J. Mol. Biol. (1975) 92, 207-223

Organization and Function of the Tail of Bacteriophage T4 II.? Structural Control of the Tail Contraction MASAYUKIYAMAMOTO AND HISAO UCHIDA The Institute of Medical Science The University of Tokyo P.O. Takana,wa, Tokyo, Japan (Received 8 July 1974) The interaction of proteins, during their operation in the mature T4 bacteriophage particle, was studied by examining the function of various mutant phage particles incorporating genetically altered protein(s) or those that lack the component protein(s). Thus, the contraction behavior of the tail of the phage was studied by heating phage particles which carried various combinations of heatsensitive @ES) mutations and structural defects produced by amber (am) mutations. The following phenomena were observed: (1) Elimination of the long tail fibers from the viral suppressed tail contraction in many situations, provided retained protein P9$.

particle completely the phage particle

(2) Elimination of P 12, the short tail fibers, induced tail contraction treatment at WC, which does not affect intact phage particles.

after heat

(3) Beat denaturation of the carboxyl terminus of the short tail fiber inhibited the process of tail contraction. The inhibition was relaxed by superimposed denaturation of P5, as indicated by the behavior of double mutants h,a in genes 5 and 12. (4) Major constituent during tail contraction.

proteins of the tail, including P12 and P9, were conserved

Based on these f?ndings, a possible model of the baseplate was constructed in which P6, P7, P8, PlO, P25, P27 and P29 were visualized to constitute one functional unit acting as the skeleton of the baseplate whose hexagon-hexagram transition was controlled by “the outer proteins” which included P5, P9, Pll and both the short and long tail fibers.

1. Introduction after attachment to the host cell, The contraction, which eventually leads to the injection of the viral DNA into the cell (Hershey & Chase, 1952), is a highly co-ordinated phenomenon displayed by a complex structure composed of genetically defined proteins. The process of the contraction was morphologically studied in detail by electron microscopy using both thin-sectioning and negative-staining techniques (Simon & Anderson, 1967a,b). The The bacteriophage

T4 contracts its tail immediately

t Paper I in this series is Yamamoto & Uchida, 1973. $ Abbreviations used: P before the number of a gene indicates the product of that gene; am, amber; hs, heat sensitive. When necessary, the gene name is inserted between square brackets before the isolate number of a mutant; thus, &[G]Y 122 should read as hs mutant Y 122 in gene 6. 207

208

M. YAMAMOTO

AND

H.

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final stage was aharacterized by contraction of the tail sheath, which pulls up the baseplate along the central tail core, with concomitant transfiguration of the baseplate from a hexagon to a hexagram. At the same time, the short tail fibers connect the baseplate to the cell wall. Although these changes are dramatic, functional organization of proteins constituting the tail structure has not been well understood. In a previous communication, we have described the isolation and partial characterization of heat-sensitive T4 tail mutants, which produce viral particles more sensitive to heating at 55°C than the wild-type T4. The mutants were classified into three groups A, B and C according to the types of morphological alterations of the heat-inactivated particles (Yamamoto & Uchida, 1973). The classification was mostly gene-specific, indicating that each gene product was assigned a specific function in the morphological change of the phage tail. Genes 5 and 12 belong to group A, genes 18, 25 and 48 to group B, and genes 6, 7,10,25 and 27 belong to group C. Study of various hs? mutant particles has an advantage in elucidating the structural control of the tail contraction because we can examine the phenomenon without interaction with the host cells. Various genetic techniques, including in vitro complementation of defective particles (Edgar & Lielausis, 1968), can also be employed. Here we describe experiments to show that gene products of group A, whose hs mutations caused heat inactivation of the particle without any obvious morphological change, have important regulatory functions in triggering contraction of the tail. The long tail fibers were also found to participate in the contraction mechanism; a possible model for the structural control of the tail contraction will be discussed.

