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A mutation which bypasses the requirement for p24 in bacteriophage T4 capsid morphogenesis
1. AJol. Biol. (1977)
116, 261-283
A Mutation Which Bypasses the Requirement for p24 in Bacteriophage T4 Capsid Morphogenesis LOI& ANNE MCNICOL~-,LEE D. SIMON~ 1 n.sfit,ute for Cancer Research,
Philadelphia,
Pent&. 19111, ll.8.d.
AND
LINDSAY W. BLACK of Biological School of Medicine,
Department
Ut~ivwsity
of Maryland
(Receivecl 15 Novender
Chenuktry
Bnltimwe,
1576, and in revised form
3d
Y1201, U.S.A.
5 May
1977)
A mutation (byp24) affecting the N-terminal region of ~23 will suppress the lethal cffccts of urn and ts mutations in gene 24. In t,he presence of normal ~24, the byp24 alteration causes a delay in t,he cleavage of capsid proteins and the assembly of a high percentage of isometric, short-headed particles; thttrefore, the byp24 mutation can affect the length of the T4 capsid. In the absence of ~24, 24- byp24 tlouble mutants show a reduced rate of cleavage of capsid precursor proteins, and a reduced rate of virus assembly. Immunoprecipitation wit’h anti-p24 serum has shown the presence of both p24 and ~24~ irk wild-type phagc particles. The 24-byp24 particles contain no p24 or 1)24~, as determined by immunoprecipitation, urca/acrylamidc gel electrophoresis, and two-dimensional isoelectric focusing, urea/acrylamidc gradient gel electrophoresis. They have a normal electron microscopic appearance, pH stability, and heat stability; but they are more resistant to osmotic shock than wild-type T4. We suggest that p24 normally functions in the initiation of phage T4 capsid I)rotcin cleavage reactions.
1. Introduction The head of bacteriophage T4 is a complex structure, composed of many protein species, which is formed through the successive intermediates outlined in Figure 1. In the first step, the precursor form of the major structural protein of the head, p23§, is thought to form membrane-bound aggregates or lumps (Laemmli et al., 1970; Simon, 1972; Coppo et al., 1973). From these lumps proheads are organized. These t Present address: Division of Riology, California Institute of Technology, Pasadena,, Calif. 91125, U.S.A. $ Present address: Waksman Institute of Microbiology, Rutgers University, New Brunswick, NJ. 08903, U.S.A. $ Xbbreviat,ions used: p23 is the protein product of gene 23, and a similar terminology is used for thr other gene products. IP stands for internal prot.oin. ~23~, pIPIG, et,c., represent the cleavtxd form of these gpne produ& found in the mature phage head. 261
202
IA. ,I. M~!NIc’olJ, 1,. IJ. SIMON .\SI) Shell components 0’0 ~23 core components P21 P22 PIPI
~~~~
I,. w. HL:\(‘K
Shei I components P20 P23c p 24 p 24’
Core components pepttdesil plPIC c
il N A P2C’ p23c
BDl
c
SC?
Q
fisD8~
P49 Lump
r-portlcle
Empty head
Full head
1. Schematized outline of bacteriophage T4 head assembly. For references and description FIG. see the text. It is not known exactly when ~24, phoc, or psoc are added to the nascent head; nor is it known whether p21 or p40 are transiently incorporated into the structure. The origin of peptide II is obscure.
resemble the T-particles formed in gene 81 or gene 24 defective infections. The 7particle shell is formed of p23 and ~20, and surrounds an assembly core composed of ~22, pIPI, pIPI1, and pIPI (Laemmli & Favre, 1973; Luftig & Lundh, 1973; Showe & Black, 1973; Bijlenga et al., 1974; Black $ Brown, 1976). Assembly of the T-particles requires a host fa,ctor, ~20, ~21, ~22, and p31 (Laemmli et al., 1970; Coppo et al., 1973). When genes 21, 24, and 40 are functional, the structural proteins of the r-particle are cleaved: ~23, ~24, pIPI, pIPII, and pIPI have an N-terminal fragment removed to form p23”, ~24~, pIPIe, pIPII”, and pIPIIIc (Laemmli, 1970; Tsugita et al., 1975; Black 6 Brown, 1976). The core protein p22 is totally degraded to small fragments; and one of these, peptide VII, remains within the mature particle (Showe & Black, 1973; Goldstein & Champe, 1974). At least ten other T4 proteins are required after cleavage for late steps in the completion of a mature, active head. Of these, ~16, p17 and p49 are required for DNA packaging (Luftig & Ganz, 1972), while the remainder (~2, p4, ~13, ~14, ~50, ~64, and ~65) affect the activity, but not the apparent morphology, of the mature head (Laemmli, 1970; Hamilton & Luftig, 1976). T-pa,rticles are assembled in 21- and 24- infections, and protein processing reactions do not occur under such conditions (Laemmli et al., 1970). The 24- T-particles are more labile to isolation procedures than the 21- r-particles, but in vivo they can be conservatively matured into normal heads when p24 activity is restored (Bijlenga et al., 1973), whereas 21- T-particles are inert. Therefore the function of p24 is associated with cleavage of pre-assembled proteins, although p21 appears to be the actual protease (Goldstein & Champe, 1974; Onorato & Showe, 1975). In addition, p24 plays a structural role in the finished head, controlling osmotic shock sensitivity (Leibo & Mazur, 1966). And it is required to prevent abberant head formation, since mutations in gene 24 give rise to closed polyheads and giant phages (Bijlenga et al., 1976; Paietta et al., 1976). To investigate the morphogenetic role of ~24, we have isolat,ed an cxtragenic mutation which suppresses gene 24 deficiencies. This paper demonstrates that the supressor is an altered gene 23 protein, which can be processed and assembled into viable particles in the absence of ~24.
~24
BYPASS
263
MUTATlON
2. Materials and Methods (a) Bacteria
and bacteriophage
strains
T4D wild-type; gene 24 umE303, anaE355, amN65, amNG373, amNG433, tsL90, and t&IO; gene 23 amB17, amH11, amE506, and arnE1236; gene 21 anzE322; gene 22 amB270 and anzE209 ; gene 31 amN 111; gene 44 amN82; gene 55 amBL292 ; and gene e anaH are from tlie collection of Dr R. S. Edgar. The gene 24 osmotic shock-resistant mutant ‘I’4BOp is from Dr A. W. Kozinski. Escherichia coli BE is from Dr R. H. Epstein and fl. CO/%R is frotn Dr 1~. S. Elgsr. E. co/i 11,,wT and suII arc from Dr 1’. Stringini. (b) ,%Iedia and cultural Pliago stocks were grown in H broth or M9 rnctlium rising nt,aridartl t,c!clmiqucs (Adams, 1959).
techniq7ces and plnt,ing
was done on EHA
ngar
(c) Osmotic shock resistance Osmotic shock resistance was measured using a variation of the procedure of Anderson et al. (1953). Samples, each containing 10s phage in 0.1 ml of 4 ivr-NaCl at 4°C in a 25 mm x 200 mm screw-cap test tube, were equilibrated for 15 min. Then 10 ml distilled water at 4°C: was “dumped” into the t,ube, and the survivors assayed by dilution through cokl brot,li (d) &r$cation i4C-labeled Kellciihorger
pliage (1968).
were
purified
by
of phage particles the
(c) Electron
CsCl step-gradient
procedure
describrd
1))
microscoy y
The fixation, dehydration, embedding, sectioning and staining of infected cells I>nvtt been described (Method I in Simon, 1972). Phage were negatively stained in 2 steps, first for 25 min with saturated aqueous many1 acetate adjusted to pH 3.5 with HCl, then followed by 2% aqueous many1 acetate (pH 4*3), which was immediately removed by blotting with filter paper (A. H. Doermann. personal communication). Specimens were examined in a Siemens Elmiskop I electron microscope. (f ) Electrophoresis
and immunoprecipitation
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate \vas carried out according to Laemmli (1970) on a slab gel apparatus described by Studier ( 1973). Urea/acrylamide gradient, sodium dodecyl sulfate gels were as described by Castillo et al. ( 1977). Two-dimensional isoelectric focusing, urea/acrylamide gradient gels, sodium dodeeyl sulfate were modified from the method of O’Farrell (1975) as described by C.-L. Hsiao (manuscript in preparation). Electrophoresis of immunoprecipitates was as follows : 60,000 cts/min of CsCl-purified [14C]amino acid-labeled phage, or lysates of phagu infected, [i4C]amino acid-labeled bacteria, were mixed with 1011 unlabeled T4D+ phage in 1% sodium dodecyl sulfate electrophoresis sample buffer and heated to 100°C for 5 min. After cooling, the volume was increased lo-fold with 0.01 M-Tris-HCl (pH 7.6) and antiserum against p24 was added to equivalence with respect to the dissociated carrier phage. After 18 h at room temperature, the precipitate was centrifuged from suspension at 2600 revs/min for 30 min, washed once with 0.02 M-TriseHCl (pH 7.6), recentrifuged as above, resuspended in sodium dodecyl sulfate sample buffer at lOO”C, and submitted to electrophoresis. Radioactive phage or lysat,es were similarly disrupted in the presence of cold carrier phage and applied to double-diffusion gels against antiserum prepared to purified ~24. Autoradiography of dried sodium dodecyl sulfate and immunodiffusion gels was as previously described (Black & Ahmad-Zadeh, 1971). Antiserum against purified p24 was kindly provided by Dr M. I(. ShOWf.~.
I,. A. Mc;“;I(‘OL,
264
L.
D. SIMON
AND
I,.
