Proc. Nat. Acad. Sci. USAVol. 69, No. 4, pp. 907-911, April 1972

Infection of Escherichia coli by T2 and T4 Bacteriophages as Seenin the Electron Microscope: T4 Head Morphogenesis*

(capsids/plasma membrane/tau particles/phage heads/DNA)

LEE D. SIMON

The Institute for Cancer Research, Philadelphia, Pennsylvania 19111

Communicated by Thomas F. Anderson, January 17, 1972

ABSTRACT Bacteriophage T4 capsids seem to beassembled on E. coli protoplasmic membranes. Thisprocess seems to involve "lumps" of head protein, whichconvert to r particles, which in turn give rise to emptyheads. The empty heads leave the bacterial membraneand are then filled with DNA in the central region of thecell. T4 gene 16 and 17 products appear to be necessary forhead filling.

Many of the steps in the morphogenesis of T-even bacterio-phages have been demonstrated in extracts of bacteria infectedwith various conditionally lethal phage mutants (1-3). Someof the more complex assembly reactions, however, such asthe formation of phage heads, have not been observed in vitro.The proper functioning of at least eight phage genes (genes

23, 31, 22, 20, 40, 66, 21, and 24) is required for the produc-tion of normal T4 phage heads (4). If particular gene productsare missing, aberrant head-related complexes may be formed.Gene 23 product (major head protein) produces the simplestvisible complexes, so-called "lumps" bound to the cell mem-brane (5). Production of single-layered polyheads requires theproducts of three genes, 23, 31, and 22. The formation ofanother, more intricate, head-related structure, the elongatedTparticle, depends on the products of six genes, 23, 31, 22, 20,40, and 66 (4). Despite this type of information pertaining toso-called "aberrant" head forms, the assembly process fornormal T4 capsids has not yet been described.The data presented below are concerned with the in vivo

morphogenesis of T4 phage heads, as seen in the electronmicroscope.

MATERIALS AND METHODSUnless specified otherwise, liquid cultures were grown at 370with aeration in L-broth containing 10 g NaCl, 1 g glucose,10 g Bacto-tryptone, and 5 g yeast extract per 1000 ml ofH20, adjusted with 1.0N NaOH to pH 7.0.Phage Strains. T4D (referred to as T4+) was originally

obtained from R. S. Edgar. T4amN66 (gene 16), T4amN56(gene 17), and T4tsA70 (gene 31) were obtained from F. R.Frankel. Amber mutants were propagated on the permissivebacterial host, Escherichia coli CR63, originally obtainedfrom R. S. Edgar.

Bacteria. E. coli B, used in all experiments reported below,was obtained originally from R. S. Edgar. It is nonpermissivefor amber mutants. Only mid-log phase bacteria were used.

Electron Microscopy. Most procedures have been described(6). The few changes involve use of an epoxy embeddingmedium (7), of lead citrate (8) for staining sections, and, insome experiments, of an initial glutaraldehyde fixation.Material to be fixed with glutaraldehyde was poured into anice-cold tube containing enough 2.5% glutaraldehyde in 0.06M cacodylate buffer at pH 7.2 to bring the final glutaralde-hyde concentration to 0.5%. This ice-cold suspension wasaerated for 5-10 min. After 45 min, the bacteria were pelletedand then post-fixed in 1% osmium tetroxide-0.5% uranylacetate in veronal acetate buffer (6) overnight. This fixationprocedure will be referred to as method I.

In other experiments the glutaraldehyde fixation wasomitted. In these cases, the materials to be fixed were chilledat the appropriate times in an ice bath with aeration for 2 min.The bacteria were then rapidly pelleted and fixed overnight ina 1% osmium tetroxide-0.5% uranyl acetate solution. Thisfixation procedure will be referred to as method II. Theresults are essentially the same with either method; however,more accurate time points are obtained by method I.

Quantitative determinations of different types of structureswere made directly from thin sections by observation in theelectron microscope. Each bacterium that entered the fieldof view was carefully examined and every clearly distinguish-able phage head-related structure was scored. Counts arereported only for material fixed by method I.