2. Materials and Methods (a) Phage and bacterial straim T4D phage mutants amN128 in gene II and amN69, amNG262, amNG555, amNlO4 and amNG75 in gene 12 were kind gifts from Dr W. B. Wood. Other am and hs T4D mutants have been described in a previous report (Yamamoto & Uchida, 1973). The ha character is defined as produoing phage particles more sensitive to heating at 55°C than the wild-type T4D particle. Eschekchia ~0% B was a non-permissive host, and E. coli CR63 was a permissive host for amber mutant phages. Various multiple mutant T4 phages used in the present experiments were prepared by genetic crosses. A double mutant Iis in arbitrary T4 genes a and 6 was constructed by a genetic cross between the two double mutants am[a] *hs[b] and h@a]*um[b], selecting for a recombinant having am+ and hs characters, The proof of the genotype ha[a] -ha@] was based on ir, viva complementation tests with am[a], am[b] and other appropriate single mutant am phages. Procedure for the in viva complementation test has been described (Yamamoto & Uchida, 1973). A triple hs mutant, hs[5]Y213*hs[6]Y122*hs[12]Y217, was constructed in a similar manner: hs[5]Y213*hs[6]Y122 was crossed with am[E’]N69 and recombinants showing both hs and am phenotypes were selected. Among the recombinants, those which retained two ha mutations in genes 5 and 6 were selected by in viva oomplementation tests using E. coli CR63 as indicator bacteria in the assay of complemented phages. The triple mutant hs[5]Y213-hs[6]Y122*am[12]N69 thus obtained was crossed with am[6]B251*h~[IZ]Y217, and am+ phages were selected. Again, the recombinant phages were individually tested by in v&o complementation tests for the presence of the three hs mutations. (b) Media and buffers Bactopenassay broth M3 (Difco) was used for growing bacteria. 3XD medium and M9 buffer were the same as those described previously (Yamamoto 87;Uchida, 1973). Tris/ t See footnote $, page 207.

TAIL

CONTRACTION

glucose medium used for preparing radioactive the formulation of Hershey (1955).

209

OF T4 PHAGE

defective phage lysates was according to

(0) In vitro complementat~on Defective extract of Tlam[7]BlG was prepared according to the procedure described by Edgar & Wood (1966), except that the extract was centrifuged at 25,000 g for 20 min to remove bacterial debris and active phage, if any. Complementation with particles laoking Pll, P12 or long tail fibers was carried out by incubation at 30°C for 2 h with an excess amount of the defective extract. The titration was made by using E. coli CR63 as plating bacteria. (d) Preparation of T4 particles uGth 14C-labeEedtails i4C-labeled hs mutant tails were prepared by infecting, at time zero, E. coli B cells growing at 30°C in Tris/glucose medium with either um[23]Bll .hs[G]Y 122 or am[23]Hll. b[25]Y109 double-mutant phages at a multiplicity of infection of about 5 per cell. 5 min after the infection, the cells were superinfected with the same double-mutant particles at the same multiplicity of infection. At 13 min, n-[U-i4C]leucine (2’70 Ci/mol; Dai-ichi Pure Chemicals Co., Tokyo) was added to give a final radioactivity of 1 &i/ml. At 60 min, the cells were sedimented by centrifugation at 3500 revs/min for 5 mm. The pellet was freeze-thawed to prepare defective extract which served as the radioactive tail donor, and the in. vitro complementation with unlabeled am[7]Bl6-defective extract was carried out at 30°C for 3 h. Complete particles thus obtained were concentrated by differential centrifugation and purified by fractionation with sucrose density centrifugation at 18,000 revs/mm for 30 min using a Spinco SW27.1 rotor. After oentrifugation, samples were collected from the bottom of the tube, and portions were assayed for infective particles and radioactivities. A typical preparation contained 20,000 cts/min per 3 x 109 particles. (e) Sodizlm dodecyl sulfate-acrylamid gel electrophoresis Slab gel electrophoresis was carried out by the technique of Studier (1973). The aorylamide concentration of the resolving gel was 15%, and electrophoresis was carried out at a constant current of 10 mA per slab for 5 h at room temperature. After electrophoresis, the gel slab was dried to a flat sheet bonded to filter paper, and a radioautogram was made with industrial X-ray film (Konishiroku Photo Co.). (f) Electron m&oswpy Samples for the electron microscope were fixed with 2% formaldehyde, and negatively stained with 2% phosphotungstic acid. A JEM-100B electron microscope (JEOL, Japan) was used for the observation.