$5.. I
3. Results (a) Isolalion
of a su~jmwor
of qeue 84 mutut~ts
Gene H-specific suppressor mutations were isolated from spontaneous pst,udo-rt:\-clrt ants of gene 24 amber mutants, which had a cold-sensitive (cs) phenotype on fi. ~oli BE. Such mutants were found in stocks of 2P(amE303), 2J(amNG433). and 24(~~xNifi5), at frequencies of abut, lo-“. as phnge ahlr to plaque on E. coZi BE. at 12°C’ (\\,itll :I
Cross I amNG433
byp24(ll
amNG433
X
X omd65
am+ weld-type
om1(165
byp2412)
om+byp24
recomblnonts
recombmonk
2!iiq=g
(a)
omE209
am8270
omH I I
am017
Gene 31
Gene 24
Gene 23
Gene 22
amE omNG373
amE
amE
amE amNG433
-rlI.2 __
77
amNlll
fSSl0
byP24 o,i-.
amN65
I.0 2.6 4.2
l-8
6.8 8.4
(bl Fro. 2. Isolation, temporaturo sensitivit,y, and map position of tho byp24 mutation. (a) In crosses I and 2, the percentage of wild type or byp24 progeny phage was determined by comparing the number of plaques on E. coli BE at 30°C to the number on E. coli B4OsuI at 3O’C. Burst sizes of the wild type and the byp24 progeny are compared at 30°C and 42°C. (b) The positions of amber mutations within gene 23 are from Sarabhai et al. (1964). The mutants 24(amE366) and 24(arnNG373) are at the same site; both 24(amE303) and 24(amNG433) are located at a second site; and 24(amE356) is to the left of 24(amE303) (A. H. Doermann, personal communication). From our crosses we find that 24(amN66) is located to the right of 24(anaNG433). This agrees with the relative sizes of the amber fragments of p24 which are produced during infection with these two mutants (Fig. 3). The positions of the two ts24 mutations are not established, although they appear to be non-allelic. The distances between byp24 and the various am. markers are given as 200 x (the number of wild-type phage on E. coli BE at 42’C/the total number of phage in BIOsuI at 3O’C). The map distances are underestimated since no correction is made for T41)+ phage, which give small plaques at 42°C (approx. 10 to 20%).
p24
BYPASS
265
MUTATION
TABLE 1
Ejkiency I~acterial
strain
II
1~4OsuI
of plating and burst size of Tdz4(amE303)byp24t Temperature (“(3
Titer
Efficiency of plat ing$
42 37 30 24
2.1 x 1.7 x 2.4 x 6.5 x
10” 10” lo7 106
1.0 0.63 x.9 s 10 -5 2.4 y IO-5
42 37 30 24
3.0 3.8 2.5 3.2
x x x x
10’” 10” 10” 10’1
I.0 1 .:1 0.83 1.1
15 “.4x
10-Z
120 430
t The phage stock was prepared at 42°C on a B40suI host. $ For each bacterial host), the 42°C titer was taken as 1.0.
burst size of 15) but not at 25°C (burst size of 0.024) (Table 1). Although the pseudorevertants were cs on E. co& BE, they were viable at all temperatures on E. coli B4Osu1, which contains a nonsense suppressor. Under suppressed conditions, these phage were slightly temperature-sensitive, as judged by burst size. In addition, the cs mutants displayed at low temperature the suppression pattern of the amber mutants from which they were derived. For example, both 24(am.N65) and its cs derivatives grew on E. coli B40suI at all temperatures, and neither grew on B40sulI at low temperature. At 42°C only the cs grew on B4OsuII or E. coli BE. These data suggested that the cs isolates might actually be double mutants containing the original 24(amN65) and a second mutation, able t,o compensate for the gent: 2;C deficiency at high, but not at low temperatures. To test this hypothesis we backcrossed cs mutants to wild-type phage. The progeny contained some phage which behaved like the original amber : able t’o grow on E. coli B40suI at both low and high t,emperature, unable to grow on E. coli BE at any temperature, and unable to give recombination or complelnentation with the original amber mutation. Therefore, we concluded that the original 24(am) mutat,ions were indeed present, and being suppressed by a second mutation, which we have called b,yp24: for bypass. We observed relatively close linkage of byp24 to 24(am) markers in the backcrosses to wild type. To isolate the byp24 suppressor(s) we crossed tw.0 independent cs mutants, derivtbd from 24(amNG433) and from 24(amN65), which are separated by approximateIS 0.8 map units (Fig. 2(a), cross 1). From the cross of 24(amNG433)byp(l) with 24 (nmN65)byp24(2) we selected phage able to grow on E. cdl: BE at 25°C (Fig. 2(a), cross 2). All the progeny selected from this cross had apparently wild-type plaque morphology at 30°C or 37”C, and minute plaque morphology at 42°C. Burst size measurement in liquid culture confirmed that these byp24 mutants produced a large burst, at 30°C and grew poorly at 42°C (Fig. Z(a)). One such recombinant, hereafter simply referred to as byp24, was selected for further study. The byp24 was combined with 24(am) mutants 24(amNG433), 24 (am.E303), and 24(amN65) (located at three different loci) and the ts mutants 24(tsL90) and 2l(tsSlO). These double mutants were able to grow on E. coli BE at 42°C. These
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Flc:. 3. Head protein processing and p24 synthesis in nmP4 and 24(um)by~24 infections. E. wli R” or HCOsuI growing in M9 medium wwe infected at 42°C or 30°C at a multiplicit,y of 5, antI superinfectcd at a multiplicity of 5 at 5 min (42°C) or 12 min (30°C). [14C]Amino acids were adtkd from 12 min t.o 18 min (42°C) or 21 min to 30 min (3O”C), and then an excess of non-radioactive amino acids added from 18 min to 30 min (42T) or from 30 min to 60 min (30°C). Bacteria wwc centrifuged from solution at 30 rnin or 60 min, frozen and thawed once in 1 rmx-MgSO,, treat,etl with 4 pg pancreat,ic DNase/ml for 10 rnin at 37”C, boilctl in sample buffer, and t,hen applied to a urea/acrylarnide gradient, sodium dodecyl sulfate-containing gel (samples a to S) or to a 12.5% acrylamide gel containing sodium dodrcyl sulfak (samples t to v). Autoradiographs were: prepared from the dried gels.
p24
BYPASS
MUTATION
267
genetic data suggest that the byp24 suppressor is not specific for mutations located at particular sites within gene 24, but is able to overcome any gene 24 deficiency. In addition, the suppression of 24(ts) mutants makes it unlikely that the byp24 suppressor accomplishes phage production by either incorporating an active amber fragment or by suppressing 24(am) mutations through transcriptional or translational suppression mechanisms. The 6~~24 was next crossed with various am mutants situat)ed near gene 24 (Fig. 2(b)), taking advantage of its ts plaque phenotype to measure tjhe frequency of wild-type recombinants. As the mapping results indicate, byp24 is located in gene 23, very near 23(amHll) the most PT-t,erminal mutation known within gene 23 (Sarahbai et al., 1964). It is likely that there are a very limited number of byp24 sites. (1) Revertants of 24(am) able to grow on E. cc& BE at 42°C occur in the ratio of one wild type to about one to two 24(am)byp24. Assuming equal mutation rates at the amber and at potential byp sites, this rat,io suggests that the number of suppressor sites is roughly equal to the number of amber reverting sites; (2) five isolated byp24 phage from cross 2 (Fig. 2(a)) gave the same recombination frequency with 23(amHll), suggesting that at least byp24(1) and byp24(2) are located near the same site; and (3) there did not appear t’o be any wild-type phage resulting from cross 2. In summary, the genetic results suggest that there is one, or a small number of closely linked sites, in gene 23 which can mutate to make p24 dispensable at high temperature, The location of the suppressor close to W(amH11) shows that it is located within the region that codes for the N-terminal fragment which is removed by cleavage during head assembly (Celis et al., 1973; Tsugita & Isobe, personal communication; C.-L. Hsiao, unpuhlished data). (b)
Head protein
processing
and p24 synthesis
in am24 and am24byp24
infections
Since genetic analyses demonstrated that byp24 overcomes the lethal effect of both and ts mutations in gene 24, we examined phage-specific proteins synthesized during 24(am) and 24(am)byp24 infection. Figure 3 shows autoradiographs of urea/ acrylamide gradient, sodium dodecyl sulfate gels of extract’s from [14C]-amino acid-labeled, infected cells. Comparison to wild-type infection (with normal head protein processing) (Fig. 3e) and to 21(amE322) infection (with no head protein processing) (Fig. 3f) shows that 24(amNG433) and 24(amN65) do not synthesize p24 when grown on E. coli BE at either 42°C or 30°C (Fig. Sa,c,g and i). Cleavage of ~22, ~23, pIPII1, and other head precursor proteins does not occur; no ~23~, pIPI”, or pIPIITC are produced (Fig. 3a,c,g and i). When grown on E. coli B4Osut at 32°C. 24(unhNt%) and 24(amNG433) 1)ro d uce p24 and head protein processing occurs (Fig. 3m and 0). The apparent amber fragments are detected in E. coli BE, M, ,-22,000 in tho 24(amN65) infection, and -16,000 in the 24(amNG433) infection (Fig. 3, am
Growth conditions: samples are designated, e.g. T4D ’ B4L’, for ‘I-41) + grown on Ii:. co/j R” at. 42’C, in this and subsequent Figures. a, 21(omNG43R).B42; b, 24(smNG433)by~24.B42; c, 24(rtmNGB) .B4”; d, 24(wnN65)hy~xl4. H4”; c, T.&D+ -B42; f, 21(~7nE322)~B42; g, 24(nmNG433) .B30; h, 24((tmNG433)by~24.B30; i, 24(atmN66).B30; j, 24(amN65)[email protected]; k, T4D+ -B30; 1, ?l(vmE322).B30; m, 24(an~SG433)~ ~~142; n, 24(umNG433)byp24~suI42; o, 24(nmN65)-~~142; p, 24(rcmN65)byp24.~uI42; q> T4D’ . ~11142; r, ~l(ancE322)~su142; s, purified phagc particles T4D + .B42; t, [email protected]; u, Dyp”4.B:W; v, T4D + .B30.
