JOURNAL

OF VIROLOGY, Oct. 1977, p. 201-210 Copyright © 1977 American Society for Microbiology

Vol. 24, No. 1 Printed in U.S.A.

Properties of "Diplophage": a Lipid-Containing Bacteriophage RUBENS LOPEZ,' CONCEPCION RONDA,' ALEXANDER TOMASZ,2* AND ANTONIO PORTOLES' Instituto de Immunologia y Biologia Microbiana, Madrid, Spain,I and The Rockefeller University, New York, New York 100212

Received for publication 21 January 1977

We describe the purification and properties of Dp-1, a bacteriophage isolated from Diplococcus pneumoniae. The phage was sensitive to the organic solvents deoxycholate and Sarkosyl, and its infectivity was reduced by treatment with phospholipase C. Electron microscopy indicated the presence of a double-layered coat around the phage particles. Purified phage preparations contained lipid amounting to about 8.5% of the dry weight of the phage, and thin-layer chromatography resolved the lipids into four components. The phage had a buoyant density in CsCl of 1.47 g/cm3, and a sedimentation constant in 0.1 M NaCl of 313S. Analysis in acrylamide gel electrophoresis indicated the presence of three major proteins. Dp-1 DNA shows a density of 1.681 g/cm3. Neutralizing antisera against the phage have a low potency (K < 120/min). Successful isolation of pneumococcal bacteriophages has been recently reported by three independent groups (15, 21, 29). In a previous paper (15), we pointed out a number of difficulties, such as low titers and poor stability upon storage, in the handling of one of these phages (Dp-1). Another peculiar and interesting property of this phage is the apparent requirement for host murein hydrolase activity in the release of progeny bacteriophage particles from the infected bacteria (22). These unique features of Dp-1 have prompted us to characterize this bacteriophage in more detail. Our observations suggest that Dp-1 contains lipids. Lipidcontaining phages have not been described in gram-positive bacteria so far (6).

tained catalase (15). SPP1, a phage of Bacillus subtilis, was obtained, purified, and plated as previously described (14). Preparation of highly purified Dp-1. One of the main problems in dealing with Dp-1 is the difficulty in obtaining a high titer of phages in the crude lysates and in avoiding the dramatic drop of the titer once it has been purified. The method we finally adapted for purification of Dp-1 is similar to the one used by Franklin for Pm-2 (6): S. pneumoniae R36A was grown in CpH 8; when the culture reached a titer of 1.7 x 107 cells/ml, the bacteria were infected with Dp-1 (multiplicity of infection [MOI] = 1:40). At a cell concentration of 1.2 x 108 cells/ml, an equal volume of prewarmed medium was added, and the culture was incubated until the completion of lysis. NaCl was added to the lysate (to give a final concentration of 0.5 M), and cell debris was removed by centrifugation (5,000 x g at 4°C for 15 min). Polyethylene glycol 6000 was added to the supernatant at the final concentration of 10% (vol/vol) and stored at 4°C for at least 36 h. The precipitate was collected by centrifugation (5,000 x g, for 15 min). The pellet was resuspended in NTM (1% of the original volume) and layered on the top of a three-step CsCl gradient (p = 1.70, 1.50, and 1.30 g/ml). The phage was centrifuged at 26,000 rpm for 210 min in an ultracentrifuge (Beckman L350), using an SW27-1 rotor. The clear phage band was collected and ultracentrifuged overnight in an SW501 rotor at 35,000 rpm. The phage band was isolated and dialyzed against NTMM. Buoyant density determination. Purified Dp-1 was centrifuged to equilibrium in an ultracentrifuge (Beckman L3-50) using an SW50-1 rotor. Dp-1, 0.4 ml (2.5 x 1010 PFU), was layered onto the top of a threestep cesium chloride gradient (28). After 24 h of centrifugation at 35,000 rpm at 20°C, fractions were

MATERIALS AND METHODS General. Streptococcus pneumoniae R36A (Rockefeller University stock) was the host strain. The bacteria were grown in C-medium at pH 8 (CpH 8) (30). K-CAT (21) and K-CpH 8, a medium in which Na+ was completely replaced by K+ in the basic CpH 8 medium, has also been used for obtaining crude lysates of Dp-1. Phage stocks were kept either at -70 or 4°C in NTMM buffer (0.5 M NaCl; 10 mM Tris-hydrochloride, pH 7.8; 10 mM MgCl2* 7 H2O; 10 mM 2-mercaptoethanol) (6). Other buffers used in our experiments were: TBT (0.1 M NaCl; 0.1 M Trishydrochloride, pH 7.8; 10 mM MgCl2), NTM buffer (NTMM without mercaptoethanol), and SSC (0.15 M NaCl and 0.015 M sodium citrate). Plating of the phage Dp-1 was performed in a slightly modified CpH 8 medium (0.15 M phosphate buffer instead of the usual 0.05 M) at 32°C by the usual soft-agar method (1), except the soft agar con201

