Morphology of bacteriophage P22 as seen in thin sections of pelleted phage

VIROLOGY

71, 434-443

Morphology

(1976)

of Bacteriophage

GLENN ’ L?epurtment of Cellular

A. ZORN’

P22 as Seen in Thin Sections Phage AND

MICHAEL

of Pelleted

GOUGH2

and Comparative Biology, and 2 Department of Microbiology, New York at Stony Brook, Stony Brook, New York I1 794

State University of

Accepted January 16, 1976 The tails of adsorbed P22 in thin section appear longer than tails seen on negatively stained phage. Phage were pelleted and sectioned to determine tail length on free phages. The tail length of both adsorbed and unadsorbed phage in thin section were nearly identical, approximately 240 A. This represents a tail length about one-third longer than that observed in negatively stained specimens. Areas of phage pellets were in highly ordered arrays (paracrystalline arrays). The phage head in thin section is sliced into shapes that are consistent with the head being an icosahedron. INTRODUCTION

Salmonella phage P22 has an isometric head and a short tail (Yamamoto and Anderson, 1961). The diameter of the phage head is approximately 550 A (arithmetic mean of measurements of Yamamoto and Anderson, 1961; Botstein, Waddell, and King, 1973). King, Lenk, and Botstein (1973) determined that eight proteins make up the phage particle. Of the eight proteins, the one specified by gene 5 is present in the largest number, about 400 copies per phage. This protein is the major structural protein of the phage head and probably accounts for its characteristic shape and size. The number of gene 5 products is in good agreement with the expected number of 420 subunits for an icosahedron (Botstein et al., 1973). The product of gene 1 is a minor structural protein of the head, present in about 20 copies per phage. The genes 16 (10 copies per phage) and 20 (2030 copies per phage) specify products contained in the phage head that are required for DNA injection (Hoffman, 1973; R. Griffin, personal communication). At one apex of the head is a necklike structure. The gene (genes) which specify the neck of the phage has not been identified, although 434 Copyright All rights

Q 1976 by Academic Press, Inc. of reproduction in any form reserved.

the product of gene 26 (30 copies per phage) is a likely candidate. Micrographs published by Israel, Anderson, and Levine (1967) show that the neck is a structure distinct from the rest of the tail and that it is present on particles which lack the pins and spike. Protein X is contained in the phage particle but has not been associated with a specific structure. The tail of P22 in negatively stained preparations appears to be a rather simple structure. It consists of a single protruding spike and six pins (Israel et al., 1967; King et al., 1973; see also Fig. 1). No gene so far has been associated with the spike. The product of gene 9 is responsible for “serum blocking power” and reacts with heads which have a neck to form infectious particles in vitro (Israel, Rosen, and Levine, 1972; Israel et al., 1967). The pins seen on the completed particle are the product of gene 9 (20 copies per phage). The apparent base plate may be made of gene 9 products; perhaps the 9 protein folds so that part of it is involved in the base plate structure and the remainder forms the pins. Alternatively, different gene products might be responsible for the base plate and the pins, but this idea is difficult to reconcile with the in vitro assembly of phages from heads

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OF

(which lack the base plate) and purified 9 product. The 9 product has endorhamnosidase activity which hydrolyzes the O-antigen of Salmonella typhimurium, enabling the phage to penetrate the polysaccharide surrounding the cell and reach a receptor on the cell surface (Iwashita and Kanegasaki, 1973). At the time of phage adsorption, gross alterations in phage morphology are known to occur in some phages (T-evens) while other phages remain relatively unchanged (lambda). This study began with thin-section electron microscopy of P22 to see if any alteration in phage structure was associated with the adsorption process. The length of the adsorbed phage tail appeared longer than that seen in negatively stained electron micrographs of free phage. We considered that the difference in tail length could be an adsorption associated alteration. To examine the tail length of unadsorbed phage, we thin-sectioned pellets of P22. The tail length of the phages is the same as that of adsorbed phages in thin section. A model is presented to explain the discrepancy between tail lengths observed in negative staining and thin sectioning. Unexpectedly we found that particles in some areas of the pellet are in paracrystalline arrays. The arrangements of particles in the paracrystalline arrays and the profiles of thin-sectioned phage heads are consistent with P22 having an icosahedral head.