RESULTSTo observe the process of T4 head morphogenesis, E. coli Bcells were infected with T4+ phages at a multiplicity ofinfection (MOI) of 5. At various times thereafter, aliquotswere withdrawn from the culture and fixed. Thin sectionsthrough a sample fixed by method II 12 min after infectioncontain several types of intracellular T4 head-related struc-tures (Fig. la and b). The most prevalent form is headspartially filled with fibrillar material. This fibrillar materialhas the same morphology as the DNA seen in the heads ofadsorbed virions that only partially injected their nucleicacid before fixation (see Figs. 7 and 12 in ref. 6). The fibrillarcontent of the partially filled heads described in this paper isassumed to be phage DNA. Partially filled heads are notusually present at the very periphery of the cell; they arelocated in the central, relatively electron-transparent region ofa bacterium. The partially filled heads in Fig. la vary fromnearly empty to nearly full with regard to DNA content.Three types of head-related structures can be discerned

on the E. coli protoplasmic membranes (Fig. 1): (i) lumps,

* This is paper IV in the series, "The Infection of Escherichia coliby T2 and T4 Bacteriophages as Seen in the Electron Micro-scope."

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Proc. Nat. Acad. Sci. USA 69 (1972>

FIG. 1. Sections through E. coli B bacteria after T4 + phage infections. Bars in these and subsequent micrographs represent about 100nm. (a and b) Cells, fixed by method II 12 min after infection, contain several membrane-bound, head-related structures, including emptyheads (E), lumps (L) and r particles (T). Heads that vary from full to empty are present in the central region of the cell. (c) Cells, fixedby method I 20 min after infection, contain many mature T4 particles. A pair of X particles are shown on the bacterial membrane. Theinset shows another T particle from this preparation. Several thin fibers (arrow) extend from the highly organized core to the cell membrane.