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LOPEZ ET AL.

collected by needle puncture from the bottom of the tubes. The refractive index of several fractions was determined by an Abbe refractometer at 250C. The fractions obtained were diluted 1:10 with 0.15 M NaCl, and their UV absorption spectra were determined in a spectrophotometer (Beckman model 25). Finally, the fractions were tested for specific infectivity. Acrylamide gel electrophoresis. Gel preparation and sample staining were done as previously described (34). A suspension (0.5 ml) of purified phage (50 /ig to 1 mg/ml) in TBT was disrupted by addition of 50 gl of 1% sodium deoxycholate and 1% 2-mercaptoethanol, and 25 1l of tracking dye solution and drop of glycerol were added to the disrupted phage. Of the above mixture, 100 to 200 1l was added to the top of 10% polyacrylamide gels. Electrophoresis was carried out at room temperature overnight using 5 mA per tube. Gels were stained for 2 to 12 h at room temperature in 0.16% Coomassie brilliant blue and destained in several changes of 0.5% methanol0.75% glacial acetic acid in water (vol/vol). DNA extraction. Dp-1 [3H]DNA labeled as described below was extracted and purified by either phenol treatment (11) or after the proteinase K procedure (8). p29 ['4C]DNA (a gift from M. Salas) was purified by using the procedure of Gross-Bellard et al. (8). CsCl density centrifugation of Dp-1 DNA. The conditions used were similar to those described by Hirokawa (11). Dp-1 [3H]DNA plus 029 [14C]DNA were dissolved in CsCl solution (3.83 g of CsCl in 2.6 ml of 1/10x SSC). Centrifugation was done in an ultracentrifuge (Beckman L3-50) using the SW50-1 rotor at 35,000 rpm and 10'C for 60 h. About 40 fractions were collected on Whatman glass fiber filters (Whatman GF/A) under vacuum, dried, and directly counted using a toluene-based cocktail with 0.3% 2,5-diphenyloxazole and 0.01% 1,4-bis-12]-(5phenyloxazolyl)benzene. As a density reference 429 ['4C]DNA (p = 1.703 g/cm3) was used at the same time. This is a value close to the one recently found by Borst (3) and M. Salas (personal communication) using a scale in which Escherichia coli DNA is assigned a value of p = 1.710 g/cm3. Adsorption rates and one-step growth experiments. A procedure similar to that used by Mahony and Easterbrook (16) was followed. Bacteria were grown in CpH 8 medium at 37°C. At a cell concentration of 1 x 107 to 7.5 x 107 colony-forming units (CFU)/ml, Dp-1 was added at an MOI of 1:5 to 1:10. One-milliliter samples were withdrawn at different time intervals and centrifuged, and the free phage titer was determined in the supernatant. For one-step growth experiments, bacteria growing at mid-log phase (5 x 107 to 7.5 x 107 cells/ml) in CpH 8 medium were infected with phage at an MOI of 1:10 (16) (under these conditions, 80% of the phage was adsorbed in 10 min). Preparation of purified 32P-labeled Dp-1. To prepare 32P-labeled Dp-1, the host cells were grown in a modified CpH 8 medium in which the concentration of phosphorous in the normal medium (50 mM, as buffer phosphate) was reduced to 2.5 mM, and 47.5 mM Tris, pH 8, was added (CpH 8-TP medium).

J. VIROL. Bacteria were grown in this medium overnight. The next morning, the culture was diluted to a concentration of 1.7 x 107 CFU/ml with 750 ml of fresh CpH 8-TP medium containing 1 ,uCi of [32P]phosphoric acid per ml. When the culture reached a cell concentration of 2.5 x 107 cells/ml, Dp-1 was added at an MOI of 1:3, and 300 ml of CpH 8-TP medium (containing 1 ,uCi of [32P]phosphoric acid per ml) was added at the same time. Since the replacement of phosphate by Tris as a buffer increases the time needed for the lysis of the cultures (unpublished observations), this experimental procedure was needed in order to avoid the chance of the phage remaining trapped inside the cells as the result of a drop in the pH of the medium (22). Dry-weight determinations. Purified bacteriophage isolated from the isopycnic centrifugation was dialyzed and concentrated by pelleting in the ultracentrifuge. The phage was resuspended in distilled water to give an optical density of 24 (at 260 nm). Two milliliters of such a suspension (containing 1012 PFU/ml) was treated as indicated by Espejo and Canelo (5) and Vidaver et al. (33) to obtain the total virus weight and the lipid dry weight. A microanalytical balance (model 414/51, Sauter and Elinger, Germany) was used, and the mean value of four independent weight determinations was used to calculate the weight of the lipid. Lipid extraction. Lipids were extracted and purified from unlabeled or 32P-labeled Dp-1 following the procedures described by Schafer et al. (25). The chloroform extracts were concentrated under a stream of N2 at room temperature and stored at - 200C. Chromatographic procedures. Chloroform extracts were analyzed either on precoated Silica Gel F-254 thin-layer plates (Merck, Darmstadt) or on Silica Gel H (0.25 mm thick) impregnated with 1 mM sodium tetraborate (17). A developing tank (21.5 by 21.5 by 60 cm) lined with filter paper was filled with 140 ml of the adequate solvent and allowed to equilibrate for at least 3 h and then was used for developing the chromatograms. For the phospholipids, chloroform-methanol-water (65:24:4, vol/vol/vol) was used as solvent (12). Neutral lipids (fatty acids plus neutral lipids) were developed with a mixture of petroleum ether (bp = 60 to 70°C)diethylether-acetic acid (90:10:1, vol/vol/vol) as previously described (26). Total lipids were visualized on chromatograms by iodine vapors or by spraying the plates with ammonium-molybdate-perchloric acid (26). Labeled components on thin-layer plates were detected by exposure to Kodak NoScreen X-ray film for 4 days. After the film had been developed, regions on the chromatography plate containing the [32P]phospholipids were scraped and added to methanol as previously described (23). Lipid extracts were also prepared from the centrifugal pellet of about 2 x 109 pneumococci (harvested from a logarithmically growing culture). The solvent was evaporated under a stream of nitrogen gas; the residue dissolved in several hundred microliters of chloroform-methanol (2:1) and was used for the analysis of lipids. Thin-layer chromatography was performed on Silica Gel G plates (Merck, 0.025-