BACTERIOPHAGE

P22

435

lox cells/ml in TB and infected at a multiplicity of infection (m.o.i.1 of 5 phage P22 at 37”. After 60 min several drops of chloroform were added. The bacterial debris was removed by low-speed centrifugation at 12,000g for 10 min, and the phage pelleted at 35,000 g for 90 min. The pellet was resuspended in TB and the phage was purified in a CsCl step gradient as described by Smith (1968). Two bands could be seen in the CsCl. Each band was collected by puncturing the side of a nitrocellulose centrifuge tube with a hypodermic syringe and withdrawing the visible material. The upper band was found to contain whole bacteria and bacterial debris in addition to free phage and will not be described further. The lower band was primarily phage with little contaminating material and was prepared for electron microscopy. Method I. One microliter of phage was diluted into 9 ml of 0.01 M MgSO,. Twoand-one-half milliliters of 2.5% glutaraldehyde (Simon, 1972) in 0.06 M Na cacodylate was added to bring the final concentration of glutaraldehyde to 0.5%. The material was recentrifuged at 1200 g for 10 min. The supernatant was subjected to high-speed centrifugation at 35,000 g for 2l/2 hr. The phage pellet was immediately fixed with a solution of 1% 0~0, in R-K buffer (Simon, 1972) and left overnight at room temperature. Method II. The phage were treated identically to Method I with the exception that the prefixation with glutaraldehyde was omitted. MATERIALS AND METHODS Preparation of adsorbed phage for thinBacterial and phage strains. Salmosection microscopy. MG3 was grown to 2 x nella typhimurium MG3, a wild-type S. 10H cells/ml and infected with P22wt. The typhimurium, was used for all infections phage were allowed to adsorb for 3 min and as indicator bacteria on plates for deand fixed and embedded according to the termining phage titers. Phage P22 c+ procedures of Simon (1972) using glutaral(wild-type) and P22 cl were used throughdehyde as a prefixative. out this study and were originally from the Embedding of phage pellets. The phage collection of M. Levine (1957). pellets were dehydrated in a graded aceMedia. MG3 was grown in Tryptone tone series and infiltrated with Spurr’s Broth, TB (10 g of Tryptone, 5 g of NaCl, 1 Low Viscosity Epoxy as modified by Simon liter of distilled water) and plated on Tryp(1972). tone agar, TA, which is TB plus 15 g of Preparation of thin sections. Thin secagar. tions were cut using a diamond knife (purPreparation of phage pellets for thinchased from DuPont and Co., Wilmington, section microscopy. MG3 was grown to 2 x Delaware) on an LKB Model 8800 Ultrami-

436

ZORN

AND

crotome III microtome. The sections were silver or gold in color. The thickness of the sections was easily distinguishable in the electron microscope. Silver sections were, on the average, about the thickness of a phage head, while gold sections were thicker than the diameter of the phage head. The sections were picked up on copper grids. Formvar support films with a thin carbon coat were sometimes used for added stability. The sections were stained with 2% aqueous uranyl acetate overnight. To enhance the contrast of the phage tail, some sections were stained for 72 hr. The grids were rinsed in distilled water and stained with Reynolds lead citrate (Reynold, 1963) for lo-12 min. The grids were then rinsed in distilled water and allowed to dry. Negative staining. Grids were stained with 2% uranyl acetate using the procedures of Simon and Anderson (1967). Electron microscopy. The grids were examined on a Siemens IA, a JEM-GC, or a Hitachi HU-12 at 80 kV or a JEM-100B at 60 kV. RESULTS

Negatively stained P22. Three negatively stained P22 are shown in Fig. 1. We measured the tail length on 50 negatively stained phages. Assuming that the diameter of the head is 550 A, we found the tail length to be 176 A from the beginning of the neck to the end of the pins, but not including the spike. This measurement is in rough agreement with Yamamoto and Anderson (1961) who reported that the length of the neck plus base plate plus pins was 140 A. The range of lengths among the 50 tails was 120 to 200 A. The variability of these measurements is discussed later. Thin-sections of adsorbed phage. Figure 2 is a thin section of a wild-type P22 adsorbed to its host. Inspection of this photograph and comparison to the negatively stained preparations (Fig. 1) show that the tail of the adsorbed phage appears longer. The average tail length of 25 adsorbed phages was 236 A and the range was from 220 to 240 A. We considered that the apparently longer tail could be a result of expansion upon adsorption or differences

