective, whereas spontaneously contracted 9-4 are not. UT12-4, like UT4, differ from adsl2-4 in t,he type of spheroplast they will infect, DNase sensitivity, PG sensitivity, etc. Despite these differences, al1 of these particles are indistinguishable from UT4 in gross morphology. The exquisite sensitivity of infectivity and DNA release from adsl2-4 to PG suggests a physiological role of PC in DNA ejection during or after penetration. Jesaitis and Goebel (1952) also showed that LPS preparations released DNA from phage, and this was corroborated by Wilson et al. (1970). Thus, this t’ype of phenol-ext’racted cell wall preparation must contain all the components required for the various stages of phage adsorption, including DNA ejection. Jesaitis and Goebel (1953) also showed that this DNA release depended upon an identified (et,hanol-extractable) lipid. Both UT+ and c&12-9 were inactivated by the various membrane preparations at a calculated PG content of lo+’ M (see Figs. 4 and 5). This inactivation probably does not reflect their PC content only. The PC in the membrane could be ineffective, and both types of particles could be equally sensitive to some membrane receptor site. We would like to find, therefore, a PGfree membrane preparation which inactivates contracted particles without causing them to release their DNA. It would therefore be of interest to test the potency of various cell envelope fractions for their ability to release DNA as well as to adsorb phage particles at various stages of infection. The fact that contracted particles like UT+ and u&12-4 cannot infect bacteria directly but can infect spheroplasts implies that the bacterial surface must be altered to form or expose receptors used in late stages of infection. It seems clear that phage adsorption involves levels of interaction between the phage and the host envelope and that these mutual interactions must occur in an ordered sequence. The chemistry of these interactions is not yet fully understood. The isolation of new phage adsorption intermediates and different preparations of alt’ered cell envelopes should help toward this end.
ADSORPTION
OF MODIFIED
ACKNOWLEDGMENTS We thank Patsy Arscott for valuable help in preparing this manuscript and especially for her help in naming the stages of adsorption, Dr. J. Dawes for permission to quote her results before publication, Sherry Sass for technical help, and Dr. Andrew Wright for continuing interest and discussion. This work was supported in part by N.I.H. Grant No. GM13511 and N.S.F. Grant No. GB-27601. E.B.G. is a Career Development Awardee of the N.I.H. REFERENCES BALTZ, R. H. (1971). Infectious DNA of bacteriophage T4. J. Mol. Biol:62,425437. BAUMANN, L., BENZ, W. C., WRIGHT, A., and GOLDBERG, E. B. (1970). Inactivation of ureatreated phage T4 by phosphatidylglycerol. Virology 41, 356-364. BENZINGER, R., KLEBER, I., and HUSKEY, R. (1971). Transfection of Escherichia coli spheroplasts. I. General facilitation of double-stranded deoxyribonucleic acid infectivity by protamine sulfate. J. Viral. 7, 646-650. DAEMS, W. TH., V24N DE POL, J. H., and COHEN, J. A. (1961). Some remarks on the morphology of bacteriophage T4B. J. Mol. Biol. 3,225-227. EDGAR, 12. S., and LIELAUSIS, J. (1968). Some steps in the assembly of bacteriophage T4. J. Mol. Biol. 32, 263-276. FLATGAARD, J. E. (1969). The role of the gene 9 product in the assembly and triggering of bacteriophage T4. Doctoral Thesis, California Institute of Technology. JESAITIS, M. A., and GOEBEL, W. F. (1952). The chemical and antiviral properties of t,he somatic antigen of phase II Shigella sonnei. J. Exp. Med. 96, 409-438. JESAITIS, M. A., and GOEBEL, W. F. (1953). The interaction between T4 phage and the specific lipocarbohydrate of phase II Shigella sonnei. Cold Spring Harbor Symp. Quant. Biol. 18, 205208. KABX!K, H. R., and DITTMER, J. (1970). Quoted in KABACK, H. It., The transport of sugars across isolated bacterial membranes. in “Current Topics in Membranes and Transport” (F. Bronner and A. Klein, eds.), Vol. 1, p. 51. Academic Press, New York.
T4 PARTICLES
235
KABACK, H. R., and STADTMBN, E. R. (1966). Proline uptake by an isolated cytoplasmic membrane preparation of Escherichia coli. PTOC. Nat. Acad. Sci. USA 55,92C-927. KING, J. (1968). Assembly of the tail of bacteriophage T4. J. Mol. Biol. 32, 231-262. KING, J. and LAEMMLI, V. K. (1972). Bacteriophage T4 tail assembly: structural proteins and their genetic ident’ification. J. Mol. Biol. (in press). KING, J., and MYKOLAJEWYCZ, N. (1972). Bacteriophage T4 tail assembly: proteins of the sheath, tube, and baseplate. J. Mol. Biol. (in press). MASON, W. S., and HAZELKORN, R. (1972). Product of T4 gene 12. J. Mol. Biol. 66, 445469. OSBORN, M. J., GANDER, J. E., PARISI, E., and CARSON, J. (1972). Mechanism of assembly of the outer membrane of Salmonella typhimurium: Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247, 39623972. SIMON, L. D., and ANDERSON, T. F. (1967). The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. I. Attachment and penetration. II. Structure and function of the baseplate. Virology 32, 279-297, 298-305. SIMON, L. D., Swa;v, J. G., and FLATGAARD, J. E. (1970). Functional defects in T4 bacteriophages lacking the gene 11 and gene 12 products. ViroZogy 41, 77-90. VANARKEL, G.A., VAN DE POL, J.H.,and COHEN, J. A. (1961). Genetic recombination and marker rescue of urea-disrupted bacteriophage T4 in spheroplasts of E. co&. Virology 13, 546-548. WAN, A. C. (1970). Growth and transformation of phage T4 in Escherichia, Salmonella, Aerobatter, Proteus and Serratia. Doctoral Thesis, Tufts University. WAIS, A. C., and GOLDBERG, E. B. (1969). Growth and transformation of phage T4 in Escherichia coli B/4, Salmonella, Aerobacter, Proteus and Serratia. Virology 39, 153-161. WILSON, J. H., LUFTIG, R. B., and WOOD, W. B. (1970). The interaction of bacteriophage T4 tail components with purified E. coli lipopolysaccharide. J. Mol. Biol. 51, 423-434.