PROPERTIES OF DIPLOPHAGE

VOL. 24, 1977 thickness). Chloroform methanol - water (65:25:4, vol/vol/vol, solvent A) and chloroformmethanol-acetic acid (80:15:8, vol/vol/vol, solvent B) were used as solvents, and the plates were either exposed to iodine vapor or were sprayed with the ammonium molybdate-perchloric acid reagent (followed by heating at 80'C) in order to visualize the lipid components. Commercially available lipids were chromatographed along with the unknown samples as reference standards. Five major lipid components were detected in the pneumococcal extracts, and these were tentatively identified (in the order of their increasing mobilities in solvent B) as a glycolipid, phosphatidylglycerol, cardiolipin, a second glycolipid, and neutral lipids. A more detailed characterization of pneumococcal lipids will be described in another context in a forthcoming publication (D. Horne, R. Hakenbeck, and A. Tomasz, submitted for publication). Sedimentation velocity measurements. The sedimentation coefficient of Dp-1 was calculated from data obtained using an analytical ultracentrifuge (Spinco model E) at 11,071 rpm; the temperature of the run was 20'C; a 12-mm-high Kel-F centerpiece was used. Antigen preparation. Purified bacteriophages were treated as described by Nowinski et al. (19) for lipid-containing viruses. An equal volume of a phage suspension in TBT (about 5 x 10'° PFU/ml) was added to cold TBT containing Tween 80 (0.2%, final concentration), and the preparation was mixed thoroughly and kept in ice; then 4 volumes of ethyl ether were added, and the mixture was kept at 40C for 10 min with occasional shaking and centrifuged (5,000 x g, 5 min). The aqueous phase was treated with ether three more times. Finally, the aqueous phase was dialyzed for 24 h against several changes of TBT. Antibody preparation. For each injection, solubilized antigens were emulsified with an equal volume (0.4 ml) of incomplete Freund adjuvant (Difco Laboratories, Detroit, Mich.). To immunize the rabbits, both the intramuscular and intradermal routes were used. Injections (0.3 mg of protein) were given for 3 weeks, followed by a new injection (0.4 mg) 20 days later and a final one (0.8 mg) 40 days after that. Rabbits were bled, and the blood was left to clot for 20 h at 40C. The serum was removed by low-speed centrifugation and was stored in small portions at -20'C. Antiserum titer was determined by the method of Adams (1). Electron microscopy. Purified Dp-1 suspensions spread on Formvar-carbon films were either stained with 2% phosphotungstate at pH 7.2, or, alternamm

-

Purification step

(1) Crude lysate (2) After PEG 6000 precipitationa (3) After CsCi I (4) After CsCl II a PEG, Polyethylene glycol.

tively, were fixed with 2.5% glutaraldehyde in 0.1 M NaCl, pH 7.5, and negatively stained with 2% uranyl acetate solution as described by Hinnen et al. (10). All grids were examined at 80 kV with a Siemens electron microscope, or a Hitachi H-1 electron microscope at 75 kV accelerating voltage. All chemicals used were commercially available analytical grade products: [methyl-3Hlthymidine was purchased from New England Nuclear, Boston, Mass.; 32p was obtained from the Junta de Energia Nuclear, Madrid, Spain. Phospholipase C was purchased from Worthington Biochemicals Corp., Freehold, N.J. The phospholipid standards, phosphatidylethanolamine, cardiolipin, and phosphatidylcholine, were from Sigma Chemical Co., St. Louis, Mo. RESULTS