GOUGH

between negatively stained preparations (Fig. 1) and thin sections (Fig. 2). Tail length in thin section. If the apparent lengthening of the phage tail in Fig. 2 is a result of adsorption, we would expect unadsorbed phages in thin section to have the same length tail as that of negatively stained unadsorbed phage (Fig. 1). Figure 3 shows pictures of three phages which were obtained by thin section. They are representative of 25 phages on which the tail length was measured. The average length of such tails was 240 A with a range of 225 to 255 A. When a phage lies in a plane such that the microtome cuts parallel to the phage tail, the apparent length of the tail is maximal. If the phage lies at an angle to the plane of sectioning, part of the tail may be removed by the cut. For this reason we selected phages which appeared to have “long” tails and baseplates to make the measurements which yielded the 240-A length. Many particles have shorter tails or no visible tails. We ascribe the shorter tail or absence of tails on these particles to be the result of the tail lying out of the plane of sectioning. The good agreement between the tail length of adsorbed and unadsorbed phages prepared by thin section led us to conclude that no gross morphological changes occur at the time of adsorption. Figure 4 is a drawing of a regular icosahedron resting on one face with an attached projection that rests on the same level as the polygon. The length of the projection to the maximum diameter of the icosahedron is the ratio 240:550. This represents an idealized P22 prepared for negative staining. We can, by making one assumption, reconcile the difference in negatively stained and thin-sectioned tail length. The assumption is that the tip of the tail rests on the substrate; that is, the tip of the tail is down, as shown. We assigned the length of the tail in thin section 240 A, to the hypotenuse (a-b). By measuring polystyrene icosahedrons we find that the vertical distance from the substrate to the origin of the projection (b-c) is equal to 0.3 of the maximum diameter of the icosahedron. Assuming the head is 550 A in

FIG. variable FIG. baseplate baseplate FIG. appear C shows

1. Negatively stained P22. The baseplates appear in close association with the phage head and are in length. Bar represents 0.1 pm. 2. P22 adsorbed to S. typhimurium in thin section. DNA of the phage is partially injected. The is not closely linked to the head. Plane of section is not perfectly parallel to phage tail, and the can be seen at the end of a narrow tube which connects to the head (bar represents 0.1 pm). 3. Unadsorbed phage in thin section. Micrographs were obtained by sectioning pelleted phage. Tails similar in length to those of adsorbed phages and longer than tails in negatively stained specimens. two phages with tails connected. Bar represents 0.1 pm. 437

438

ZORN

AND

C FIG. 4. An idealized phage. The head is an icosahedron in profile, and the tail a line. The phage rests on a face and the tail tip. The line a-b is the tail; the line a-c its horizontal projection.

diameter, we obtain the value 165 A for line b-c. Having lengths a-b and b-c we can solve the Pythagorean theorem for the distance a-c. The calculated distance a-c is 174 A. Distance a-c is the horizontal projection of a-b and is the apparent length of a-b when viewed from above. The calculated value of 174 A and the observed value of 176 A are in good agreement. The great variability of tail lengths on negatively stained phages (120-200 A) may be due to some flexibility in the tail. A bend between points a and b would reduce the apparent length of the tail. Whether the head was lying on an apex or a face of the icosahedron would affect the length of b-c and, thus, the apparent length of the tail (a-c). Some tails may not be sticking to the substrate (formvar film) and would appear longer. Differences in stain thickness should also affect the apparent tail length. Thin sections of prefixed pelleted P22. Phage P22 was prefixed with glutaraldehyde before being pelleted and prepared for thin-sectioning (Method 1). Figure 5 is an electron micrograph of the resulting pellet. Most of the phage particles are randomly oriented with respect to each other, but in some regions the phages are more closely packed in para-crystalline arrays. The term para-crystal is used simply to mean an ordered array in three dimensions. It does not imply true crystallization. Thin sections of nonprefixed pelleted P22. Phages prepared by Method II are