3. Results (8) Effect of long tail $ber elimincction 0% the heat sensitivity of hs mutants The long tail fiber of T4 phage is known to assume various conformational states, and the adsorbability of the phage to the host cell depends on the extended state of the fibers (Anderson, 1945, Kellenberger et al., 1965). Previously, we have reported that indole, which is known to reduce the adsorbability of some T4 mutants as well as T4D wild-type phage, specifically suppresses the heat sensitivity of the group C!, and only group C, TLSmutant particles (Yamamoto & Uchida, 1973). This finding suggests that the conformation

of long tail fibers affects the state of group C gene

products in the tail structure. Genes 6, 7,10,25 and 27 belong to group C, and heat inactivation of any one of these gene products results in an immediate contraction of the tail.

In order to demonstrate

the interaction

group C gene products, and the co-ordination

between

long tail

fibers

and

of group C?gene products within the

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M. YAMAMOTO

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tail structure, we examined the effect of tail fiber elimination on the heat sensitivities of various Its mutant particles. Long tail fibers were eliminated from hs mutant phage particles by infection of the non-permissive cell, E. coli B, with various mutants harboring the amN52 mutation in tail fiber gene 37 in addition to the Izs mutation to be studied. After heat treatment at 55”C, the activity of defective particles was assayed by in vitro complementation of tail fibers (Edgar & Wood, 1966). The efhciency of complementation was very high, converting almost all of the fiber defective particles into complete particles. When the am[37]N52 -hs double mutant was grown in an am permissive host cell, hs particles with tail fibers were obtained, which served as a control in the experiment. Figure 1 shows the inactivation kinetics at 55°C of am[37]N52.lzs[6]Y122 mutant particles with or without long tail fibers. As indicated by the results, there was a pronounced suppression of the heat sensitivity of hs[6]Y122 particles upon removal of long tail fibers by introduction of the am mutation. Slight inactivation of the fiberless particle was comparable to the inactivation of the wild-type T4 particles at the same temperature. Similar experiments were performed with various hs mutations, and the results obtained are summarized in Table 1. The results indicated that heat sensitivities of all group C, and only group C, hs mutant particles were suppressed by the elimination of long tail fibers from the viral particles. In particular hc$7]Y202, which was the only exception to the indole suppression observed in group C mutations (Yamamoto & Uchida, 1973), was also suppressed by the elimination of fibers. Therefore, the present observations reinforced our previous inference that the co-ordinated stability of group C gene products in the tail structure is strongly

.

lo-3-

,

\,

.. 20

IO Time

30

im~n)

FIG. 1. Inactivation kinetics of am[37]N&Z.hs[6]YlZZ particles with or without long tail fibers. Double mutant partioles either with or without long tail fibers were prepared by the growth of ana[37]N62*hs[KjY122 on either E. COGCR63 or E. coli B, respectively. Particles were purified by sucrose density-gradient centrifugrations, and dialyzed against M9 buffer. After hoat troatmont at 65W, the defective particles were complemented in vitro by incubation with the donor extract of tail fibers. Plaque assays were made with CR63 ae indicator bacteria. -+-a-, Complete fiberless particles. particles ; --O--O-,

TAIL

CONTRACTION

211

OF T4 PHAGE

TABLE 1

Heat sensitivities of various hs mutant particles with or without tail Jibers

Group

A

B

C

Mutation

Time at 55OC (min)

Surviving Complete

5 12 12

Y213 Y120 Y217

10 30 30

0.08 0.3 1.0

18 48

Y102 Y234

10 10

6 7 7 10 25 27

Y122 Y202 Y216 YlOl Y109 Y226

30 30 30 30 30 30

hs mutation Gene

Each mutant

harbors

anz[37]N52

fraction

0.3