abcdibf~ghijk~mno
P !I
r
s’- t
Vlt:. 4. Protein composition of gene 2t mutant phagc,. [‘41’].\mino acitl-lahcltvl l)hag~~ \v(‘r’ prcparetl and purified on C&l band gradient,s as described in Materials and MvtJhotls. l’hagc or i,nn,unoprecipit,ates prepared from 60,000 ct,s/min of each purifictl mutant phagc (Jlateriuls awl Methods) w-ere boiled in sample buffer and applied to 8% (samples a t,o o) 01’ !)o;, (samples 1’ to t) ncrylamide gels containing sodium dodecyl sulfat,c. Infected ccl1 lysatrs WC‘I‘Pprepar~tl at 37’(‘. I’hage: a, T4D+ .B42; b, ZC(omNG433)byp24~suI42; c, 24(rcmNG433)61/~~.R42; p, 24. ‘9 (rcmNG433)byp24.d30; g, 24(nmNG43R)by$4.suI42; I‘, 24((~mNG433)~~yp_4.B43; ,s. “4. (trmNG433). ~11142; t, T4D + . B4”. Immunol)recipitate~: d, T4D + .B42; e, byp24.B42; f, 24(wnNG4:S3) .su14”; g, ZqrcmNU4:l:l). by~~24~suIBO; h, 24(nmNG439)b~f/‘t4.suI42; i, 24(tcmN6.5)6y~.suI42: j, ~d(nmNG433)b?/p24.H4:!: k, 2Z(rtmEXOS)~B, info&xl cell lysate; I, 2:S(n~nH11)24(nmN65)~B, infoct,cd ccl1 lysatc; m, 25(~mHll)~.B, info&cd cell lysato; II, 24(trm65) .B, infected cell lysat,o; o, 2f(rtrn~90) .B, infoctc*d cell lysatc.
arrows a and c). When 24(amN65)byp24 or 24(amNG433)byp24 are grown in E. coli BE at 42”C, p24 cannot be detected but protein processing does occur, and a large amount of ~23” is produced (Fig. 3b and d). During infection with these mutants at the restrictive temperature (30°C) no ~24 is detected and the formation of ~23” a.nd other processed head proteins is not significant (Fig. 3h and j). Both 24(amN65)byp24
~24
BYPASS
MUTATIOS
%!I
P22
FI(:. 5. Kinetics of head prot.ein processing in 2J(nmNG433)byp24 and T4D + infections. E. coli 11” grooving on M9 medium at. 42°C was infected at a multiplicity of 5, and superinfected 5 min litter. The infect.ed cells were mixed with “C-lebeled amino acids at 12 min, and excess non-radioactive amino acids were added at 14 min. Samples were pipeted onto ice at various times, the bacteria centrifuged from solution, and quickly frozen. (For samples a to n the radioactive pulse was between 12 min and 18 min, and the bacteria were centrifuged from solution at 2.5 min.) Infected bacteria were boiled in sample buffer and applied to fly0 acrylamide gels containing sodium dodecyl sulfate. Samples were prepared after infection with: a, T4D+ : b, PZ(nmE322): c, 24(nmNG433); d, )‘S(nmHll); e, 23(amH11)24(amN65). ?4(mtXG433byp24): f, 14.5 min; g, 16.5 min; h, 18.5 min; i, 20.5 min: j, 24.5 min; k, 29-5 min. l’4ll+ ; 1, 14.5 Inin; m, 16.5 min: 11, 155 min; o. 20.5 min; 1,: 24.5 min; q, 29.5 min.
urea/acrylamide gradient, sodium tlotlwyl YIC. 6. Two-dimensional isoelectric focusing, sulfate gel electrophoresis of T4D + and 24(nmNG433)byp24 phago. Two ixoclect,ric focusing gels of dissociated phage, with migration from the center out: a, 24(amNG433)6yp24~42, 30,000 cts/min applied and c, T4D + .R42, 60,000 cts/min applied were submitted to electrophoresis in the vertical direction on a urea/acrylamide gradient gel containing sodium dodecyl sulfate (Materials and Methods). Track b contains for comparison disrupted T4D+ phage particles with no isoelectric focusing. It appears that ~24~ underlies ~23~ in the second dimension and is absent from the 24(nm)byp24 phage. The 2 smallest proteins from phage particles (pIPI” and pIPIF) appear to have been lost from the basic end of the isoelectric focusing gel in this run.
p24
BYPASS
MUTATION
271
and 24(amNG433)byp24 produce the p24 amber fragments on E. coli BE at 30°C and 42”C, and produce intact p24 during infection of B4OsuI (Fig. 3n and p). Therefore, by analysis of infected cells on acrylamide gels containing sodium dodecyl sulfate. 24(amNfS)byp24 and 24(amNG433)byp24 contain the original amber mutation: and are able to accomplish head protein processing in E. coli BE at 42°C in the absence of ~24. When the single byp24 mutant is grown on E. coli BE at 3O”C, cleavage is inefficient when compared to a wild-type infection. This effect is more pronounred when byp24 is grown at the more restrictive temperature (Fig. 3t,u and v). (c) Protein composition of 24(amNG433)byp24 phage purticles ~24 is cleaved during normal head assembly, and the processed protein ~24” is reported to be a major component of the capsid (Laemmli, 1970). Since acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of E. coli BE infected with 24(am)byp24 showed that p24 was not synthesized at 42”C, although head protein cleavage and phage production occurred (Fig. 3), we wanted to determine the protein composition of the phage produced. Preparations of [14C]amino acidlabeled phage were purified in CsCl step gradients, and their proteins separated on sodium dodecyl sulfate-containing acrylamide gels (Fig. 4). The following phage were purified and examined in this way : T4D + (Fig. 4a and t), byp24 (data not shown), and 24(amNG433)byp24 (Fig. 4c and r) grown on E. coli BE at 42°C; 24(anzNG433) (data not shown) and 24(amNG433)byp24 (Fig. 4p) grown on B40suI at 30°C ; and 24(amNG433) (Fig. 4s) and 24(anzNG433)byp24 (Fig. 4b and q) grown on B40suI at 42OC.We were not able to detect the absence of a protein band from any one of these\ phage preparations.
T!me 01 42OC hn!
Fro. ‘7. Single-step growth kinetics of T4D+ and 24(nnaNG433)byp24 in E. coli BE at 42’i’. E. coli BE growing exponentially at 42°C were infected with T4D+ or 24(amNG433)byp24 at a multiplicity of 4. At 2.6 min the bacteria were superinfected with the same phage at the same multiplicity, and at 4 min anti-T4 serum was added. At 8 min the infected bacteria were diluted from antiserum into fresh broth at 42’C, and the concentration of infected cells was cetermined. Samples of infected bacteria were removed at the indicated times and lysed with CHCl, to determine the phage yield per infected bacterium. -x -X -, T4D + ; -a--•--, 24(amNG433)byp24.
27’
L.
A. McNICOL,
L.
D.