GOUGH

shown in Fig. 6. This micrograph is at the same magnification as Fig. 5. It can be seen that the random areas of the phage pellets in Figs. 5 and 6 are very similar. Observations of many sections have convinced us that the para-crystalline regions are larger in pellets prepared by Method II (no prefixation) than those prepared by Method I. Figures 7-9 are sections through some of the larger para-crystalline arrays found when phage are prepared by Method II. A number of different stacking patterns are evident in Fig. 7. Six different patterns can be identified each representing separate “mini” crystals. It is not known if each of the patterns observed represents different ways of closely stacking phage particles or whether each pattern represents a different plane cut through similar crystals. We have made no attempts to distinguish between these possibilities. Figures 8 and 9 are micrographs of large regular arrays found in Method II preparations. Both of these patterns suggest helical arrangements of phage particles. We expect that helices viewed end-on would appear as circles of particles. We have never observed such a pattern and doubt that the patterns in Figs. 8 and 9 are helical. The helical appearance could result if the plane of sectioning is at an angle with respect to the planes of virus particles. The shapes of phage heads seen in thin section. The phage head has an overall projection of a hexagon when viewed in negative staining (Yamamoto and Anderson, 1961; Israel et al., 1967; see also Fig. 1). The majority of the phage particles in thin section also have the shape of a hexagon as seen in Fig. 3 and enlarged in Fig. lOa-f. Using polystyrene models, we found that most slices through an icosahedron yield figures that have an overall hexagonal projection; an example is shown in Fig. 11. This is true even for some cuts which produce faces that contain seven, eight, or more sides. However, some slices were obtained that gave rise to figures that projected more than six sides. Such shapes could not be convincingly identified in thin sections of the phage. Projections that were round or amorphous in shape were

MORPHOLOGY

FIG. 5 Micrograph of thin randon 111r oriented phage with FIG. 6 Micrograph of thin preparf :d by Method I (Fig. 5).

OF

BACTERIOPHAGE

P22

section of phage pellets prepared by Method I. The pellet is coml several small para-crystalline arrays. Bar represents 2 pm. section of phage pellet prepared by Method II. The pellet is similar Bar represents 2 pm.

)OSf

to

,d of that

440

ZORN

FIG. 7. Para-crystalline pattern represents different Bar represents 1 pm.

arrays illustrating ways of stacking

AND

GOUGH

six different phage particles

seen; these may represent those figures that contain more than six sides. Pentagonal shapes could easily be identified in thin section (see Fig. log-l). Pentagons were consistently less dense than most of the hexagons. Five-sided figures were obtained from our polystyrene models of icosahedra (see Fig. 11). It can be seen that the only way to obtain a pentagon is to make a thin cut through the icosahedral model that includes one of the apices close to the center of the cut. The observations of others (Yamamoto and Anderson, 1961) that the head is isometric are consistent with the head being a regular polygon. The two identifiable shapes that we see in thin section, hexagons and pentagons, are consistent with the head being an icosahedron. DISCUSSION

The length of the P22 tail. Our data show that the tails of adsorbed and unadsorbed phage P22 are the same length (Figs. 2 and 3) in thin sections. In thin section, the tail measures 240 A, as com-

stacking or different

patterns. planes

It is not known through one major

if each crystal.

pared to 176 A seen in negatively stained preparations (Fig. 1). We concluded that the short tail in negatively stained preparations is an artifact. We propose that the tail is always “down,” that is, on the substrate, in negatively stained preparations. Inspection of Fig. 4 shows that this would cause the tail to be seen at an angle in negatively stained preparations and would result in its shorter appearance. Par-a-crystalline arrays of P22. An unexpected result of this investigation of centrifuged phage P22 is the observation of para-crystalline arrays in the phage pellet. Speyer and Khaurakkach (1973) grew crystals of phage T4. Electron micrographs of those crystals show very ordered arrays of particles. The phages are arranged head-to-head in repeated bilayers, with the phage tails interdigitated between the head bilayers. In another plane, when the section passes through the tails, it can be seen that the tails are arranged on a regular hexagonal pattern. Our para-crystalline arrays formed by centrifugal forces lack the preciseness of the T4 crystals.

MORPHOLOGY

FIIGS. 8 and 9. Higher of S()me of the paracrystals.

magnifications

OF

BACTERIOPHAGE

of two of the stacking

patterns

P22

observed,

illustrating

the Ial

-ge size

442

ZORN

10. (a-f) section.

FIG.

thin

Hexagonal

head shapes

AND

observed

in thin

2 I

--,’ I’ I-------L--IO 0 FIG. 11. Illustration gonal and hexagonal models of icosahedra.

2

0

GOUGH

2’

of ways to obtain pentashapes by slicing through

Nonprefixed particles (Method II; see Figs. 6-9) are arranged into larger crystals than the prefixed particles (Method I; see Fig. 5). We think that this difference results from increased rigidity of the tail as a result of prefixation in Method I. Higher magnification electron micrographs of para-crystalline regions show that the tails are arranged randomly with no preferred orientation. The random arrangement of the tails in all the ordered regions suggested to us that the tails are squeezed into small crevices or deformities of the para-crystalline lattice. We assume that the greater flexibility of nonprefixed tails would facilitate such squeezing and allow larger para-crystalline regions to be formed. We have observed that prefixed pellets produced more easily observable tails in the random parts of the pellet. The phages on which tail lengths were measured (Fig. 3) were prefixed.

section.