63, 225435
interactions
Department
(1973)
Between
Modified
WENDY
C. BENZ’
Phage AND
T4 Particles
EDWARD
and
Spheropiasts
B. GOLDBERG
of Molecular Biology and Microbiology, Tufts University School of Medicine, 1% Harrison Avenue, Boston, Massachusetts 02111 Accepted February
9, 1973
Two types of contracted phage particles with full heads were compared. Artificially contracted T4 phage particles were produced by treatment with urea (UT+), and naturally contracted T4 phage particles lacking gene 12 product were recovered after temporary adsorption to bacteria (adslZ+). Both types can infect T4-resistant bacteria if the bacterial envelope is altered by penicillin or lysozyme-EDTA treatment. Both are inactivated by cell membrane preparations and by low concentrations of phosphatidylglycerol which cause the particles to release DNA. Both are sensitive to trypsin. Differences in the particles as to the type of spheroplasts they can infect and the degree of their sensitivity to phosphatidylglycerol and DNase are correlated with mode of contraction rather than with phenotype. The properties of these contracted phage are described in terms of the properties expected for particles at intermediate stages of adsorption. Several mutants in other baseplate genes were tested, and it was found that genes 9,11, and 12 are unnecessary for infection of spheroplasts. We conclude that baseplates, as well as long tail fibers and their corresponding receptors in the bacterial envelope, do not play a part in the infection process of these contracted particles. This suggests to LIS that these organelles are necessary only during the initial stages of infection.
richia coli B (c&12-$1) (Simon et al., 1970). Their morphological properties and interactions with bacteria and spheroplasts correspond in many respects to particles at the contracting and penetrating stages, and they appear to function as expected for all stages of adsorption after the sheath contracts.
INTRODUCTION
The interaction between T4 phage and the bacterial envelope during infection can be divided into several stages (see Fig. 1). Phage adsorption starts with landing on the cell surface and ends with the injection of DNA into the cell. Some postulated intermediate stages of this process are described in the legend to Fig. 1. In an attempt to study the details of the scheme and to identify intermediates in the adsorption process, we have examined the properties of two types of phage particles which resemble particles at inbermediate stages of adsorption: particles treated by urea (UT4) (van Arkel et al., 1961; Wais and Goldberg, 1969; Baumann et al., 1970) and particles with a baseplate mutation in gene 12 which were recovered after adsorption to Esche1 Present address: Department of Biology, The Johns Hopkins University, Baltimore, Maryland, 21205.
MATERIALS
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Media. P broth, washing fluid, LSTG, V broth, spheroplast dilution medium (SDM), and SDM top agar were described by Wais and Goldberg (1969). L Broth contained 10 g Bacto-tryptone (D&o), 5 g yeast extract (Difco), and 5 g NaCl in 1 liter distilled water and was brought to pH 7.0 by addition of 1 ml 1 N NaOH. 0.1 M Salt Solution contained 5.85 g NaCl, 3.40 g MH=J’O, , 3.55 g Na2P04 in 1 liter distilled water. All media (except SDM) were sterilized in the autoclave for 20 min at 121°C. SDM was sterilized by filtration and stored frozen. 225
Copyright All rights
AND
BENZ
AND
GOLDBERG
CONTRACTINQ
PENETRATING
FIG. 1. Stages of T4 phage adsorption. This drawing, adapted in part from Simon and Anderson (1967), represents our current view of the stages of adsorption, although much of it is still conjecture. Landing. The phage particle attaches to the LPS layer of the bacterial envelope by long tail fibers. The attachment of each fiber is reversible, and each can be repositioned as the particle moves over the surface of the bacterium once it has landed (the landing site is shown here in relation to a stationary bump drawn on the surface). Pinning. The landed particle continues to move un and down while wandering over the bacterial surface until the baseplate is in the proper position, close enough to the appropriate surface receptor, to make contact. Further movement is restricted and the particle is pinned down. Contracting. The pinned particle undergoes a series of morphological changes. The baseplate pins elongate; the baseplate itself expands and detaches from the distal end of the tail tube and is drawn away from the bacterial surface as the sheath contracts to expose the tail tube. Penetrating. The contracting particle, held taut against the bacterial envelope by elongated baseplate fibers, penet.rates the layers of the envelope with the tail tube. Unplugging. The penetrating particle reaches a tail tube receptor on the plasma membrane and is positioned for final unplugging of the tail tube. Injecting. The unplugged particle ejects DNA from the head through the tail tube. The DNA then passes into the bacterial cell.
Bacteria. E. coZi B was from the collection of Dr. A. D. Hershey, E. coli 011’ (B/K hybrid) with amber suppressor II (Bsuf) from Dr. E. Stahl, E. coli B40SuI with amber suppressor I from Dr. P. Strigini, E. coli CR63 (X) with amber suppressor I from Dr. L. Simon, E. coli CR63 from Dr. R. Edgar and E. coli B/4 from Dr. H. Revel. E. coli B40suI/4 was isolated in this laboratory from E. coli B40suI. It permits growth of T2 wild type and amber mutants, but not T4 phage.