Purification of Dp-1. Table 1 shows the reof Dp-1 during the different steps of purification. We have confirmed the previous observation (29) that the dilution of Dp-1-infected cultures, when the cell concentration reaches 2 x 108, can increase from four to five times the phage concentration in the lysate. In most experiments, this titer was about 1 x 109 to 3 x 109 PFU/ml. This relatively low concentration of phage in the lysate may be due to the dependence of the phage upon the autolytic system of the host (22). Similar lysate titers were found when CpH 8 medium was replaced by K-CAT or by K-CpH 8 as proposed by Porter and Guild (21) for other phages of pneumococcus (data not shown). The degree of purification is best illustrated by data obtained with 3H-labeled phage. A 300-ml culture of bacteria at a cell concentration of 1.4 x 107 cells/ml was infected with Dp-1 (MOI = 1:40). Five minutes later, 2'-deoxyadenosine (200 ,ug/ml) and [methyl-3H]thymidine (10 ,uCi/ ml) were added. The culture was incubated at 37°C until complete lysis occurred. The lysate was processed in the same manner as described for unlabeled Dp-1. The fractions collected after the three-step CsCl gradient centrifugation were counted and titrated (Fig. 1A). The density distribution of infectivity and total radioactivity after the equilibrium centrifugation in CsCl was as shown in Fig. 1B. Specific activity of the purified Dp-1 [3H]DNA was 2 x 104 to 3 x 104 dpm/,ug.

covery

TABLE 1. Purification of bacteriophage Dp-1 Total PFU Titer (PFU/ml) Volume (ml) 3.0 x 1012 109 3,000 2.8 x 1012 5.5 x 10'° 44

8 4

203

1.2 x 1011 1.5 x 101"

9.6 x 101l 6.0 x 1011

Recovery (%)

100 93 32 20

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E2 0 0

E

0~

10

FRACTION NUMBER

15

20

25

30

FRACTION NUMBER

FIG. 1. (A) Density distribution of radioactivity (a) and infectivity (0) of partially purified Dp-1. Phage precipitated with polyethylene glycol and suspended in NTM was layered on the top of a CsCl step gradient containing three steps: p = 1.70 g/cm3, p = 1.5 glcm3, and p = 1.50 glcm3. (B) Equilibrium analysis of 3Hlabeled Dp-1. The phage band obtained in (A) was collected and directly centrifuged to equilibrium in an SW50-1 rotor at 35,000 rpm for 24 h. Symbols: PFU per milliliter, 0; total radioactivity, 0.

TABLE 2. Stability of Dp-1 phage during treatment with different reagents Additions

Phage infectivity (%)a after 8 days at: -70aC 4°C

44.3 67 None 11.4 48.5 1 M NaCl 7 8.6 2 M NaCl 93 86 10% Glycerol 53 10% Me2SO4 100 a Titer of infectious phage in the pure preparation at the beginning of the experiment (1010 PFU/ml) was taken as 100%.

0)1 C

.C

40 O

Stability of the phage. To test the stability of the phage, 0.4-ml portions of phage (in NTM, containing 1010 PFU/ml) were stored for 8 days at either -70 or 40C in the presence of different additives. The loss of biological activity was measured by the plaque test (Table 2). Adsorption rates and one-step growth experiments. When bacteria were infected at the mid-log phase (7.5 x 107 CFU/ml), 80% of the phage were adsorbed in 10 min (Fig. 2); the adsorption rate was substantially lower when the host cells were infected at the beginning of the log phase (107 CFU/ml). Mahony and Easterbrook (16) have pointed out the difficulty in performing the typical "one-step growth" experiment (1) with anaero-

80

E o \ " 0)1 60u O.. \

20 _

I_ 0

5

10

15

I

20

Time af ter infection (min.) FIG. 2. Kinetics of adsorption of bacteriophage Dp-1 onto cells of S. pneumoniae R36A at a cell density of 107 cellslml (@) or 7.5 x 107 cellslml (0). The MOI was 0.1 to 0.2. At different times, samples were withdrawn and centrifuged, and the number of free phage was determined in the supernatants.

bic or microanaerobic organisms. We have confirmed previous results about the variations in the burst sizes of phages isolated from S. pneumoniae as a function of the cell densities (29). The dependence of Dp-1 upon the host autolytic