(g-1)

Pentagonal

head

shapes

observed

in

The shape of the P22 head. Careful examination shows that the rows of particles in the para-crystaIIine regions are not straight; while the phage tails may contribute to these discontinuities, the packing pattern of phage P22 in Fig. 9 appears similar to that seen in a thin section of crystallized reovirus (Bernhard and Tournier, 1962). Reovirus, of course, have no tails. Other stacking patterns (not shown) were obtained that are identical to reovirus crystals. Reovirus are icosahedrons. We interpret the similarities in the stacking patterns of the two viruses as being consistent with P22 also being icosahedral. Casjens and King (1974) prepared thin sections of P22-infected S. typhimurium. The intracellular particles are arranged in a regular array, and this may be related in its structure to the para-crystalline arrays that we see. We have compared the outlines of sliced polystyrene icosahedrons with the profiles of sliced phage heads. As already mentioned, the six- and five-sided profiles shown in Fig. 10 can be generated by slicing icosahedrons. The isometric measurements of P22 and the profiles seen in thin section are consistent with the idea that P22 is a regular icosahedron. The occurrence of only one major protein in the head (Botstein et al., 1973) suggests that the protomers of the phage are identical and a regular polyhedral shape is the most likely arrangement of such subunits. All of these data point to the P22 head being an icosahedron. The tail is known to have sixfold

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symmetry (Yamamoto and Anderson, 1961). This raises the question, as with many other phages, of how the tail (with sixfold symmetry) is attached to the head (with fivefold symmetry). ACKNOWLEDGMENTS This research was supported by a grant from the National Science Foundation, No. BO 39274 (to M.G.). The authors also wish to express their gratitude and appreciation to Dr. L. Simon for his training of G.A.Z. REFERENCES W., and TOURNIER, P. (1962). Ultrastructural cytochemistry applied to the study of virus infection. Cold Spring Harbor Symp. Quant. Biol. 27, 67-82. BOTSTEIN, D., WADDELL, C. H., and KING, J. (1973). Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. J. Mol. Biol. 80, 669-695. CASJENS, S., and KING, J. (19741. Catalytic head assembling protein in virus morphogenesis. J. Supramolec. Struct. 2, 202-224. HOFFMAN, B. (1973). Ph.D. Thesis, University of Michigan, Ann Arbor, Michigan. ISRAEL, J. V., ANDERSON, T. F., and LEVINE, M. (1967). In vitro morphogenesis of phage P22 from heads and baseplate parts. Proc. Nat. Acad. Sci. USA 57, 284-291. ISRAEL, J. V., ROSEN, H., and LEVINE, M. (1972). Binding of bacteriophage P22 tail parts to cells. J. Viral. 10, 1152-1158.

BERHARD,

BACTERIOPHAGE

P22

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S., and KANEGA~AKI, S. (1973). Smooth specific phage adsorption: Endorhamnosidase activity of tail parts of P22. Biochem. Biophys. Res. Commun. 55, 403-409. KELLENBERGER, E., and EDGAR, R. S. (1971). Structure and assembly of phage particles. In “The Bacteriophage Lambda” A. D. Hershey, (ed.), pp. 271-295, Cold Spring Harbor Press. KING, J., LENK, E. V., and BOTSTEIN, D. (1973). Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. J. Mol. Biol. 80, 697-731. LEVINE, M. (1957). Mutation in the temperate phage P22 and lysogeny in Salmonella. Virology 3, 2241. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17, 208-212. SIMON, L. D. (1972). Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope: T4 head morphogenesis. Proc. Nat. Acad. Sci. USA 69, 907-911. SIMON, L. D., and ANDEMON, T. F. (1967). The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope I. Attachment and penetration. Virology 32, 279-297. SMITH, H. 0. (1968). Defective phage formation by lysogens of integration deficient phage P22 mutants. Virology 34, 203-223. SPEYER, J. F., and KHAURAKKACH, L. H. (19731. Crystalline T4 bacteriophage. J. Mol. Biol. 76, 415-417. YAMAMOTO, N., and ANDERSON, T. F. (19611. Genomit masking and recombination between serologically unrelated phage P22 and P221. Virology 14, 430-439. IWASHITA,