Bacteriophage strains. T4Dac41 was from the collection of Dr. A Doermann and is the wild type phage (w.t.) in this work. T4D mutants amN69 (gene 12), am N93 (gene ll), ana El7 (gene 9), and am N5.!? (gene 37) were from Dr. R. Edgar. T4DamB17amNl20rdj (which makes neither heads (gene 23-) nor tails (gene 27-) and has a deletion in rI1) was from Dr. L. Simon. The amEl’7amN52 (gene 9, gene 37) double mutant was constructed in this laboratory. Nomenclature. Phage are viable particles
ADSORPTION
OF MODIFIED
which will infect bacteria, whereas phage particles (4) denotes only the physical entities and does not imply viability. Wild type phage is abbreviated w.t., Urea-treated wild type phage is abbreviated UT& Active UT+ are those few UT+ which can infect spheroplasts. Phenotypically altered phage particles are referred to according to the number of the mutant gene and the strain of E. coli on which they were grown. For example, an 11 .B40suI phage is mutant in gene 11 and grown on E. coli B40suI. 11-d indicates phage particles grown under restrictive conditions which lack the product of gene 11 (Pll). 11-4 also lack P12 since PI1 is an obligatory precursor to P12 addition on the morphogenetic pathway (King, 1968). Phage stock Wild type and am-B&: Bacteria were aerated overnight in P broth, diluted 20-fold into P broth, and incubated at 37°C for 30 min without aeration. One overnight phage plaque per 25 ml bacterial suspension was suspended in 1 ml P broth (with a few drops of chloroform) and then added to the bacteria. The infected culture was aerated for 5 hr at 37”C, chloroform was added, and the debris sedimented at 7000 g for 5 min. The supernatant was incubated at 37°C with DNase (10 pg/ml) and RNase (100 pg/ml) for 20 min, with pancreatin (100 pg/m.l) for another 20 min, and then filtered through a O.&pm Millipore filter. The phage in the filtrate were concentrated by sedimentation at 35,000 g for 45 min and washed 3 times with washing fluid. Before the last wash, the phage were passed through a 0.45~pm Millipore filter. The phage were finally resuspended in a few ml of washing fluid. 11 .B and 12.B: Bacteria were grown to 4 X lo8 cells/ml in P broth. The culture was cooled to room temperature, and 11. B40suI or 12.B40suI phage were added t,o a m.o.i. of 3-5. The culture was aerated by bubbling for 10 min at room temperature, sedimented at 7000 g for 3-5 min at cold or room temperature, and the cells resuspended in the original volume of P broth. This procedure removed most of the unadsorbed phage. The culture was bubbled vigorously at 30°C for 145 hr, chloroform was added, and the phage particles purified by low and
T4 PARTICLES
227
high speed centrifugation as for w.t., and resuspended in a few ml of washing Auid. 9. B and 9,S7. B: This procedure was modified somewhat from preparation of 11 .B. L Broth was used instead of P broth t,o retard spontaneous inactivation. After chloroform treatment, the lysate was centrifuged at 7000 g for 3 min at room temperature, and the supernatant centrifuged at 35,000 g for 45 min. The phage were then resuspended in 0.1 M salt solution in about Woo the lysate volume and stored at room temperature. In the case of 9.B or 9, 37 .B, freshly prepared phage stocks were used for each experiment. Radioa&ve phuge stocks. 32P-labeled phage stocks of w.t., am.BsuI, and 11 .B were made as above, except that lo-15 &i/ml 32P was added to the bacteria immediately before the addition of phage. In making 9.B, 12-B, and 9, 37-B, the 3eP was added after low speed centrifugation and resuspension in fresh medium. 35S-labeled w.t. stocks were made as above, except that LSTG was substituted for P broth, the bacteria were initially aerated at 37°C for 1 hr, and 100 PCi 35S was added immediately before addition of phage. Reagents. Phosphatidylglycerol (PG) was made from E. coli B/4 (Baumann et al., 1970). It was bubbled out of organic solvent into 0.1 M sodium phosphate buffer, pH 7.0, before addition to phage. Crystalline deoxyribonuclease I and lysozyme were from Worthington Biochemicals. Trypsin and trypsin inhibitor were from Sigma. Special biological preparations. Ureatreat’ed phage particles (UT4) were prepared as described by Wais and Goldberg (1969). Spheroplasts were either lysozyme-EDTA spheroplasts from E. coli B40suI/4, as described by Baumann et al. (1970), or penicillin spheroplasts of E. coli B40suI/4 as described by Wais and Goldberg (1969) for E. coli B, except that P broth was used instead of LSTG, and the cells were grown to a titer of about 8 X 10s cells/ml before adding V Broth, sucrose, and penicillin. Adsorbed i2- phage particles (adsld-4): E. coli B was grown in P broth to a titer of l-2 X lo8 cells/ml The cells were harvested and resuspended in one-tenth the original volume
228
BENZ
AND