SIMON
AND
L. W.
13l,.\~‘Ji
Our results suggested that ~23” and ~24” overlap on the sodium dodecyl sulfat’econtaining acrylamide gel system. Urea/acrylamide gradient gels containing sodium dodecyl sulfate or low concentration acrylamide gels containing or lacking urea did not show differences in composition between T4D+ and 24(am)byp24, although these changes in the gel did shift the relative positions of ~23” and ~24. The problem
p?4
BYPASS
MUTATION
“53
appeared to be due to the very large amount of ~23” in phages, which causes spreading of the p23c band; occasionally a band which may be ~24~ emerges from t’he ~23” hand, but, its identification is quite uncertain. The p24 band is readily identifiable on virt,ually any sodium dodecyl sulfate-containing acrylamide gel of T4-infected cells, \et there is considerable disagreement in identifying ~24” on sodium dodecyl sulfate,-containing acrylamide gels of phage particles. For example, the identifications of ~24” by Laemmli (1970), Vanderslice & Yegian (1974), Coppo et al. (1973) and Aehi et al. (1974), differ considerably in both position and amount relative to ~23”. fiincc any capsid formation-gene defect prevents ~24” formation, and since somcx ot,her proteins near ~23” also appear to be derived from p23 (Fig. 5a and e), it is difficult t,o identify ~24~ with certainty in phage particles. It is likely that some of the bands in purified phage previously identified as ~24” might be derived from p23 OI’ c~ultl be the hoc protein or other proteins of unknown origin (Ishii & Yanagida, 1!)75). We were able to precipitate ~24~ from CsCl-purified, sodium dodecyl sulfatcdisrupbcd phage, using antiserum directed against p24 (kindly provided by Dr M. K. Showe). Either through autoradiography of disrupted radioactive phage in immunodiffusion gels (data not shown) or through electrophoresis of the immunoprecipitates on sodium dodecyl sulfate-containing acrylamide gels in the absence of p23”, it was possible to identify ~24” within the phage, and to show that p24 is missing ouly from 2d(am)6yp24 phage grown on E. co.5 BE at 42°C (Fig. 4a and j). The immunoprecipitation is specific for ~24, since a band of radioactive protein is prccipitsted from cells infected with amber mutants in genes 21, 22 or 83 (Fig. 4k,m >rlltl 01. but not from cells infected with amber mutants in gene 24 or genes 23. %J (Fig. 11 and n). Using equal amounts of radioactive phage, it is apparent that only %(ant)hyp24 phage grown on E. coli BE at 42°C lack radioactive protein bands pr+ cipihat)ing with antiserum directed against p24 (Fig. 4a and j). There appear to 1~ two vc~ry slightly separated forms of p24 incorporated into phage particles, althougl~ only one hand is observed in capsid formation gene-defective phage-infected cells; both these bands are located under the ~23” band in purified phage (Fig. 4c and 0). Therefore, it appears as though p24 and its processed form ~24” are found in thcb T4 capsid. but the processed form is difficult to distinguish from p24 on standard gels. WC also determined that 24(amE303)byp24 does not incorporate any host protein into its ca.psid in the absence of ~24. E. coli B was prelabeled with 14C-labeled amino acids. The radioactive cells were washed twice and infected with wild type or %3(attt. E303)h;qp24 at a multiplicity of infection of five in the presence of cold medium. At cell Isis the phage were purified by differential centrifugation and their radioactive prot)eins examined by acrylamide gel electrophoresis in the presence of sodium dodec*?;l sulfate. There was no difference between the banding patterns of the wild type ;rntl mutant phage (data not shown). Twc~dimensional isoelectric focusing in urea/acrylamide gradient gels containing sodium dodecyl sulfate was used to confirm our observation that 24(am)byp24 phagc lack 1’24~. The ~24” is apparently more acidic than p23”, so the two proteins can l)e rcsolvcad in the first, isoelectric focusing, dimension even though they have t,he same rnobilit~y in tho second, apparently molecular weight-dependent, urea/acrylamido dimension (Fig. 6). It appears from this and other two-dimensional gels that Z-l(attt NG433)hy~~24 grown on E. coli BE at 42°C (Fig. 6a) are lacking the ~24” band present in ‘l’-tl) + phage (Fig. 6c). The conclusion that ~24 is missing from 2~(cm)bj/p%
~24
BYPASS
MUTATION
276
particles is thus supported by the combined evidence from gels of infected cell lysates (Fig. 3), gels of immunopreeipitat,es of disrupted phage (Fig. 4)) and isoelectric focusing of labeled particle proteins. A very rough estimate can be made of the amount of ~24” in the capsid. There are 144 molecules of ~18 in the T4 particle and the molecular weights of p18 (80,000) and p24 (45,000) have been estimated (Laemmli, 1970; King & Mykolajewycz, 1972). From densitometer tracings of autoradiographs of the p24 bands following immunoprecipitation (Fig. 4d and i) and of the ~18 bands in the phage which were used fo1 the p24 precipitation (Fig. 4a,b and c), it can be estimated that there are 30 molecules of p24 and ~24~ in the phage head. This figure is likely to be an underestimate of the actual amount of ~24” in the phage head, since losses of ~24~ are likely during preparabion of the immunoprecipitates, and it is obvious that such a measurement is subject to considerable variability. We note that only gr0wt.h on E. co& BE at 42°C. does 24(am)byp24 produce phage lacking p24 (Fig. 4j). The 24(am)byp24 phaga grown on E. coli B4OsuI or b;yp24 grown on E. coli BE at 42°C contain p24 in amounts comparable to wild type (Fig. 4d and i). Apparently, the byp24 mutant can utilize ~24 if it is available at any temperature; and when p24 is available, phage assembly is considerably more efficient. (d) Physiology of 24(am)byp24 infection Infection of E. c&i BE with 24(awP)byp24 phage at 42°C leads to the production of phage, polyheads, and T-particles (see section (e), below). The efficiency of phage formation is reduced considerably compared to wild-type infection, perhaps due to this production of aberrant head structures. We examined the time of phage appearance by measuring single-step growth at 42°C (Fig. 7). The 24(amNG433)byp24 progeny appear at a considerably reduced rate and final yield. In the wild-type infection, the first phage is produced at about 15 minutes, and phage production is 50% complete by 20 minutes, whereas in the 24(nwSG433)byp24 infection bhe first phage do not appear until 20 minutes. The reduced rate of phage formation in the 24(amNG433)byp24 infection is correlated with a greatly reduced rate of head precursor protein cleavage, as is shown in Figure 5. In the 24(amNG433)byp24 infection at 42”C, pulse-labeling of proteins from 12 to 14 minutes followed by a chase with cold amino acids, shows that ~23” appears to a significant extent only in the sample collected at 20.5 minutes (Fig. 5i). The large increase in ~23” concentration at 24.5 and 29.5 minutes (Fig. 5j and k) shows that, p23 cleavage in the 24(amNG433)byp24 infection is continuing at these late times. Thus, following its synthesis, p23 can be normally processed, for an extended period, although the rate of cleavage is slow. In a parallel infection with T4D + , cleavage is already considerable at 14.5 minutes and appears to be essentially complete at 16.5 minutes (Fig. 51 and m). When E. coli BE bacteria are infected with 24(aw&G433)byp24 at non-permissive t’emperature (30”(J), and then shifted to 42°C in the presence of chloramphenicol, we observed a significant increase in the number of phage. However, the yield amounted
1’24
BYPASS
277
MUTATION
t.o only about one phage per cell, suggesting that the 24(am)byp24 or completed inefficiently in the absence of protein synthesis. (e) Electron microscopy
of mutant-infected
heads arc
assembled
cells
We have taken electron micrographs of thin sections from infected cells to det,ermine what structures the mutant phage are able to assemble. When 24(amE303)byp24 is grown under permissive conditions (42°C) the cells contain a mixt’ure of full heads, T-part,icles, and polyheads (Fig. 8). However, at 24°C morphogenesis is blocked and no normal heads are seen (Fig. 9(a) and (b)). The byp24 mutant produces finished heads when grown at either 24°C or 42°C. In agreement with the large amount of uncleaved p23 seen in Figure 3~. the mutant assembles relatively large amounts of polyhead at the higher, partially restrictive temperature (Fig. 10). (f) Osmotic shock resistance of the am24byp24 phage particles WC were interested in the biological consequences of assembling a capsid in the ahscnce of ~24. Since gene 24 has been reported to control the osmotic shock phenotype of T4 capsids (Leibo & Mazur, 1966), we examined the osmotic shock phenotype of the mutant particles. Table 2 lists the survival of various phage following osmotic shock. The 24(amE303)byp24 phage prepared on E. coli B at 42°C are about 15fold more resistant to osmotic shock than were the wild type, suppressed 24(amE303). or 61~~24phage. However, there degree of resistance is not so marked as that of thrh T4BOi mutant, which shows a 96-fold higher survival than wild-type phage. Although t,he 24(amE303)byp24 particles displayed altered osmotic properties. their* ca.psids had normal stability to pH (data not, shown) and to heat inactivatioll D’ig. II). -
I IO
I
20
I
30
TIM hn)
FIG. 11.
Heat stability of wild type and 24(amNG433)byp24 phage. A mixture (>f 108 2~(r~mNG433)by~24.B42 and 10 lo 44(umN82)55(amBL292)e(nmH26)~suI42 phage was incuhetl>tl at, 67°C. At. various times samples were plated on BQOsuI at 37°C for total remaining phagc a~itl 011 1%” at. 42°C for remaining 24(amNG433)byp24 phage. - -i ;- I_ ‘-. %J(omNG433)b?yp24: ,’ “, , ~;((~rmN82)55(amHLL’94)e(amH26).
FIG:. 12. Electron micrographs of mgatimly hy ccntrifugation t.hrough a CsCl step gradient (B) T4D + .B42.
stained phagc pwticles. ‘I’ht- phrtgc wwv l)urifkl t+4’L; and cliroct,ly stained. (A) %1( ~lnLE:~(l:l)h!/l”‘4.
1’24 BYPASS
279
MUTATIOK
TABLE
2
Osmotic shock resistance of mutant phuge particles
Host
I’hage
T4D 24(amE303) byp24
B B40suI B
24(antE303)byp24
B
Ratio (mutant survival/ wild-type survival)
Temperature (“C)
2.5(amE303)byp24
B4OsuI
42 42 24 42 42
T4R24(0;)
B
42
(2.38&O+J2) x 10-s (5.03k3.7) x 10-S (2.36-~:0.34) x 1O-2 (3.57kO.65) x 1O-2 (2.35*1.6)x 1O-3
1.0 2.1 0.99 15 3.5
(2.29kO.14)
96
t This value represents the average of 3 independent for thv experimental details.
(g) Electron
microscopic
appearance
x 10-l
experiments.
See Materials
and Methods
of mutant particles
Figure 12 illustrates negatively stained preparations of various mutant phage particles. Tn the absence of ~24, the phage produced by 24(amE303)byp24 on E. co& B at) 42°C have a normal appearance (Fig. 12(A)). Compared to wild-type phage (Fig. 12(B)), the double mutant has a full-size capsid. However, in the presence of normal ~24, the byp24 mutation causes the assembly of abnormally sized heads. Under permissive conditions, E. coli B at 3O”C, byp24 produces a significant fraction of short-headed particles (Fig. 13). Table 3 presents quantitative data from scans of many fields. At 3O”C, 21% of the byp24 phage have an isometric or two-thirds-sized head, and 7’5 have a three-quarter length capsid. In contrast, the wild-type phage preparation showed 2% and the 24(amE303)byp24 grown on E. coli B at 42°C showed 4o/o short,headed phage. When the byp24phage were grown at the more restrictive temperature 42°C. even more particles were abnormal, since 56(x, of the population had isometric heads. From these observations it is clear that the byp24 mutation can affect cap&l lengt’h determination.