VOL. 24, 1977

PROPERTIES OF DIPLOPHAGE

205

system for its liberation (22) might be a critical factor contributing to this phenomenon. For the particular conditions described above, the burst size of Dp-1 was about 40 phage per cell, and the

latent period was 25 min (Fig. 3). Buoyant density of Dp-1. The virion has a buoyant density in cesium chloride at 20'C of 1.47 g/cm3 (Fig. 4). As can be seen, the profiles of PFU and absorbency at 260 nm coincided when the phage was centrifuged to equilibrium in CsCl gradients. Acrylamide gel electrophoresis. The proteins of the disrupted purified virus can be resolved into three main bands (Fig. 5). Some analyses indicate the presence of an additional minor polypeptide migrating close to the origin. Sedimentation coefficient of Dp-1. Several phage dilutions were prepared in 0.1 M NaCl to calculate the extrapolated sedimentation coefficient (we have taken 313S as the zero concentration value). After correcting this value to standard conditions, an s20, value of 318S was obtained. The sedimentation boundary pattern of the purified phage is shown in Fig. 6. Buoyant density of Dp-1 DNA. The radioactivity profile of 3H-labeled Dp-1 in a CsCl density gradient is shown in Fig. 7. 429 ['4C]DNA was used as a reference. The buoyant density of Dp-1 DNA was 1.681 g/cm3, which would correspond to a guanine-cytosine content of 27 mol% if Dp-1 DNA contained the four common bases.

D

i

I E

E

4,1incu

10

20

30

40

FRACTION NUMBER

FIG. 4. Equilibrium density gradient of Dp-1. Purified Dp-1 was centrifuged to equilibrium in CsCl. After 24 h of centrifugation in an SW50-1 rotor at 35,000 rpm, at 20°C, fractions were collected from the bottom of the tube and analyzed. II

0 o

.e z

,

-J

c-i 0

loa8

/

3

ORIGIN

5

7 Frqr

E

0

U.

FIG. 5. Electropherogram of Dp-1 proteins run according to (34), stained with Coomassie brilliant blue, and scanned with a spectrophotometer (Gilford model 2400) equipped with a linear transport system (model 2410S). Roman numerals refer to the viral polypeptides.

S6-

S

u --l -0

-.25

-.50

.75

..100

-.-

125

150

TIME (min.)

FIG. 3. One-step growth curve ofDp-1 on S. pneumoniae R36A. Dp-1 (MOI about 0.1) was added at a cell density of 7.5 x 107 cell/ml. Incubation was at 370C.

This value is similar to that found for phage 44A, which was isolated from Staphylococcus aureus (27). Immunodiffusion tests and K value. Immunodiffusion studies were carried out using the Ouchterlony technique (20). When whole, purified Dp-1 phage reacted with Dp-1 antiserum,

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C.

40

FIG. 6. Sedimentation patterns of Dp-1 after centrifugation for 20 min (A) and 36 min (B) at 11,071 rpm at 20°C in 0.1 M NaCI. To obtain the patterns, pictures were taken in an analytical ultracentrifuge, (Spinco model E) using UV light of wavelength 265 nm, and were scanned with a Chromoscan (JoyceLoebl) recording and integrating densitometer. 1000

,

II

1700

800k 0

a

-j1650

E

1 1.

0

1 600

4600 ?r

1.

E I:

400L

1600

It

E

1 I

0

'E

1400

0

I tk

bottom

5

10

15

-200

20

25

30 top

FRACTION NUMBER

FIG. 7. Buoyant density of Dp-1 DNA and 29 DNA. One-tenth milliliter of Dp-1 [3H]DNA (1.4 x 104 cpmlpg; 7.5 pg/ml) and 20 pi of 429 ['4C]DNA (1.5 x 105 cpmlpg; 2.5 Mg/ml) were added to 3 ml of CsCl solution in 1/10x SSC. Centrifugation was performed in a Beckman L3-50 ultracentrifuge (SW50-1 rotor) at 35,000 rpm for 60 h at 100C. Symbols: (-) 029 DNA; (0) Dp-1 DNA.

band was observed (Fig. 8A); no lines of precipitation occurred when whole S. pneumoniae 36A or unrelated phage (SPP1) were tested against anti-Dp-1 serum. Tween 80- and ethertreated virus reacted with the Dp-1 antiserum,

one

forming at least two precipitin bands (Fig. 8B). The antiserum prepared against Dp-1 gave a maximum value of K = 90 to 120/min. Evidence for the presence of a lipid envelope in Dp-1. (i) Inactivation of Dp-1 by organic solvents and detergents. The sensitivity of Dp-1 to organic solvents and detergents is shown in Table 3. The B. subtilis phage SPP1 treated in the same manner did not show any significant decay in titer. (ii) Lipase sensitivity. The infectivity of purified Dp-1, incubated with phospholipase C, was only partially affected by the enzyme after 210 min of incubation. No effect was detected for SPP1 (Fig. 9). (iii) Electron microscopy of Dp-1. When Dp1 is stained on the electron microscope grid with 2% phosphotungstate (pH 7.2), the phage shows a polyhedral head and tail (Fig. 10). The presence of a baseplate previously described by us (15) could not be confirmed with pure preparations of the phage. In some empty phages, the tail appears to have a platelike structure near the junction to the phage head (see arrow in Fig. 10 and reference 21). The diameter of the phage particle is 67 + 3 nm. In contrast to the pneumococcal omega phages, no tail fibers are apparent on Dp-1. Franklin (6) has pointed out that PM-2 and many lipid-containing viruses are damaged by phosphotungstic acid, and Dp-1 treated as previously described (10, 24) gives a morphological image reminiscent of some of the particles of PM-2 shown by Harrison et al. (9) and those obtained for iridescent virus (12). The particles of Dp-1 show a double-line boundary at both low- and high-power magnification electron micrographs (Fig. 11). (iv) Chemical analysis of the lipid. The dry weight of the lipid extracted with methanolchloroform and soluble in chloroform accounts for a minimum of 8.5% (average value of four independent determinations) of the dry weight of purified Dp-1. Lipid extracts prepared from the purified phage and from uninfected S. pneumoniae cells were analyzed by thin-layer chromatography on plates coated with Silica Gel G. After development in chloroform-methanol-water (65:25:4, vol/vol/vol), the lipids were stained with iodine vapor. Four spots were detected in both the phage and in the cellular lipid extracts. On the basis of Rf values, the four lipid components of the phage and cellular lipid extracts appeared to be similar or identical. Additional, more extensive analytical work has been done with the pneumococcal lipids. The use of several solvent systems allowed the