of P broth. 12- phage were added to a m.o.i. of 3-5. The mixture was incubated at 30°C for lo-12 min and then centrifuged in the cold at 7000 g for 3 min. The supernatant was either used directly as a&12-4, or was diluted into P broth as required. Infective center assay for UT4 and Adsl2-4. UT+ and a&12-4 infectivity were tested in a spheroplast infective center assay. Lysozvme-EDTA spheroplasts of B40suI/4 were diluted 1:l with P broth after the addition of MgSOc (Baumann et al., 1970). UT+ were added to a m.o.i. of 0.1-1.0. After 10 min at 37”C, aliquots were plated directly with 3 ml SDM top agar and B40suI indicator bacteria onto plates previously spread with 3 ml SDM top agar. AdsW9 were added in similar multiplicities, but after 12 min incubation at 30°C T4D antiserum (K = 5) was added to reduce the background of about 10m5plaques/particle on B40suI. Three minutes later, aliquots were plated as for UT@. P9, Pll, and P12. Extracts containing P9, Pll, and P12 were prepared according to the method of Edgar and Lielausis (1968) as modified by Flatgaard (1969). E. coli B was grown in L Broth at 30°C to a concentration of about 4 X lo8 cells/ml. Phage amB17amNl20rclj~ CR63 were added to a m.o.i. of 5, and the culture aerated at 30°C. At 20 ‘min an equal amount of the same phage was added. At 50 min the culture was chilled in ice water and centrifuged at 7000 g for 10 min. The cells were then resuspended in >{oo the original volume of BUM buffer (Edgar and Lielausis; 1968) and frozen in small aliquots with acetone/dry ice. To test in vitro complementation, the freshly thawed extract was diluted 1: 1 with BUM buffer. One microliter of phage to be tested was then diluted into 0.20.3 ml of this extract, incubated for 2 hr at 3O”C, and titered on CR63(h). All 9-B, 9, 37*B, ll*B, and 12.B stocks used were activatible with such extracts, and this titer was used as a measure of total phage particles in the stock. DNA release from UT+ and AdslF4. TO measure DNA release from 32P-labeled phage particles after adsorption or treatment with urea of PC, samples were treated with DNase (60 pg/ml with 6 mM MgC12) for 7
GOLDBERG
min at 37°C (UT+) or 30°C (a&12-4). In the case of acZ.s12-4, 1 &I of 30% bovine serum albumin (Pentex) was added per 1 ml sample. After acid precipitation in cold 3 % perchloric acid for 5 min and sedimentation at 7000 g for 5 min, radioactivity in the supernatant was measured in a low background (2-3 cpm) flow counter. RESULTS
AND
DISCUSSION
Role of the Baseplate in Attachment of UT4 to Spheroplasts UT@ infection requires neither long tail fibers nor tail fiber receptors in the lipopolysaccharide layer of the cell envelope (Wais and Goldberg, 1969; Baumann et al., 1970; van Arkel et al., 1961). Therefore, the landing stage can be bypassed. To determine whether the baseplate is required, several baseplate mutations (King and Laemmli, 1970) were examined. Phage particles, lacking only the product of gene 12 (Mason and Hazelkorn, 1972), were completed when necessary by adding P12 in vitro (Edgar and Lielausis, 1968). 9-4 and 11-4 were completed by the same method. 9-4, 11-4, and 12-4 were tested before and after urea treatment on lysozyme-EDTA spheroplasts of E. coli B40suI/4. Table 1 shows that untreated particles did not infect. The urea-treated particles, however, proved to be as infective as urea-treated wild type phage, whether grown on permissive or restrictive hosts. These findings show that the products of genes 11 and 12 (associat’ed with baseplate pins-Simon et al., 1970) and the product of gene 9 (associated with outer parts of the baseplate-Flatgaard, 1969; and J. King, personal communication) are not required in UT4 infection of spheroplasts. P9, Pll, and P12 may be atypical since, unlike other baseplate proteins, they are not required for tail morphogenesis. The product of gene 10 is required for tail morphogenesis, and it has been shown that urea-treated phage with altered, inactivated PlO are as infective as UT+ (Dawes, J., unpublished data). This further strengthens the argument that the baseplate is not required for UT4 infection of spheroplasts. Between the times of landing and DNA in-
ADSORPTION TABLE UT4
1
INFECTION OF B40suI/4 Phage particle
w.t. 11. Bsu+ 1l.B 12.B40suI 12.B 9. B40suI 9, 37.B”
OF MODIFIED
adsorbed and subsequently detached (Simon were tested on lysozyme-EDTA spheroplasts of B40suI/4 and assayed for infective centers. Table 2 shows that &12-4 are as infective as UT4 (about 0.1%). Lysozyme seemed to be the most important part of the spheroplast treatment, since treatment without lysozyme (EDTA alone) decreased infectivity of ads12-4 to 4% of that on lysozyme-EDTA spheroplasts. In comparison, infectivity of UT+ on cells treated with EDTA alone remained relatively high. This suggests that destruction of murein in the cell membrane, which is caused by lysozyme treatment, may be more necessary for ads12-4 infection than for UT4 infection. Infectivity of adsl2-4 on penicillin spheroplasts is less than 1% that of UT& Ib is possible that infection is inhibited by noncrosslinked murein or some other cell envelope component. On the other hand, such infection may require a component present on lysozyme-EDTA spheroplasts that is lacking or altered on penicillin spheroplasts. A third possibility is that ads12-9 are inactivated by a Mg*+ dependent DNase released by penicillin treatment (Balte, 1971). This is unlikely, however, because adsl2-4 do not infect penicillin spheroplasts even when excess EDTA (0.014M is present (Table 2). Since adsl2-4 infect spheroplasts of B40suI/4 (normally resistant to T4 infection but made infectible by lysozyme-EDTA treatment), they too bypass t’he landing stage. The particles lack P12, so that component of the basepate is not required either. It was not possible to test the requirement for P9 and Pll since 9-4 and II-+ would not adsorb to bacteria to produce contracted particles.