TABLE
3
Capsid length of various mutant phage particles Host
Temperat,ure P-3
:h Particles with capsid length?
l/l T4D ktp24 byp 2 4 21(mNG433)byp24 -t ‘L’h~~[)vvrentagtls
B B B B
30 30 42 42
98 72 37 96
3/4 0 7 7 0
are based on tcrtal counts of 118 10 341 total l~avticlcs.
2/3 2 “1 56 4
~24 BYPASS
MUTATION
281
4. Discussion In wild-type T4 infections p24 is essential for normal capsid assembly. It is an integral component of the capsid shell (Aebi et al., 1974) and controls, at least in part’, the shock resistance of the capsid (Leibo & Mazur. 1966). We have osmotic found that both p24 and ~24” are present in the capsid of wild-type T4 phage (Fig. 4a). Although these proteins can be identified on standard gel electrophoresis of infected cell extracts, they are difficult to detect in phage particles, where they are obscured by large amounts of ~23” (see Results, section (c)). Either clect,rophoresis of immunoprecipitation or two-dimensional isoelectric focusing, urea/ acrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate is required for the separation of ~24~ and ~23”. In addition, there seems to be a requirement, for p24 to prevent aberrant capsid development, since gene 24 missense mutants cause formation of polyheads and maturation of giant phages (Paietta et al., 1976). However, p24 does not seem to play an essential role in shape determination, since 24 defective mutants also accumulate s-particles, which can be directly converted to mature phage when 2d function is restored (Bijlenga et al., 1973). The capsid protein cleavage reactions which occur during normal T4 morphogenesis are blocked in 24- infect’ions, although p24 is not directly implicated in the protein processing reactions (Onorato & Showe, 1975). Our results establish the nature of a specific suppressor of the requirement for ~24 in the morphogenesia of T4 capsids: a particular mutation within gene 23. called 117~~24,permits the production and maturation of T4 heads in the absence of p24 (Figs 3: 4> and 6). The map position of this mutation near 23(amHll) (Fig. 2) strongly suggests that it alters a portion of p23 which is removed during capsid morphogenesis. A pept’ide corresponding to this portion of p23 which is excised has not been found. and it is not known to have a function following cleavage. Therefore, the hyp24 supprcssiort of gene 2d mutants should precede the stage of head assembly at which p23 is cleaved. It is surprising that a single mutation in gene 23 can overcome the requirements for ~24 in capsid assembly. Mutations bypassing normally essential gene products of phage T4 have recently been described, but these gene products are not structural components of the phage particle (Bishop $ Wood, 1976; Revel el al., 1976). Existence of the h;qp24 mutation is an additional exampIe of the great plasticity of the T4 phage head. Elimination of major structural components of the assembly core (p.lPI, pIPI1, and pLP[II) and of the capsid shell (hoc and sot) has litt,lr or no effect upon active head formation (Showe & Black, 1973; Ishii & Yanagida, 1975). What is the mechanism of the byp24 suppression ? It is clear that the mut’ation leads t,o a structural change in ~23, which is most extreme at high temperature. In the absence of normal ~24, the byp24 protein allows capsid production at 42”C, but not at lower temperatures (Table 1). When p24 is present, the byp24 mutation is tompc,rature-sensitive and produces a high frequency of short-headed particles, rspccially at 42°C (Tables I and 3). Thus the byp24 mutation clearly affects hcbnd lcbngth determination, although it maps at a site different from the other known gene 23 hnad length mutations (Aebi et al., 1974; Doermann, personal communication). ‘L’hc* hyp24 suppression of various amber and temperature-sensitive mutants in gem: 24, and the normally stoichiometric requirement for p24 in head assembly (Aebi et d.. 1974) make it ext,remely unlikely that small amount,s or fragments of 1’24 arc: used for phage product’ion. Therefort~, an import,ant unanswered question is
282
I,.
A.
McNICOL,
L.
I).
SIMON
ANI)
L.
LV. BLAC’K
whether in Z-l(nm)byp24 phage p24 is substituted or is simply missing. WC havcl apparent’ly eliminated new phage or host proteins as substitutes for 1124. However, an obvious replacement’ for ~24” is p23”, the molecular weights of t,he prot8eins art’ very similar, and the 0~~24 mutation maps in gene 23. UnfortunatIely, it, is very difficult to resolve experimentally whether the amount of ~23” increases very slightly in phage lacking p24”, because ~23” is present in such excess (1000 copies per head compared with an est’imated 30 copies per head of ~24”; Results, section (c)). Two arguments can be made in favor of a ~23” substitution for ~24” : (1) the 24(am)byp24 head has wild-type stability toward heat and pH (Fig. ll), and (2) the 24(am)byp24 phage head shows an osmotic shock resistance which is greater than wild type, but less than 24(0:) (Table 2). One reasonable model for the osmotic shock phenotype is that in the 0: mutant, p24 structure is altered so that the capsid becomes more porous to small ions, and bherefore less subject to osmotic shock. If, (1) this is indeed the basis for osmotic shock resistance, and since, (2) a p24 “hole” should be more porous than any type of structure containing an altered p24 protein, it can be argued that an intermediate porosity requires a p24 substitute, perhaps ~23”. Neither of these arguments is especially persuasive, since we lack certain knowledge of the location or structural role of p24 in the capsid and of the physical basis for the osmotic shock resistance. However, these considerations lead to an interest,ing speculation about the relationship between genes 23 and 24. This question arises naturally from t,he observations that mutation in gene 23 can make gene 24 non-essential, that the products of the two genes found in the capsid are very similar in molecular weight,, and that the two genes are adjacent to each other on the genetic map. It is possible that gene 24 arose as a duplication of gene 23, whose structure was eventually altered to allow more efficient assembly. Greater knowledge of the structure and interaction betlveen t,hese genes and proteins could provide an answer to this speculation. Tllis work was supported in part by grant AI11863 from the National Institutes of Allergy and Infectious Diseases t,o one of ns (L. D. S.) ; by a National Institutes of Health Postdoctoral Fellowship to another author (L. A. M.); and by grant ,4111676 from the Pu’ational Institutes of Allergy and Infectious Diseases to the third author (L. W. B.). We tllank Dr William Mason for his extremely helpful criticism of the manuscript, Dr Michael K. Showo for the generous donation of anti-p24 serum, and Pat Coon for excellent assistance with some of these experiments, REFERENCES Adams, M. H. (1959). Bacteriophages, Interscionce, New York. F., KellenAebi, U., Bijlenga, R. K. L., van den Broek, R., van den Broek, J., Eiserling, berger, C., Kellenberger, E., Mesyanzhinov, V., Muller, L., Showe, M., Smith, R. & Steven, A. (1974). J. Suprarnol. Stmct. 2, 253-275. Anderson, T. F., Rappaport, C. & Muscatine, N. A. (1953). An%. Inst. Pmt. 84, 5-10. Bijlenga, R. K. L., Scraba, D. & Kellenberger, E. (1973). Virology, 56, 250-267. Bijlenga, R. K. L., van den Broek, R. & Kellenberger, E. (1974). Nature (London), 249, 825-827. Bijlenga, R. K. L., Aebi, U. & Kellenberger, E. (1976). J. Mol. Biol. 103, 469-498. Bishop, R. J. & Wood, W. B. (1976). ?‘i’iroZogy, 72, 244-254. Black, L. W. & Ahmed-Zadeh, C. (1971). J. Mol. Biol. 57, 71-92. Black, L. W. & Brown, D. T. (1976). J. Vi7iroZ. 17, 894-905. Castillo, C. J., Hsiao, C.-L., Coon, P. & Black, L. W. (1977). J. Mol. BioZ. 110, 585-601. Celis, J. E., Smith, J. D. & Brenner, S. (1973). Nature New BioZ. 241, 130-132. Coppo, A., Manzi, A., Pulitizer, J. F. & Takahashi, H. (1973). J. Mol. BioZ. 76. 61-87.
p24
BYPASS
MUTATION
Xl
Goldstein, J. & Champe, S. P. (1974). J. 17iroZ. 13, 419-427. Hamilton, D. L. & Luftig, R. B. (1976). J. Birol. 17, 550-567. Ishii, T. & Yanagida, M. (1975). J. Mol. Biol. 97, 655 660. Kellenberger, E. (1968). Virology, 34, 549-561. King, J. & Mykolajewycz, N. (1972). J. &IoZ. BioZ. 75. 33!& 358. Laernrnli, U. K. (1970). Nature (London), 227, 680-685. Laemrnli, U. K. & Favre, M. (1973). J. Mol. Biol. 80, 57%59!). Laemmli, U. K., Molbert, E., ShoLve, M. & Kcllenberper, E. (1970). J. Mol. Bid. 49, !W 113. Leibo. 8. P. & Mazur, P. (1966). Biophys. J. 6, 747-ii2. Luftig, R. 13. & Ganz, C. (1972). J. ViroZ. 10, 545-554. Luftig, H. B. & Lundh, N. P. (1973). Proc. Nat. AC&. Sci., c!.S.d. 70, 1636-1640. O’Farrell, P. H. (1975). J. Riol. Chem. 250, 4007-4021. Onorato, L. & Showe, M. K. (1975). J. Mol. Biol. 92, 395-412. Paietta, J. V., McDonald, T. L. & Doermann, A. H. (1976). J. rirol. 18, 785-787. Revel, H. It., Herrmann, R. & Bishop, R. J. (1976). T’iroZog?/, 72, 255-265. Sarahhai, A. S., Strettou, A. 0. W., Brenner, S. & I(olle, A. (1964). 12:c&re (Lo~ldorc). 201, 13-71. Showr, M. K. & Black, L. W. (1973). Nature Sezcl Bid. 242, 70~m75. Simon, 1~. D. (1972). Proc. Nat. i2cacZ. Sci., U.S.A. 69. 907.!)ll. Studier, F. W. (1973). J. Mol. Biol. 79, 237-248. Tsugit,a, A., Black, L. W. & Showc, M. K. (1975). J. &fol. Wiol. 98, 271-275. \‘au&rslicc, K. W. & Yegian, C. D. (1974). l~irolog~. 60, 265~-275.