PROPERTIES OF DIPLOPHAGE

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207

II

FIG. 8. Peripheral wells 1, 2, and 3 were filled with whole purified Dp-1 phage at three different dilutions; wells 4 and 5 were filled with whole S. pneumoniae R36A cells, and well 6 contained purified SPP1, a phage of B. subtilis. Center well contained Dp-1 antiserum. (B) Peripheral wells (1-6) filled with antiserum (dilution 1:1, 1:2, 1:4, 1:8, 1:16, 1:32). Center well: Tween 80-ether-treated Dp-1.

TABLE 3. Inactivation of Dp-1 by organic solvents and detergents Treatment

Dp-1 phage None 0.25 volume of chloroform 5% sodium deoxycholate, 20 min 0.05% Sarkosyl, 20

Titer (PFU/

ml)

treateontreated/nn treated

2.4 x 108 2.2 x 104

9.1

4.1 x 104

1.7 x 10-4

3.8 x 104

1.6 x 10-4

4.4 x 107

0.2

2.5 x 108 2.8 x 10"

1.0 1.1

1.8 x 10"

0.7

10"

0.9

1.0 X 10-5

min

0.25 volume ofether SPP1 phage None 0.25 volume of chloroform 5% sodium deoxycholate, 20 min 0.05% Sarkosyl, 20

2.2 x

min

1.3 3.2 x 108 0.25volumeofether a Phages Dp-1 or SPP1 in TBT, pH 7.8, were treated with chloroform or ethyl ether for 24 h at 4VC according to the procedure of Andrews and Horstman (2). Samples for assay were withdrawn from the aqueous layer. The values are given as percent (volume to volume).

separation of five major classes of lipids: two glycolipids, two phospholipids, and neutral lipids (see Materials and Methods). The rather limited quantities of phage lipids available did not allow a similar, more extensive analysis. The four lipid components detected in the phage extract had Rf values (in the order of increasing mobilities) corresponding to those of the pneumococcal phospholipids (I), glycolipid A (II), glycolipid B (III) and neutral lipids (IV). In lipid extracts prepared from 32P-labeled, purified bacteriophage, two of the lipid components (components I and II) were found to contain radioactivity. Further work will be needed for the identification of the phage lipids. DISCUSSION To avoid problems of contamination with host cell products, we have followed most of the methods of purification and criteria of purity recommended by Franklin (6) for lipid-containing bacteriophages. The results described suggest that our phage preparations are reasonably pure: (i) equilibrium centrifugation of labeled and unlabeled preparations yielded similar profiles in cesium chloride gradients. (ii) In the final stage of

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purification, only three major (plus possibly one minor) polypeptides were detected by polyacrylamide gel electrophoresis, and additional rounds of purifications by isopycnic CsCl centrifugation did not change this pattern. (iii) The antigenic studies by Ouchterlony immunodiffusion show the specificity of the viral proteins, and no host antigens were found. (iv) The only nucleic acid obtained from the peak of infectious material gave a single peak in isopycnic centrifugation and showed a density (p = 1.681 g/cm3) very different from that of the host cell DNA (p = 1.701 g/cm3) (13). The data shown indicate that the purified bacteriophage Dp-1 contains lipids. The sensitivity of Dp-1 to organic solvents and detergents is consistent with the presence of lipids. Elec-

at --4- --I----Q5OOpg/ml 0g/ro _S --

E lop

L. 20 pg/ml 500pg/ml

60

ISO

0.02M Tris, pH 7.8, 7.5 x 10-3M CaCl2, and 1O-3 M MgCl2. Samples were removed at various times and ) Dp-1; ( ---) tested for infectivity. Symbols: ( *) O) and (0-----0) no lipase; (@ SPP1; (0 A) and (A---A) 500 20 pg of lipase per ml; (A pg of lipase per ml.