SPHEROPLASTS' et al., 1970). These adsl2-4
Relative infectivity of baseplate mutants untreated
urea-treated
SO.07 0.01 <0.002 SO.05 0.03 10.17 c
1.00 0.45 0.95 0.65 1.45 0.60 0.50
Q Infectivity was measured by the spheroplast infective center assay. T4D antiserum (K = 5) was added to untreated phage a few minutes before plating infective centers. Infectivity is expressed as the number of infective centers formed divided by the total number of phage particles. This number is about 1 X lo+ for wild type (w.t.) UT+ and is defined as a relative infectivity of 1.00 to which all the mutant preparations are compared. b A 9,37.B double mutant was used instead of a 9.B single mutant because 9.B had too high a background of phage to be useful in this system. Since 99+ lack long tail fibers and 37 is a tail fiber gene, this double mutant has the same morphology as 9- alone. c The infective phage in a 9,37.B stock were 4% as responsive to T4D antiserum as wild type T4. Therefore, not enough viable phage could be removed to make accurate measurement of spheroplast infection.
jection, phage show marked changes in appearance (Simon and Anderson, 1967). Their baseplates expand and sheaths contract to expose the tail tube. UT4 resemble phage at those intermediate stages (Daems et al., 1961). However, only 0.1% of UT4 are infective, and so electron microscopic observations of the majority may not characterize the few which are infective. Nevertheless, UTC$ fulfill the prediction that tail fibers and functional baseplates are not required for a phage infection after contraction. Infectivity
229
T4 PARTICLES
of AdslP~
To obtain more naturally contracted parCcles for comparison with UT+, 12-4 were mixed with E. coli B. Most of the particles
DNase Sensitivity
qf UT4 and Ads 12-4
The firmness and specificity of the adsorption of UT+ to spheroplasts was tested with DNase. In order to determine whether DNA from infective UT+ is exposed at any stage between pinning and final injection, UT4 and mixtures of UT+ and spheroplasts were treated with DNase. Table 3 shows that UT+ are insensitive to DNase up to 100 pg/ml and UT+spheroplast mixtures up to
230
BENZ TABLE
INFECTIVITY
OF UT+
B40suI/4
2
AND adS12-+ SPHEROPLASTP
Spheroplasts
Lysozyme + EDTA Lysozyme alone EDTA alone Penicillin Penicillin + 0.014 iM EDTA Penicillin + 0.0007 M EDTA
AND
ON E.
COk
Relative infectivity UT4
a&12-$lb
1.00 1.00 0.70 0.10 0.08 0.13
0.50 0.10 0.021 10.001 <0.0015 lO.001
a Infective center assays, lysozyme-EDTA and penicillin spheroplasts were spheroplasts, made as described in Materials and Methods. 0.1 mg/ml lysozyme and 0.7 mM EDTA were used unless noted otherwise. Relative infectivity is defined in Table 1. b The concentration of adsl2-4 was determined from the amount of 3zP in the supernatant solution after adsorption of 32P-labeled 12-4 and from the known specific activity of the particles as determined by in vitro complementation and is, therefore, a minimum estimate. Normally 25-35y0 of the 32P was found in the supernatant fluid. However, 30-40’% of this z2P was acid soluble (see also Fig. 8). Thus, the actual number of potentially active adsl2-4 is probably >$ to $6 the amount determined from the crude estimate, and the actual infectivity might be 1.5-2-fold greater than shown. Simon et al. (1970) reported that most 12- phage detached after adsorption and almost no DNA was released. However, our method of preparation differed in that our bacteria were more concentrated and a smaller fraction of the particles may have reversibly adsorbed, yielding an even higher infectivity. In any case, we chose those concentrations of phage and bacteria that gave the most infective particles on spheroplastjs.
10 Hg/ml (or 100 pg/ml DNase, P. Arscott, unpublished experiments). This insensitivity to DNase and the rapid and specific adsorption of active UT+ to spheroplasts supports the idea of a firm and specific union leading to a direct, protected transfer of DNA from particle to host. These findings also rule out transfection as the mechanism of DNA transfer of UT& Table 3 also shows that udsl2-4 are sensitive to 0.1 fig/ml DNase under conditions in which UT+ and 12-4 are unaffected
GOLDBERG
by lo4 times that concentration. In order to determine if DNase attacks DNA while still in the phage particle or during injection, a&12-4 were incubated with 0.1 pg/ml Dn’ase and diluted into spheroplasts at various times. Figure 2 shows that inhibiTABLE INHIBITION
OF a&12-+
DNase concn Gww
0 0.01 0.1 1.0 10.0 100.0
Fraction
3 BY DNASE~
INFECTIVITY
of infectivity
remaining
UT4
&12-4
12-4
1.00 0.93 1.00 0.93 1.00 1.00
1.00 1.00 0.37 0.10 0.04 0.01
1.00
0.92 0.97
a UT+, adsl2-4, and 12-+ were incubated with various concentrations of DNase and 0.006 M MgClz at 30’ C for 7 min. Infectivity of adsl2-4 and of UT4 was then assayed by the spheroplast infective center assay, in which the DNase concentration was diluted lo-fold. Infectivity of the (unadsorbed) 124 was assayed by the ability to be activated in the in vitro complementation assay.
-
PREINCUBATION
TIME
(MIN)
FIG. 2. Effect of preincubation with DNase on infectivity of adslr+. Samples of adslic4 were incubated with 0.1 fig/ml DNase in 0.006 E4 MgCls at 30’ C for the times indicated and then assayed by the spheroplast infective center assay. The ads124 particles are somewhat unstable at 30” C; therefore the experiment was controlled with parallel incubations of the ads124 at 30” C with MgC12 only (-DNase).