116, 261-283
A Mutation Which Bypasses the Requirement for p24 in Bacteriophage T4 Capsid Morphogenesis LOI& ANNE MCNICOL~-,LEE D. SIMON~ 1 n.sfit,ute for Cancer Research,
Philadelphia,
Pent&. 19111, ll.8.d.
AND
LINDSAY W. BLACK of Biological School of Medicine,
Department
Ut~ivwsity
of Maryland
(Receivecl 15 Novender
Chenuktry
Bnltimwe,
1576, and in revised form
3d
Y1201, U.S.A.
5 May
1977)
A mutation (byp24) affecting the N-terminal region of ~23 will suppress the lethal cffccts of urn and ts mutations in gene 24. In t,he presence of normal ~24, the byp24 alteration causes a delay in t,he cleavage of capsid proteins and the assembly of a high percentage of isometric, short-headed particles; thttrefore, the byp24 mutation can affect the length of the T4 capsid. In the absence of ~24, 24- byp24 tlouble mutants show a reduced rate of cleavage of capsid precursor proteins, and a reduced rate of virus assembly. Immunoprecipitation wit’h anti-p24 serum has shown the presence of both p24 and ~24~ irk wild-type phagc particles. The 24-byp24 particles contain no p24 or 1)24~, as determined by immunoprecipitation, urca/acrylamidc gel electrophoresis, and two-dimensional isoelectric focusing, urea/acrylamidc gradient gel electrophoresis. They have a normal electron microscopic appearance, pH stability, and heat stability; but they are more resistant to osmotic shock than wild-type T4. We suggest that p24 normally functions in the initiation of phage T4 capsid I)rotcin cleavage reactions.
1. Introduction The head of bacteriophage T4 is a complex structure, composed of many protein species, which is formed through the successive intermediates outlined in Figure 1. In the first step, the precursor form of the major structural protein of the head, p23§, is thought to form membrane-bound aggregates or lumps (Laemmli et al., 1970; Simon, 1972; Coppo et al., 1973). From these lumps proheads are organized. These t Present address: Division of Riology, California Institute of Technology, Pasadena,, Calif. 91125, U.S.A. $ Present address: Waksman Institute of Microbiology, Rutgers University, New Brunswick, NJ. 08903, U.S.A. $ Xbbreviat,ions used: p23 is the protein product of gene 23, and a similar terminology is used for thr other gene products. IP stands for internal prot.oin. ~23~, pIPIG, et,c., represent the cleavtxd form of these gpne produ& found in the mature phage head. 261
202
IA. ,I. M~!NIc’olJ, 1,. IJ. SIMON .\SI) Shell components 0’0 ~23 core components P21 P22 PIPI
~~~~
I,. w. HL:\(‘K
Shei I components P20 P23c p 24 p 24’
Core components pepttdesil plPIC c
il N A P2C’ p23c
BDl
c
SC?
Q
fisD8~
P49 Lump
r-portlcle
Empty head
Full head
1. Schematized outline of bacteriophage T4 head assembly. For references and description FIG. see the text. It is not known exactly when ~24, phoc, or psoc are added to the nascent head; nor is it known whether p21 or p40 are transiently incorporated into the structure. The origin of peptide II is obscure.
resemble the T-particles formed in gene 81 or gene 24 defective infections. The 7particle shell is formed of p23 and ~20, and surrounds an assembly core composed of ~22, pIPI, pIPI1, and pIPI (Laemmli & Favre, 1973; Luftig & Lundh, 1973; Showe & Black, 1973; Bijlenga et al., 1974; Black $ Brown, 1976). Assembly of the T-particles requires a host fa,ctor, ~20, ~21, ~22, and p31 (Laemmli et al., 1970; Coppo et al., 1973). When genes 21, 24, and 40 are functional, the structural proteins of the r-particle are cleaved: ~23, ~24, pIPI, pIPII, and pIPI have an N-terminal fragment removed to form p23”, ~24~, pIPIe, pIPII”, and pIPIIIc (Laemmli, 1970; Tsugita et al., 1975; Black 6 Brown, 1976). The core protein p22 is totally degraded to small fragments; and one of these, peptide VII, remains within the mature particle (Showe & Black, 1973; Goldstein & Champe, 1974). At least ten other T4 proteins are required after cleavage for late steps in the completion of a mature, active head. Of these, ~16, p17 and p49 are required for DNA packaging (Luftig & Ganz, 1972), while the remainder (~2, p4, ~13, ~14, ~50, ~64, and ~65) affect the activity, but not the apparent morphology, of the mature head (Laemmli, 1970; Hamilton & Luftig, 1976). T-pa,rticles are assembled in 21- and 24- infections, and protein processing reactions do not occur under such conditions (Laemmli et al., 1970). The 24- T-particles are more labile to isolation procedures than the 21- r-particles, but in vivo they can be conservatively matured into normal heads when p24 activity is restored (Bijlenga et al., 1973), whereas 21- T-particles are inert. Therefore the function of p24 is associated with cleavage of pre-assembled proteins, although p21 appears to be the actual protease (Goldstein & Champe, 1974; Onorato & Showe, 1975). In addition, p24 plays a structural role in the finished head, controlling osmotic shock sensitivity (Leibo & Mazur, 1966). And it is required to prevent abberant head formation, since mutations in gene 24 give rise to closed polyheads and giant phages (Bijlenga et al., 1976; Paietta et al., 1976). To investigate the morphogenetic role of ~24, we have isolat,ed an cxtragenic mutation which suppresses gene 24 deficiencies. This paper demonstrates that the supressor is an altered gene 23 protein, which can be processed and assembled into viable particles in the absence of ~24.
~24
BYPASS
263
MUTATlON
2. Materials and Methods (a) Bacteria
and bacteriophage
strains
T4D wild-type; gene 24 umE303, anaE355, amN65, amNG373, amNG433, tsL90, and t&IO; gene 23 amB17, amH11, amE506, and arnE1236; gene 21 anzE322; gene 22 amB270 and anzE209 ; gene 31 amN 111; gene 44 amN82; gene 55 amBL292 ; and gene e anaH are from tlie collection of Dr R. S. Edgar. The gene 24 osmotic shock-resistant mutant ‘I’4BOp is from Dr A. W. Kozinski. Escherichia coli BE is from Dr R. H. Epstein and fl. CO/%R is frotn Dr 1~. S. Elgsr. E. co/i 11,,wT and suII arc from Dr 1’. Stringini. (b) ,%Iedia and cultural Pliago stocks were grown in H broth or M9 rnctlium rising nt,aridartl t,c!clmiqucs (Adams, 1959).
techniq7ces and plnt,ing
was done on EHA
ngar
(c) Osmotic shock resistance Osmotic shock resistance was measured using a variation of the procedure of Anderson et al. (1953). Samples, each containing 10s phage in 0.1 ml of 4 ivr-NaCl at 4°C in a 25 mm x 200 mm screw-cap test tube, were equilibrated for 15 min. Then 10 ml distilled water at 4°C: was “dumped” into the t,ube, and the survivors assayed by dilution through cokl brot,li (d) &r$cation i4C-labeled Kellciihorger
pliage (1968).
were
purified
by
of phage particles the
(c) Electron
CsCl step-gradient
procedure
describrd
1))
microscoy y
The fixation, dehydration, embedding, sectioning and staining of infected cells I>nvtt been described (Method I in Simon, 1972). Phage were negatively stained in 2 steps, first for 25 min with saturated aqueous many1 acetate adjusted to pH 3.5 with HCl, then followed by 2% aqueous many1 acetate (pH 4*3), which was immediately removed by blotting with filter paper (A. H. Doermann. personal communication). Specimens were examined in a Siemens Elmiskop I electron microscope. (f ) Electrophoresis
and immunoprecipitation
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate \vas carried out according to Laemmli (1970) on a slab gel apparatus described by Studier ( 1973). Urea/acrylamide gradient, sodium dodecyl sulfate gels were as described by Castillo et al. ( 1977). Two-dimensional isoelectric focusing, urea/acrylamide gradient gels, sodium dodeeyl sulfate were modified from the method of O’Farrell (1975) as described by C.-L. Hsiao (manuscript in preparation). Electrophoresis of immunoprecipitates was as follows : 60,000 cts/min of CsCl-purified [14C]amino acid-labeled phage, or lysates of phagu infected, [i4C]amino acid-labeled bacteria, were mixed with 1011 unlabeled T4D+ phage in 1% sodium dodecyl sulfate electrophoresis sample buffer and heated to 100°C for 5 min. After cooling, the volume was increased lo-fold with 0.01 M-Tris-HCl (pH 7.6) and antiserum against p24 was added to equivalence with respect to the dissociated carrier phage. After 18 h at room temperature, the precipitate was centrifuged from suspension at 2600 revs/min for 30 min, washed once with 0.02 M-TriseHCl (pH 7.6), recentrifuged as above, resuspended in sodium dodecyl sulfate sample buffer at lOO”C, and submitted to electrophoresis. Radioactive phage or lysat,es were similarly disrupted in the presence of cold carrier phage and applied to double-diffusion gels against antiserum prepared to purified ~24. Autoradiography of dried sodium dodecyl sulfate and immunodiffusion gels was as previously described (Black & Ahmad-Zadeh, 1971). Antiserum against purified p24 was kindly provided by Dr M. I(. ShOWf.~.