300

TIME (min) FIG. 9. Sensitivity of Dp-1 to phospholipase C. Purified Dp-1 (1.6 x 108 PFU/ml) was incubated with phospholipase C at 37°C in the presence of

N

A

FIG. 10. Electron micrographs of Dp-1. Purified phage (x126,000).

was

stained with 2% phosphotungstate (pH 7.2)

PROPERTIES OF DIPLOPHAGE

VOL. 24, 1977

S

209

..

0

-AS,

41.

41;.'

I

FIG. 11. Purified Dp-1 was stained with uranyl acetate as described in (10). In order to minimize optical artifacts, the microscope was carefully corrected for astigmatism, and photographs were taken at several focal settings in a through-focal series (x234,000).

tron microscopy studies of Dp-1 show that this bacteriophage has a structure that resembles PM-2 (9) and the iridescent virus type 2 (12), i.e., two well-known lipid-containing viruses. The incomplete inactivation of Dp-1 by phospholipase C may be a consequence of a problem of penetrability of the lipid envelope by the

The dependence of Dp-1 upon the host autolytic system and the extreme sensitivity of lipidcontaining phages to several reagents used during the process of purification might explain the difficulties previously encountered in attempts to obtain high-titer stocks of this bacteriophage. The procedures described here provide us with a method to obtain and preserve a reasonable titer of viable particles. Nevertheless, no enhancement in the titer of the crude lysate could be observed when Na+ was replaced by K+ in the medium as recommended by Porter and Guild (21) for some other pneu-

enzyme (18). The total lipid content has been estimated as a minimum of 8.5%. According to a previous report (12), this amount would be sufficient to envelope the surface of a phage particle of the size of Dp-1 (diameter approximately 67 nm), although there is no evidence at present sug- mococcal phages. Some phages of species taxonomically close to gesting regularity of the distribution of the lipid in the virus surface. Determination of S. pneumoniae have been found to possess a whether or not the phospholipid composition of remarkably low content of guanine plus cytoDp-1 is different from that of the host cell will sine in their DNA (27). The low buoyant density of Dp-1 DNA (p = 1.681 g/cm3) may indirequire further analytical work. Dp-1 bacteriophage appears to be a poor im- cate a similar base composition. The unique biological properties of Dp-1, as munogen, as indicated by the low K value obtained for antisera (K < 120/min). Poor immu- described in this communication, make this nogenic behavior seems to be a characteristic bacteriophage system an attractive one for shared by many lipid-containing phages (PM-2 several types ofculture studies. The possible role mycobacteriophages, etc.) as well as other lipid- of phage lipid in the triggering of host autolytic enzyme may provide insights into the cellular containing viruses (32).