ADSORPTION
OF MODIFIED
tion of infective center formation is dependent on the time of preincubation with DNase. Therefore DNase inactivated a&12-4 before any contact with spheroplasts. We do not know why the value (extrapolated from Fig. 2) for no preineubation is 25% inhibition of infective centers. It is possible that 0.01 pg DNase, under these conditions, may also interfere with transfer of DNA from UT4 after contact with spheroplasts. It is unlikely, however, that a DNA molecule as large as that of T4 could remain intact in a solution of 0.1 pg/ml DNase or even 0.01 pg/ml DNase. Yet, Table 3 shows that UT+ and 12-4 remain fully infective and lo-40 % of a&12-I#J remain infective under those conditions. Such high frequencies of transfection (> 10-4) have not yet been demonstrated (Baltz, 1971; Benzinger et ai., 1971). As further confirmation that UT4 and a&12-4 do not transfer their DNA by transfection, the particles were treated with
CTRYPSINI
pa/ml
FIG. 3. Sensitivity of UT+ and adsl2-+ to trypsin. The phage were incubated with various concentrations of trypsin for 7 min at 30” C and assayed by the spheroplast infective cent.er assay. Unadsorbed 12+ were assayed for reactivation by in vitro complementation. Wild type phage were assayed by titering on CR63. Since the various phage preparations had to be kept in different media (0.1 M PO1 , pH 7.0, 2% BSA for UT+; P broth for adsl2-4; BUM buffer for 12-4), sensitivity of UT+ to trypsin was tested in each of the media and found not to vary (unpublished data).
gg z f z
231
T4 PARTICLES
2
3 PG CONTENT
4
5
6
X IO5 M
FIG. 4. Inhibition of UT+ by different bacterial membrane layers. The four membrane fractions from S. typhimurium were separately incubated with UT+ preparations for 10 min at 37’C at the concentrations indicated. The UT+ were then assayed by the spheroplast infective center assay. The PG concentrations of the membrane fractions were calculated from their relative protein and PG content. The rmoles PG/mg protein (determined by J. Carson) are: (A) 0.052, (B) 0.032, (C) 0.031, (D) 0.029. The fractions A-D represent successive layers of the cell envelope from inner plasma membrane to outer lipopolysaccharide membrane (Osborn et al., 1972).
trypsin at various concentrations. Figure 3 shows that both are sensitive to concentrations as low as 0.5 pg/ml. The addition of trypsin inhibitor immediately after trypsin treatment and before assaying for infective centers did not alter these results. This suggests that trypsin, like DNase, acts directly on the contracted phage particles themselves. The sensitivity of UT# infectivity to low concentrations of trypsin and theinsensitivity to high concentrations of DNase prove that DNA transfer during UT4 infection is not by transfection. For a&12-4, however, it is still possible that release of DNA into the medium is triggered by a specific interaction between the particle and the spheroplast which is inhibited by trypsin action. Such a mode of infection should be somewhat sensitive to DNase, and this might also help to explain the residual inhibition of infective centers at zero time in Fig. 2.
232
BENZ
PG CONTENT
AND
X IO’ M
FIG. 5. Inhibition of UT+ and a&12-+ by membrane vesicles. S. typhimurium membrane vesicles (Kaback and Stadtmann, 1966) were incubated for 10 min at 30°C with either UT+ or a&12-6. Membrane vesicles, diluted to the appropriate concentration in the buffer used for the final stage of their preparation, were mixed with an equal volume of phage particles for incubation. Control preparations, used for determination of 100% infectivity, lacked membrane vesicles in the buffer. The PG concentration of the membrane vesicles was calculated from the protein content according to the ratio 0.09 rmoles PG/mg protein (Kaback and Dittmer, 1969).
E$ect of Membrane Components on the Injectivity of UT4 and AdslFqi The differences between UT+ and a&12-9 in sensitivity to DNase and ability to infect penicillin spheroplasts suggest’a difference in their mode of infection. Therefore, we investigated the effects of various components of the spheroplast membrane. LysozymeEDTA spheroplasts of Salmonella typhimurium were sonicated and the debris separated on a sucrose gradient containing EDTA (Osborn et al., 1972). The four fractions (a gift from J. Carson) correspond to layers of the cell envelope. Figure 4 shows that all fractions inactivate UT& although the inner layer seems to be a little less effective. Membrane vesicles of S. typhimurium (Kaback and Stadtman, 1966) were also tested, and Fig. 5 shows that they inactivate UT4 and a&12-+ to an equal degree at low concentrations. At higher concentrations, 75 % of UT+ are inactivated while 100% of a&12-4 are inactivated. The fraction of
GOLDBERG
UT+ which was not inactivated is not yet explained. Baumann et al. (1970) showed that phosphatidylglycerol (PG), alone of all components of the gram-negative cell membrane, inactivates UT+ Since all the membrane fractions and membrane vesicles tested contained PG, UT+ and a&12-9 were tested for sensitivity with purified PG. Figure 6 shows that’ a&12-4 are 100-1000 times more sensitive than UT4. To determine whether this might be due to the absence of P12 or to the method of contraction, UT12-$3 were compared with UT+. Figure 7 shows that UT+ and UT12-4 are almost equally sensitive to PG. The same was true with UT9-+ and UTll-4. Thus, these baseplate components are not involved in the inactivation of active UT+ by PG. If PG does play a role in phage infection, a&12-I$ appear to be at a stage for eflicient interaction with PG. The ektreme sensitivity of these naturally contracted particles suggests that similar interactions occur in vivo.
z $ loo- o--oodl12-q /y--/p oI--Q XX I-x UT+ ii ,I ; 80: 2 ,’ x iiy 601’ 1’ w ,’ > F ,’ 0 40lk! ,/‘O x1 z N k 20/’ 0,’ : , F , 5 I .’ /’ XIJ i O 10-7 10-G IO 10-5 10-g 10-8 z LPGI
hi
FIG. 6. Inhibition of UT+ and a&12-4 infection by phosphatidylglycerol. UT+ were incubated with various PG concentrations at 37” C for 8 min and adslZ+ at 0” for 7 min. The lower temperature was used for ads12+ because it has been shown (unpublished data) that these particles are inactivated by PG faster and as completely at 0” C as at higher temperatures and that higher temperatures tend to inactivate adsW+ even in the absence of PG. Infectivity was then assayed by the spheroplast infective center assay.