I,. A. Mc;“;I(‘OL,
264
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D. SIMON
AND
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3. Results (a) Isolalion
of a su~jmwor
of qeue 84 mutut~ts
Gene H-specific suppressor mutations were isolated from spontaneous pst,udo-rt:\-clrt ants of gene 24 amber mutants, which had a cold-sensitive (cs) phenotype on fi. ~oli BE. Such mutants were found in stocks of 2P(amE303), 2J(amNG433). and 24(~~xNifi5), at frequencies of abut, lo-“. as phnge ahlr to plaque on E. coZi BE. at 12°C’ (\\,itll :I
Cross I amNG433
byp24(ll
amNG433
X
X omd65
am+ weld-type
om1(165
byp2412)
om+byp24
recomblnonts
recombmonk
2!iiq=g
(a)
omE209
am8270
omH I I
am017
Gene 31
Gene 24
Gene 23
Gene 22
amE omNG373
amE
amE
amE amNG433
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77
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byP24 o,i-.
amN65
I.0 2.6 4.2
l-8
6.8 8.4
(bl Fro. 2. Isolation, temporaturo sensitivit,y, and map position of tho byp24 mutation. (a) In crosses I and 2, the percentage of wild type or byp24 progeny phage was determined by comparing the number of plaques on E. coli BE at 30°C to the number on E. coli B4OsuI at 3O’C. Burst sizes of the wild type and the byp24 progeny are compared at 30°C and 42°C. (b) The positions of amber mutations within gene 23 are from Sarabhai et al. (1964). The mutants 24(amE366) and 24(arnNG373) are at the same site; both 24(amE303) and 24(amNG433) are located at a second site; and 24(amE356) is to the left of 24(amE303) (A. H. Doermann, personal communication). From our crosses we find that 24(amN66) is located to the right of 24(anaNG433). This agrees with the relative sizes of the amber fragments of p24 which are produced during infection with these two mutants (Fig. 3). The positions of the two ts24 mutations are not established, although they appear to be non-allelic. The distances between byp24 and the various am. markers are given as 200 x (the number of wild-type phage on E. coli BE at 42’C/the total number of phage in BIOsuI at 3O’C). The map distances are underestimated since no correction is made for T41)+ phage, which give small plaques at 42°C (approx. 10 to 20%).
p24
BYPASS
265
MUTATION
TABLE 1
Ejkiency I~acterial
strain
II
1~4OsuI
of plating and burst size of Tdz4(amE303)byp24t Temperature (“(3
Titer
Efficiency of plat ing$
42 37 30 24
2.1 x 1.7 x 2.4 x 6.5 x
10” 10” lo7 106
1.0 0.63 x.9 s 10 -5 2.4 y IO-5
42 37 30 24
3.0 3.8 2.5 3.2
x x x x
10’” 10” 10” 10’1
I.0 1 .:1 0.83 1.1
15 “.4x
10-Z
120 430
t The phage stock was prepared at 42°C on a B40suI host. $ For each bacterial host), the 42°C titer was taken as 1.0.
burst size of 15) but not at 25°C (burst size of 0.024) (Table 1). Although the pseudorevertants were cs on E. co& BE, they were viable at all temperatures on E. coli B4Osu1, which contains a nonsense suppressor. Under suppressed conditions, these phage were slightly temperature-sensitive, as judged by burst size. In addition, the cs mutants displayed at low temperature the suppression pattern of the amber mutants from which they were derived. For example, both 24(am.N65) and its cs derivatives grew on E. coli B40suI at all temperatures, and neither grew on B40sulI at low temperature. At 42°C only the cs grew on B4OsuII or E. coli BE. These data suggested that the cs isolates might actually be double mutants containing the original 24(amN65) and a second mutation, able t,o compensate for the gent: 2;C deficiency at high, but not at low temperatures. To test this hypothesis we backcrossed cs mutants to wild-type phage. The progeny contained some phage which behaved like the original amber : able t’o grow on E. coli B40suI at both low and high t,emperature, unable to grow on E. coli BE at any temperature, and unable to give recombination or complelnentation with the original amber mutation. Therefore, we concluded that the original 24(am) mutat,ions were indeed present, and being suppressed by a second mutation, which we have called b,yp24: for bypass. We observed relatively close linkage of byp24 to 24(am) markers in the backcrosses to wild type. To isolate the byp24 suppressor(s) we crossed tw.0 independent cs mutants, derivtbd from 24(amNG433) and from 24(amN65), which are separated by approximateIS 0.8 map units (Fig. 2(a), cross 1). From the cross of 24(amNG433)byp(l) with 24 (nmN65)byp24(2) we selected phage able to grow on E. cdl: BE at 25°C (Fig. 2(a), cross 2). All the progeny selected from this cross had apparently wild-type plaque morphology at 30°C or 37”C, and minute plaque morphology at 42°C. Burst size measurement in liquid culture confirmed that these byp24 mutants produced a large burst, at 30°C and grew poorly at 42°C (Fig. Z(a)). One such recombinant, hereafter simply referred to as byp24, was selected for further study. The byp24 was combined with 24(am) mutants 24(amNG433), 24 (am.E303), and 24(amN65) (located at three different loci) and the ts mutants 24(tsL90) and 2l(tsSlO). These double mutants were able to grow on E. coli BE at 42°C. These
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Flc:. 3. Head protein processing and p24 synthesis in nmP4 and 24(um)by~24 infections. E. wli R” or HCOsuI growing in M9 medium wwe infected at 42°C or 30°C at a multiplicit,y of 5, antI superinfectcd at a multiplicity of 5 at 5 min (42°C) or 12 min (30°C). [14C]Amino acids were adtkd from 12 min t.o 18 min (42°C) or 21 min to 30 min (3O”C), and then an excess of non-radioactive amino acids added from 18 min to 30 min (42T) or from 30 min to 60 min (30°C). Bacteria wwc centrifuged from solution at 30 rnin or 60 min, frozen and thawed once in 1 rmx-MgSO,, treat,etl with 4 pg pancreat,ic DNase/ml for 10 rnin at 37”C, boilctl in sample buffer, and t,hen applied to a urea/acrylarnide gradient, sodium dodecyl sulfate-containing gel (samples a to S) or to a 12.5% acrylamide gel containing sodium dodrcyl sulfak (samples t to v). Autoradiographs were: prepared from the dried gels.
p24
BYPASS
MUTATION
267
genetic data suggest that the byp24 suppressor is not specific for mutations located at particular sites within gene 24, but is able to overcome any gene 24 deficiency. In addition, the suppression of 24(ts) mutants makes it unlikely that the byp24 suppressor accomplishes phage production by either incorporating an active amber fragment or by suppressing 24(am) mutations through transcriptional or translational suppression mechanisms. The 6~~24 was next crossed with various am mutants situat)ed near gene 24 (Fig. 2(b)), taking advantage of its ts plaque phenotype to measure tjhe frequency of wild-type recombinants. As the mapping results indicate, byp24 is located in gene 23, very near 23(amHll) the most PT-t,erminal mutation known within gene 23 (Sarahbai et al., 1964). It is likely that there are a very limited number of byp24 sites. (1) Revertants of 24(am) able to grow on E. cc& BE at 42°C occur in the ratio of one wild type to about one to two 24(am)byp24. Assuming equal mutation rates at the amber and at potential byp sites, this rat,io suggests that the number of suppressor sites is roughly equal to the number of amber reverting sites; (2) five isolated byp24 phage from cross 2 (Fig. 2(a)) gave the same recombination frequency with 23(amHll), suggesting that at least byp24(1) and byp24(2) are located near the same site; and (3) there did not appear t’o be any wild-type phage resulting from cross 2. In summary, the genetic results suggest that there is one, or a small number of closely linked sites, in gene 23 which can mutate to make p24 dispensable at high temperature, The location of the suppressor close to W(amH11) shows that it is located within the region that codes for the N-terminal fragment which is removed by cleavage during head assembly (Celis et al., 1973; Tsugita & Isobe, personal communication; C.-L. Hsiao, unpuhlished data). (b)
Head protein
processing
and p24 synthesis
in am24 and am24byp24
infections
Since genetic analyses demonstrated that byp24 overcomes the lethal effect of both and ts mutations in gene 24, we examined phage-specific proteins synthesized during 24(am) and 24(am)byp24 infection. Figure 3 shows autoradiographs of urea/ acrylamide gradient, sodium dodecyl sulfate gels of extract’s from [14C]-amino acid-labeled, infected cells. Comparison to wild-type infection (with normal head protein processing) (Fig. 3e) and to 21(amE322) infection (with no head protein processing) (Fig. 3f) shows that 24(amNG433) and 24(amN65) do not synthesize p24 when grown on E. coli BE at either 42°C or 30°C (Fig. Sa,c,g and i). Cleavage of ~22, ~23, pIPII1, and other head precursor proteins does not occur; no ~23~, pIPI”, or pIPIITC are produced (Fig. 3a,c,g and i). When grown on E. coli B4Osut at 32°C. 24(unhNt%) and 24(amNG433) 1)ro d uce p24 and head protein processing occurs (Fig. 3m and 0). The apparent amber fragments are detected in E. coli BE, M, ,-22,000 in tho 24(amN65) infection, and -16,000 in the 24(amNG433) infection (Fig. 3, am
Growth conditions: samples are designated, e.g. T4D ’ B4L’, for ‘I-41) + grown on Ii:. co/j R” at. 42’C, in this and subsequent Figures. a, 21(omNG43R).B42; b, 24(smNG433)by~24.B42; c, 24(rtmNGB) .B4”; d, 24(wnN65)hy~xl4. H4”; c, T.&D+ -B42; f, 21(~7nE322)~B42; g, 24(nmNG433) .B30; h, 24((tmNG433)by~24.B30; i, 24(atmN66).B30; j, 24(amN65)[email protected]; k, T4D+ -B30; 1, ?l(vmE322).B30; m, 24(an~SG433)~ ~~142; n, 24(umNG433)byp24~suI42; o, 24(nmN65)-~~142; p, 24(rcmN65)byp24.~uI42; q> T4D’ . ~11142; r, ~l(ancE322)~su142; s, purified phagc particles T4D + .B42; t, [email protected]; u, Dyp”4.B:W; v, T4D + .B30.