presumably of gene 23 product (5), (ii) T particles, and (iii)empty heads. Lumps vary in size, the smallest having dimen-sions similar to phage heads. A T particle consists of a dark

~~~ ~ ~ ~ ~ ~ ~ ~~1

,tc d | -.` g'

FIG. 2. Membrane-bound, head-related structures seen inT4 phage infections. An incomplete particle associated with a

lump (a), a r particle (b), intermediates between r particles andempty heads (c, d, and e), and an empty head (f) are shown.The less core material in an intermediate head, the more angularand less rounded its shell appears. All heads are oriented withtheir long axes perpendicular to the E. coli membrane. Thismaterial was fixed by method II.

FIG. 3. Empty (a), partially filled (b, c, d, and e), and fullheads (f). These heads are in the cytoplasmic area of the E.coli cell shown in Fig. 1. The partially filled heads contain coarse

fibrillar material, in contrast with the more homogeneous,granular material in the head-related structures in Fig. 2.

outer membrane surrounding a clear zone, at the center ofwhich is a dark core. r Particles have rounded vertices com-pared to the vertices of mature heads. r Particles and mem-brane-associated empty heads are oriented with their longaxes extending from the membrane toward the interior of thecell. The term empty head is used throughout this paper tomean a relatively empty head; no claim is implied that so-called empty heads possess absolutely no DNA. In fact, theopposite conclusion is probably true since these empty headsfrequently appear to contain small amounts of thin fibrillarmaterial. Since a few thin fibrils may be seen in r particles,they too may contain some DNA, although it seems clearthat very little of the material within a r particle is DNA (13).The distinction between r particles and empty heads is not

always clear. Fig. 2 shows various membrane-bound, head-related structures observed in T4 phage infections. As in-dicated by Fig. 2, lumps, r particles, and many heads whosemorphology is intermediate between 7 particles and emptyheads are evident on the inner bacterial membrane. Besideshaving less core material than normal r particles, the outershells of these intermediate head forms are usually moreangular than the T particles.

It is generally easy to distinguish these membrane-boundstructures (Fig. 2) from partially filled heads (Fig. 3). Partiallyfilled heads contain relatively coarse fibrillar material, whilethe various r structures contain material of more granularappearance.By 20 min after T4+ infection most of the heads have be-

come filled with DNA (Fig. ic), but occasionally r particlesand the other types of head-related structures mentioned

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Proc. Nat. Acad. Sci. USA 69 (1972)

above can still be seen. In the insert in Fig. ic, it is clear thatthe cores of r particles have an organized rather than amor-phous appearance. The cores have characteristic thin fibrilsthat extend to the bacterial protoplasmic membrane.To determine the relative numbers of the various head-

related structures present at different times after wild-typephage infection, T4 + particles were added to a culture of E.coli B cells (MOI = 10); aliquots were withdrawn at in-tervals and fixed by method I. These samples were sectioned-and examined in the electron microscope. The data are pre-sented in Table 1. At 10 min lumps were the only type of head-related structure observed; at 12 min the major discernablehead form was the 7 particle; by 14 min the most commonstructure was the partially filled head, and by 20 min most ofthe head structures were completely filled with DNA. Neithersingle- nor multiple-layered polyheads were observed in anyof these experiments.

T Particles (Fig. ic) and membrane-associated emptyheads (Fig. la) are frequently present in clusters. Clustersprobably result from one lump giving rise to several head-related structures. The evidence that one lump may giverise to several head-related particles is found in a temperature-shift experiment with the mutant T4tsA70 (gene 31). Inthe absence of functional gene 31 product, only lumps of head-related material are formed (4). E. coli B bacteria, growing at410, were infected (MOI = 10), and superinfected (MOI- 10) 7 min later to inhibit lysis with T4tsA70 phages.

TABLE 1. Relative frequencies of head-related structures atvarious times after T4+ infection

MINUTESAFTER NUMBER OF HEAD-RELATED STRUCTURES

INFECTION LUMPS T EMPTY ON EMPTY OFF PARTIAL FULL

10 300 0 0 0 0 0

12 244 43 13 Is 27 0

14 69 19 6 16 46 14

20 23 6 5 28 67

Counts were made until about 100 nonlump head structureswere scored (excluding the 10-min point). "Empty on" and"empty off" refer, respectively, to empty heads on and off themembrane; "partial" refers to heads partially filled with DNA.

15 Min later, the infected culture was shifted to 300; sampleswere periodically withdrawn and fixed by method I. Insamples taken 1 min after the temperature shift, only lumpsare visible. By 6 min after the shift, in addition to lumps largenumbers of T particles are seen. r Particles and partiallyfilled heads are visible 8 min after the shift. In a 12-minsample, there are numerous full, partially full, and r particles(Table 2).Consecutive serial sections through the sample 8 min after

shift frequently show many large clusters of r particles on thecell membrane (Fig. 4). The size of such a cluster is compa-

I'7 1'FIG. 4. Consecutive serial sections through an E. coli B cell infected with T4tsA70 (gene 31) phages. Bacteria were infected at the non-

permissive temperature, and 13 min later shifted to the permissive temperature. Head proteins made at the nonpermissive temperature byT4tsA70 phages may be incorporated into heads after a shift to the permissive temperature (5). This material was fixed by method I after8 min at the permissive temperature. The section thickness is about 80 nm. Head-related structures are indicated by arrows. Two clustersof T particles are visible, each cluster persisting for several sections. Neither cluster extends beyond the sections shown.

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Proc. Nat. Acad. Sci. USA 69 (1972)