ADSORPTION
DNA
Ejection from Mediated by PG
UT+
OF MODIFIED
and Ads 12-4
mechanism of inhibition of infection, since DNA was released most easily from noninfective particles. Figure 8 shows that UT9-4, UTll-r$, and UTl2-C$ release their DNA as easily as UT& However, since a smaller fraction of DNA is released by the action of PG on UTll-C#I and UTl2-9, Pll and P12 may be involved to some extent in the release of DNA from UT# by PG. A comparison of Figs. 6 and 8 shows that maximal DNA release by UT4 is complete with lo+’ M PG. This confirms the results of Baumann et al. (1970) that DNA release in UT+ is more sensitive to the action of PG than is infectivity. The existence of different classes of UT+ reinforces the possibility t’hat infective UT+ may differ structurally from the majority. Urea treatment of 9- phage particles and reversible adsorption of 12- phage particles both lead to loss of over 40 % of the DNA even before addition of PG. A&12-4 release the maximum amount of DNA (60 %) at 5 X lo+ f&l PC (see Fig. S), which is about the same concentrat,ion of PG needed for maximal loss of infectivity (100 %) (see Fig. 6). The uniformity of a&12-$ with respect to inhibition of infectivity and release of DNA by PG makes it less likely that in-
Baumann et al. (1970) reported a second action of PC on UT+: the release of DNA from about one half of all UT& They were unable to relate this observation to the
CPGI X Id%
7. Inhibition of infection by phosphatidylglycerol for UT+ of various genotypes. Ureatreated phage preparations were incubated with various concentrations of PG for 8 min at 37” C, and their subsequent infectivity assayed by the spheroplast infective center assay. The same 32P-labeled phage preparations were also used for the experiments shown in Fig. 8. FIG.
I I
I 2
233
T4 PARTICLES
I 3
I 4
I 5
I 6
I 7
CPGI X IO6 M FIG. 8. DNA release by phosphatidylglycerol from UT+ of various genotypes. The same 32P-labeled adslZ$~ as in Figs. 6 and 7 were incubated with PG as described in the legend to Fig. 6, and DNA release was determined. The DNA released in controls without PG varied from about 15% to 45% and depended upon the genotype of the phage to be treated. In addition, the amount of free DNA before PG addition for UT+, UTll-4, and UT124 varied in different preparations from 5% to 26% but had no effect on the maximum amount of DNA released with PG (unpublished data).
234
BENZ
AND
fective particles differ grossly from the maj ority. CONCLUDING
REMARKS
We have altered phage by two completely different treatments: with the general denaturing agent, urea, and by interruption of the natural adsorption of phage by alteration of the baseplate protein, P12. The resulting particles have many of the properties postulated for phage at intermediate stages of adsorption. It is interesting that both methods yield such similar particles, although the second method is more natural. Morphologically, both have contracted tails and full heads (Daems et al., 1962; Flatgaard. 1969; Baumann et al., 1970; Simon et al., 1970). Both will infect spheroplasts and can bypass the landing stage of adsorption. In both cases, only 10e3 of the particles are infective. The differences in host range and sensitivity to PG and DNase may reflect only minor differences in the particle, which may in turn belie finer distinction in the exact stage of adsorption at which the particle must commence infection of the spheroplast,. It is possible that a&12-4 are at a more advanced stage than UT+. The overall similarity of the more physiological adsl2-4 to UT#, however, lends support to the idea that UT+ also are adsorption intermediates. The existence of infective particles with contracted sheaths and full heads shows that the main purpose of contraction is probably to bring the tip of the tail tube to the inner membrane surface (Simon and Anderson, 1967) for the final joining of the tail tube with the plasma membrane. Only then, aft,er sheath contraction is complete, is DNA ejected from the phage and taken up by the bacterium. This process may well involve PG in the bacterial membrane as illustrated with UT4 and especially with adsl2-4. Not all contracted particles with full heads, however, are infective on spheroplasts. Spontaneously contracted 9-4 (Flatgaard, 1969) will not infect spheroplast,s (W.C.B., unpublished results). In fact, the means of sheath contraction is crucial to infection, since phenotypically identical particles contracted by different means show different infection properties on sphero-
GOLDBERG
plasts. Thus, UT9-4 are infective, whereas spontaneously contracted 9-4 are not. UT12-4, like UT4, differ from adsl2-4 in t,he type of spheroplast they will infect, DNase sensitivity, PG sensitivity, etc. Despite these differences, al1 of these particles are indistinguishable from UT4 in gross morphology. The exquisite sensitivity of infectivity and DNA release from adsl2-4 to PG suggests a physiological role of PC in DNA ejection during or after penetration. Jesaitis and Goebel (1952) also showed that LPS preparations released DNA from phage, and this was corroborated by Wilson et al. (1970). Thus, this t’ype of phenol-ext’racted cell wall preparation must contain all the components required for the various stages of phage adsorption, including DNA ejection. Jesaitis and Goebel (1953) also showed that this DNA release depended upon an identified (et,hanol-extractable) lipid. Both UT+ and c&12-9 were inactivated by the various membrane preparations at a calculated PG content of lo+’ M (see Figs. 4 and 5). This inactivation probably does not reflect their PC content only. The PC in the membrane could be ineffective, and both types of particles could be equally sensitive to some membrane receptor site. We would like to find, therefore, a PGfree membrane preparation which inactivates contracted particles without causing them to release their DNA. It would therefore be of interest to test the potency of various cell envelope fractions for their ability to release DNA as well as to adsorb phage particles at various stages of infection. The fact that contracted particles like UT+ and u&12-4 cannot infect bacteria directly but can infect spheroplasts implies that the bacterial surface must be altered to form or expose receptors used in late stages of infection. It seems clear that phage adsorption involves levels of interaction between the phage and the host envelope and that these mutual interactions must occur in an ordered sequence. The chemistry of these interactions is not yet fully understood. The isolation of new phage adsorption intermediates and different preparations of alt’ered cell envelopes should help toward this end.