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16. Mahony, D. E., and K. B. Easterbrook. 1970. Intracellular development of a bacteriophage of Clostridium perfringens. Can. J. Microbiol. 16:983-988. 17. Makula, R. A., and W. R. Finnerty. 1970. Microbial assimilation of hydrocarbons: identification of phospholipids. J. Bacteriol. 103:348-355. ACKNOWLEDGMENTS 18 Mizutani, H., and H. Mizutani. 1964. Action of phospholipase C on influenza virus. Nature (London) 204:781We gratefully acknowledge D. Lopez-Abella and F. Gar782. cia-Hidalgo for obtaining the electron micrographs and 19. Nowinski, R. C., N. H. Sarkar, L. L. J. Old, 0. H. J. Albendea for his technical assistance during the determiMoore, D. I. Scheer, and J. Hilgers. 1971. Characternation of the sedimentation coefficient. We thank N. Rubio istics of the structural components ofthe mouse mamand M. Espinosa for the preparation of the antiserum and mary tumor virus. II. Viral proteins and antigens. the acrylamide gel electrophoresis. Rosa Maria Saez Virology 46:21-38. (Junta de Energia Nuclear, Madrid) helped us during the 20. Ouchterlony, 0. 1967. Immunodiffusion and immunoepreparation of 32P-labeled Dp-1. We also thank Maria del lectrophoresis, p. 655-706. In D. M. Weir (ed.), HandCarmen Jimenez for her skillful technical assistance. book of experimental immunology. J. B. Lippincott Co., New York. 21. Portqr, R. D., and W. R. Guild. 1976. Characterization LITERATURE CITED of some pneumococcal bacteriophages. J. Virol. 1. Adams, M. H. 1959. Bacteriophages. Interscience Pub19:659-668. 22. Ronda, C., R. Lopez, A. Tapia, and A. Tomasz. 1977. lishers Inc., New York. Role of the pneumococcal autolysin (murein hydro2. Andrews, C. H., and D. N. Horstman. 1949. The susceplase) in the release of progeny bacteriophage and in tibility of viruses to ethyl ether. J. Gen. Microbiol. 3:290-297. the phage-induced lysis of the host cells. J. Virol. 3. Borst, P. 1971. Life, structure and information content 21:366-374. of mitochondrial DNA in autonomy and biogenesis of 23. Sands, J. A. 1973. The phospholipid composition of bacteriophage 06. Biochem. Biophys. Res. Commun. mitochondria and chloroplasts, p. 260-266. North55:111-116. Holland, New York. 4. Braunstein, S. M., and R. M. Franklin. 1971. Structure 24. Schafer, R., R. Hinnen, and R. M. Franklin. 1974. Furand synthesis of a lipid-containing bacteriophage. V. ther observations on the structure of the lipid-conPhospholipid of the host BAL-31 and the bacteriotaining bacteriophage PM2. /Nature (London) phage PM2. Virology 43:685-695. 248:681-682. 5. Espejo, R. T., and E. S. Canelo. 1968. Properties of 25. Schafer, R., R. Hinnen, and R. M. Franklin. 1974. bacteriophage PM2. A lipid-containing bacterial viStructure and synthesis of a lipid-containing bacterus. Virology 34:738-747. riophage. XV. Properties of the structural proteins 6. Franklin, R. M. 1974. Structure and synthesis of bacteand distribution of the phospholipid. Eur. J. Bioriophage PM 2 with particular emphasis on the viral chem. 50:15-27. lipid bilayer. Curr. Top. Microbiol. Immunol. 68:107- 26. Skipski, V. P., and M. Barclay. 1969. Thin layer chro159. matography of lipids. Methods Enzymol. 14:530-598. 7. Goldfine, H. 1972. Comparative aspects of bacterial 27. Szybalski, W. 1968. Use of cesium sulfate for equiliblipids, p. 1-58. In Advances in microbial physiology. rium density gradient centrifugation. Methods EnAcademic Press, Inc., London and New York. zymol. 12B:330-360. 8. Gross-Bellard, M., P. Oudet, and P. Chambon. 1973. 28. Thomas, C. A., and J. Abelson. 1967. The isolation and Isolation of high molecular weight DNA from mamcharacterization of DNA from bacteriophage, p. 553malian cells. Eur. J. Biochem. 36:32-38. 561. In G. L. Cantoni and D. R. Davies (ed.), Proce9. Harrison, S. C., D. L. D. Caspar, R. D. Camerinidures in nucleic acid research, vol. 1. Harper and Otero, and R. M. Franklin. 1971. Lipid and protein Row, New York. arrangement in bacteriophage PM2. Nature (London) 29. Tiraby, J. G., E. Tiraby, and M. S. Fox. 1975. PneumoNew Biol. 229:197-201. coccal bacteriophages. Virology 68:566-569. 10. Hinnen, R., R. Schafer, and R. M. Franklin. 1974. 30. Tomasz, A., and R. D. Hotchkiss. 1964. Regulation of Structure and synthesis of a lipid-containing bactethe transformability of pneumococcal cultures by riophage. Preparation of virus and localization of the macromolecular cell products. Proc. Natl. Acad. Sci. structural proteins. Eur. J. Biochem. 50:1-14. U. S. A. 51:480-487. 11. Hirokawa, H. 1972. Transfecting deoxyribonucleic acid 31. Tomasz, A., and S. Waks. 1975. Mechanism of action of of Bacillus bacteriophage 429 that is protease sensipenicillin: triggering of the pneumococcal autolytic tive. Proc. Natl. Acad. Sci. U.S.A. 69:1555-1559. enzyme by inhibitors of cell wall synthesis. Proc. 12. Kelly, D. C., and D. E. Vance. 1973. The lipid content of Natl. Acad. Sci. U. S. A. 72:4162-4166. two iridescent viruses. J. Gen. Virol. 21:417-423. 32. Truden, J. L., and R. M. Franklin. 1971. Structure and 13. Lacks, S. 1962. Molecular fate of DNA in genetic transsynthesis of a lipid-containing bacteriophage. IX. Seformation of pneumococcus. J. Mol. Biol. 5:119-131. rological disparity between bacteriophage PM2 and 14. Lopez, R., A. Tapia, and A. Portoles. 1975. The influits host cell components. Virology 46:808-816. ence of glutamic acid and arginine on the transfecta- 33. Vidaver, A. K., R. K. Koski, and J. L. van Etten. 1973. bility ofB. subtilis growing in a chemostat. Mol. Gen. Bacteriophage 46: a lipid-containing virus of PseuGenet. 136:87-94. domonas phaseolicola. J. Virol. 11:799-805. 15. McDonnell, M., C. Ronda, and A. Tomasz. 1975. "Di- 34. Weber, R., J. R. Pringle, and M. Osborn. 1972. Measurement of molecular weights by electrophoresis plophage": a bacteriophage of Diplococcus pneumoniae. Virology 63:577-582. on SDS-acrylamide gel. Methods Enzymol. 26C:3-27.

control of this enzyme, the activity of which has already been implied in several important phenomena, such as cell separation in division and the bactericidal action of penicillin (31).