TABLE 2. Relative frequencies of head-related structures atvarious times after temperature shift in T4tsA70

(gene-31) infection

MINUTESAFTER NUMBER OF HEAD-RELATED STRUCTURES

TEMP. SHIFT LUMPS T EMPTY ON EMPTY OFF PARTIAL FULL

200 0 0 0 0 02 200 0 0 0 0

4 200 3 0 0

6 200 62 3 0 2 0

8 200 100 7 6 20 012 200 162 20 18 108 58

Bacteria were infected and then incubated at 410 (nonper-missive temperature) for 15 min. The culture was then shifted to300 (permissive temperature) and samples were withdrawn atvarious times. Counts were made until 200 lumps had been scored.

FIG. 5. Consecutive serial sections through a cluster of T

particles contiguous with a lump. This preparation is the same

as that shown and described in Fig. 4. The T particles and lumpin the upper section apparently are associated with the bacterialmembrane at the cap of the cell, seen in tangential section in thelower micrograph.

rable to the size of a lump seen 1 min after a shift from 410 to30°. Therefore, it is likely that one lump converts to severalr particles (Fig. 5).In T4+ infections, shown in Table 1, T particles seem to be

made before other nonlump head forms. They do not appear

to accumulate in direct proportion to the number of headstructures within T4+-infected cells, as might be expected fora random aberration during head maturation. Similarly,partially filled heads appear before completed heads. Thesedata, together with the electron micrographs in Figs. 1-5,suggest that lumps of 23 product on the bacterial protoplasmicmembrane are the precursors of T particles, which in turn are

converted to empty heads on the cell membrane; the emptyheads leave the membrane and are then filled with DNA in thecytoplasm.

In support of these observations, further experimentsindicate that filling of the heads of T4 gene-16 or gene-17mutants is blocked after the empty heads come off the mem-brane, before they are filled with DNA (Fig. 6). Parallelexperiments with T4+, T416-, and T417- phages show that inT416- and T417- infections, the partially filled and fullheads visible with T4+ are not present. No partially filled or

full head has been observed in any of the T416- or T417-preparations fixed at 12 or 20 min after infection, even thoughlumps, T particles, and empty heads are seen.

DISCUSSIONThe data presented in this paper indicate that T4 bacterio-phage heads are assembled on the E. coli protoplasmic mem-brane with lumps of head protein forming first, then T par-ticles, and finally empty heads. The changes in the relativeproportions of these structures with time after infection(Table 1), as well as data from temperature-shift experimentswith gene-31 mutants (Table 2 and Figs. 4 and 5) suggestthat lumps are the precursors of r particles. Direct electronmicroscopic evidence (Fig. 2) indicates that T particles, inturn, give rise to empty heads.Lumps and T particles are found only on the bacterial

membrane, while empty heads are found both on the mem-brane and in the more central regions of the cell (Fig. 1 andTables 1 and 2). It seems that once an empty head is formed,it can readily leave the membrane. The apparent cleavage ofhead proteins during capsid maturation (9-12) may releasethe head from the protoplasmic membrane. Mutant infectionswere the only head-related structures present are lumps or rparticles contain uncleaved head proteins; on the other hand,the empty heads that accumulate in T416- or T417- infec-tions contain cleaved proteins (9). The cleavage of headproteins during maturation may also remove the "morpho-poietic cores" (13) in T particles that may be required fornormal head formation.

After T4+ empty heads enter the cell cytoplasm, they ap-parently are filled with DNA (Fig. 1). This conclusion is inagreement with the observations of Luftig and Wood (14)that in temperature-shift experiments with a T4 mutant ingene 49, partially filled heads may be converted to full heads.It seems very unlikely that heads are initially made full ofDNA, which might be subject to leakage during preparationfor electron microscopy. In parallel experiments comparingT4+ with T416- or 17- infections, no partially filled or fullheads were ever observed under 16- or 17- conditions, evenwhen most of the T4+ heads appeared partially filled (Figs.la and 6). In these experiments, if the T4+ heads lost someDNA due to leakage, the T416- and 17- heads must have lostall of their DNA essentially instantaneously. The evidence inthis paper comparing T4+ with T416- or 17- infectionsstrongly argues that the partially filled heads are in the processof acquiring DNA, and that the gene-16 and gene-17 productsof T4 are necessary to fill empty cytoplasmic capsids.The membrane-associated morphogenesis of complex

I 1.

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Proc. Nat. Acad. Sci. USA 69 (1972)

FIG. 6. E. coli B fixed by method II 12 min after T4amN66 (gene 16) infection. This preparation was made in parallel with that shownin Fig. 1. Head-related, membrane-bound structures are indicated by arrows. They include a lump (L), empty heads (E), and r particles(T). Only empty heads are visible in the cytoplasm, showing that the gene-16 product appears necessary to fill heads with DNA.

phage structures such as the baseplate has been suggested anddiscussed (15). The observation that T4 heads appear toleave the E. coli membrane and fill with DNA in the bacterialcytoplasm may relate to the attachment of capsids to newlyassembled phage tails. These tails appear to be radiallyoriented, with their baseplates near the cell membrane andtheir necks toward the interior of the cell (15). The tails,therefore, appear to be in position to interact with the newlyassembled, mature cytoplasmic capsids.

These experiments derive from fertile and provocative dis-cussions with Dr. Fred Frankel and Miss Vivian Lam. Mr. DaleSnover contributed valuable suggestions and expert technicalassistance.

This research was supported in part by Grant GB-30245 fromthe National Science Foundation, by USPHS Grants CA-06927and RR-05539 from the National Institues of Health, and by anappropriation from the Commonwealth of Pennsylvania to TheInstitute for Cancer Research.

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