ADSORPTION
OF MODIFIED
ACKNOWLEDGMENTS We thank Patsy Arscott for valuable help in preparing this manuscript and especially for her help in naming the stages of adsorption, Dr. J. Dawes for permission to quote her results before publication, Sherry Sass for technical help, and Dr. Andrew Wright for continuing interest and discussion. This work was supported in part by N.I.H. Grant No. GM13511 and N.S.F. Grant No. GB-27601. E.B.G. is a Career Development Awardee of the N.I.H. REFERENCES BALTZ, R. H. (1971). Infectious DNA of bacteriophage T4. J. Mol. Biol:62,425437. BAUMANN, L., BENZ, W. C., WRIGHT, A., and GOLDBERG, E. B. (1970). Inactivation of ureatreated phage T4 by phosphatidylglycerol. Virology 41, 356-364. BENZINGER, R., KLEBER, I., and HUSKEY, R. (1971). Transfection of Escherichia coli spheroplasts. I. General facilitation of double-stranded deoxyribonucleic acid infectivity by protamine sulfate. J. Viral. 7, 646-650. DAEMS, W. TH., V24N DE POL, J. H., and COHEN, J. A. (1961). Some remarks on the morphology of bacteriophage T4B. J. Mol. Biol. 3,225-227. EDGAR, 12. S., and LIELAUSIS, J. (1968). Some steps in the assembly of bacteriophage T4. J. Mol. Biol. 32, 263-276. FLATGAARD, J. E. (1969). The role of the gene 9 product in the assembly and triggering of bacteriophage T4. Doctoral Thesis, California Institute of Technology. JESAITIS, M. A., and GOEBEL, W. F. (1952). The chemical and antiviral properties of t,he somatic antigen of phase II Shigella sonnei. J. Exp. Med. 96, 409-438. JESAITIS, M. A., and GOEBEL, W. F. (1953). The interaction between T4 phage and the specific lipocarbohydrate of phase II Shigella sonnei. Cold Spring Harbor Symp. Quant. Biol. 18, 205208. KABX!K, H. R., and DITTMER, J. (1970). Quoted in KABACK, H. It., The transport of sugars across isolated bacterial membranes. in “Current Topics in Membranes and Transport” (F. Bronner and A. Klein, eds.), Vol. 1, p. 51. Academic Press, New York.
T4 PARTICLES
235
KABACK, H. R., and STADTMBN, E. R. (1966). Proline uptake by an isolated cytoplasmic membrane preparation of Escherichia coli. PTOC. Nat. Acad. Sci. USA 55,92C-927. KING, J. (1968). Assembly of the tail of bacteriophage T4. J. Mol. Biol. 32, 231-262. KING, J. and LAEMMLI, V. K. (1972). Bacteriophage T4 tail assembly: structural proteins and their genetic ident’ification. J. Mol. Biol. (in press). KING, J., and MYKOLAJEWYCZ, N. (1972). Bacteriophage T4 tail assembly: proteins of the sheath, tube, and baseplate. J. Mol. Biol. (in press). MASON, W. S., and HAZELKORN, R. (1972). Product of T4 gene 12. J. Mol. Biol. 66, 445469. OSBORN, M. J., GANDER, J. E., PARISI, E., and CARSON, J. (1972). Mechanism of assembly of the outer membrane of Salmonella typhimurium: Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247, 39623972. SIMON, L. D., and ANDERSON, T. F. (1967). The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. I. Attachment and penetration. II. Structure and function of the baseplate. Virology 32, 279-297, 298-305. SIMON, L. D., Swa;v, J. G., and FLATGAARD, J. E. (1970). Functional defects in T4 bacteriophages lacking the gene 11 and gene 12 products. ViroZogy 41, 77-90. VANARKEL, G.A., VAN DE POL, J.H.,and COHEN, J. A. (1961). Genetic recombination and marker rescue of urea-disrupted bacteriophage T4 in spheroplasts of E. co&. Virology 13, 546-548. WAN, A. C. (1970). Growth and transformation of phage T4 in Escherichia, Salmonella, Aerobatter, Proteus and Serratia. Doctoral Thesis, Tufts University. WAIS, A. C., and GOLDBERG, E. B. (1969). Growth and transformation of phage T4 in Escherichia coli B/4, Salmonella, Aerobacter, Proteus and Serratia. Virology 39, 153-161. WILSON, J. H., LUFTIG, R. B., and WOOD, W. B. (1970). The interaction of bacteriophage T4 tail components with purified E. coli lipopolysaccharide. J. Mol. Biol. 51, 423-434.