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Vlt:. 4. Protein composition of gene 2t mutant phagc,. [‘41’].\mino acitl-lahcltvl l)hag~~ \v(‘r’ prcparetl and purified on C&l band gradient,s as described in Materials and MvtJhotls. l’hagc or i,nn,unoprecipit,ates prepared from 60,000 ct,s/min of each purifictl mutant phagc (Jlateriuls awl Methods) w-ere boiled in sample buffer and applied to 8% (samples a t,o o) 01’ !)o;, (samples 1’ to t) ncrylamide gels containing sodium dodecyl sulfat,c. Infected ccl1 lysatrs WC‘I‘Pprepar~tl at 37’(‘. I’hage: a, T4D+ .B42; b, ZC(omNG433)byp24~suI42; c, 24(rcmNG433)61/~~.R42; p, 24. ‘9 (rcmNG433)byp24.d30; g, 24(nmNG43R)by$4.suI42; I‘, 24((~mNG433)~~yp_4.B43; ,s. “4. (trmNG433). ~11142; t, T4D + . B4”. Immunol)recipitate~: d, T4D + .B42; e, byp24.B42; f, 24(wnNG4:S3) .su14”; g, ZqrcmNU4:l:l). by~~24~suIBO; h, 24(nmNG439)b~f/‘t4.suI42; i, 24(tcmN6.5)6y~.suI42: j, ~d(nmNG433)b?/p24.H4:!: k, 2Z(rtmEXOS)~B, info&xl cell lysate; I, 2:S(n~nH11)24(nmN65)~B, infoct,cd ccl1 lysatc; m, 25(~mHll)~.B, info&cd cell lysato; II, 24(trm65) .B, infected cell lysat,o; o, 2f(rtrn~90) .B, infoctc*d cell lysatc.
arrows a and c). When 24(amN65)byp24 or 24(amNG433)byp24 are grown in E. coli BE at 42”C, p24 cannot be detected but protein processing does occur, and a large amount of ~23” is produced (Fig. 3b and d). During infection with these mutants at the restrictive temperature (30°C) no ~24 is detected and the formation of ~23” a.nd other processed head proteins is not significant (Fig. 3h and j). Both 24(amN65)byp24
~24
BYPASS
MUTATIOS
%!I
P22
FI(:. 5. Kinetics of head prot.ein processing in 2J(nmNG433)byp24 and T4D + infections. E. coli 11” grooving on M9 medium at. 42°C was infected at a multiplicity of 5, and superinfected 5 min litter. The infect.ed cells were mixed with “C-lebeled amino acids at 12 min, and excess non-radioactive amino acids were added at 14 min. Samples were pipeted onto ice at various times, the bacteria centrifuged from solution, and quickly frozen. (For samples a to n the radioactive pulse was between 12 min and 18 min, and the bacteria were centrifuged from solution at 2.5 min.) Infected bacteria were boiled in sample buffer and applied to fly0 acrylamide gels containing sodium dodecyl sulfate. Samples were prepared after infection with: a, T4D+ : b, PZ(nmE322): c, 24(nmNG433); d, )‘S(nmHll); e, 23(amH11)24(amN65). ?4(mtXG433byp24): f, 14.5 min; g, 16.5 min; h, 18.5 min; i, 20.5 min: j, 24.5 min; k, 29-5 min. l’4ll+ ; 1, 14.5 Inin; m, 16.5 min: 11, 155 min; o. 20.5 min; 1,: 24.5 min; q, 29.5 min.
urea/acrylamide gradient, sodium tlotlwyl YIC. 6. Two-dimensional isoelectric focusing, sulfate gel electrophoresis of T4D + and 24(nmNG433)byp24 phago. Two ixoclect,ric focusing gels of dissociated phage, with migration from the center out: a, 24(amNG433)6yp24~42, 30,000 cts/min applied and c, T4D + .R42, 60,000 cts/min applied were submitted to electrophoresis in the vertical direction on a urea/acrylamide gradient gel containing sodium dodecyl sulfate (Materials and Methods). Track b contains for comparison disrupted T4D+ phage particles with no isoelectric focusing. It appears that ~24~ underlies ~23~ in the second dimension and is absent from the 24(nm)byp24 phage. The 2 smallest proteins from phage particles (pIPI” and pIPIF) appear to have been lost from the basic end of the isoelectric focusing gel in this run.
p24
BYPASS
MUTATION
271
and 24(amNG433)byp24 produce the p24 amber fragments on E. coli BE at 30°C and 42”C, and produce intact p24 during infection of B4OsuI (Fig. 3n and p). Therefore, by analysis of infected cells on acrylamide gels containing sodium dodecyl sulfate. 24(amNfS)byp24 and 24(amNG433)byp24 contain the original amber mutation: and are able to accomplish head protein processing in E. coli BE at 42°C in the absence of ~24. When the single byp24 mutant is grown on E. coli BE at 3O”C, cleavage is inefficient when compared to a wild-type infection. This effect is more pronounred when byp24 is grown at the more restrictive temperature (Fig. 3t,u and v). (c) Protein composition of 24(amNG433)byp24 phage purticles ~24 is cleaved during normal head assembly, and the processed protein ~24” is reported to be a major component of the capsid (Laemmli, 1970). Since acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of E. coli BE infected with 24(am)byp24 showed that p24 was not synthesized at 42”C, although head protein cleavage and phage production occurred (Fig. 3), we wanted to determine the protein composition of the phage produced. Preparations of [14C]amino acidlabeled phage were purified in CsCl step gradients, and their proteins separated on sodium dodecyl sulfate-containing acrylamide gels (Fig. 4). The following phage were purified and examined in this way : T4D + (Fig. 4a and t), byp24 (data not shown), and 24(amNG433)byp24 (Fig. 4c and r) grown on E. coli BE at 42°C; 24(anzNG433) (data not shown) and 24(amNG433)byp24 (Fig. 4p) grown on B40suI at 30°C ; and 24(amNG433) (Fig. 4s) and 24(anzNG433)byp24 (Fig. 4b and q) grown on B40suI at 42OC.We were not able to detect the absence of a protein band from any one of these\ phage preparations.
T!me 01 42OC hn!
Fro. ‘7. Single-step growth kinetics of T4D+ and 24(nnaNG433)byp24 in E. coli BE at 42’i’. E. coli BE growing exponentially at 42°C were infected with T4D+ or 24(amNG433)byp24 at a multiplicity of 4. At 2.6 min the bacteria were superinfected with the same phage at the same multiplicity, and at 4 min anti-T4 serum was added. At 8 min the infected bacteria were diluted from antiserum into fresh broth at 42’C, and the concentration of infected cells was cetermined. Samples of infected bacteria were removed at the indicated times and lysed with CHCl, to determine the phage yield per infected bacterium. -x -X -, T4D + ; -a--•--, 24(amNG433)byp24.
27’
L.
A. McNICOL,
L.
D.
SIMON
AND
L. W.
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Our results suggested that ~23” and ~24” overlap on the sodium dodecyl sulfat’econtaining acrylamide gel system. Urea/acrylamide gradient gels containing sodium dodecyl sulfate or low concentration acrylamide gels containing or lacking urea did not show differences in composition between T4D+ and 24(am)byp24, although these changes in the gel did shift the relative positions of ~23” and ~24. The problem
p?4
BYPASS
MUTATION
“53
appeared to be due to the very large amount of ~23” in phages, which causes spreading of the p23c band; occasionally a band which may be ~24~ emerges from t’he ~23” hand, but, its identification is quite uncertain. The p24 band is readily identifiable on virt,ually any sodium dodecyl sulfate-containing acrylamide gel of T4-infected cells, \et there is considerable disagreement in identifying ~24” on sodium dodecyl sulfate,-containing acrylamide gels of phage particles. For example, the identifications of ~24” by Laemmli (1970), Vanderslice & Yegian (1974), Coppo et al. (1973) and Aehi et al. (1974), differ considerably in both position and amount relative to ~23”. fiincc any capsid formation-gene defect prevents ~24” formation, and since somcx ot,her proteins near ~23” also appear to be derived from p23 (Fig. 5a and e), it is difficult t,o identify ~24~ with certainty in phage particles. It is likely that some of the bands in purified phage previously identified as ~24” might be derived from p23 OI’ c~ultl be the hoc protein or other proteins of unknown origin (Ishii & Yanagida, 1!)75). We were able to precipitate ~24~ from CsCl-purified, sodium dodecyl sulfatcdisrupbcd phage, using antiserum directed against p24 (kindly provided by Dr M. K. Showe). Either through autoradiography of disrupted radioactive phage in immunodiffusion gels (data not shown) or through electrophoresis of the immunoprecipitates on sodium dodecyl sulfate-containing acrylamide gels in the absence of p23”, it was possible to identify ~24” within the phage, and to show that p24 is missing ouly from 2d(am)6yp24 phage grown on E. co.5 BE at 42°C (Fig. 4a and j). The immunoprecipitation is specific for ~24, since a band of radioactive protein is prccipitsted from cells infected with amber mutants in genes 21, 22 or 83 (Fig. 4k,m >rlltl 01. but not from cells infected with amber mutants in gene 24 or genes 23. %J (Fig. 11 and n). Using equal amounts of radioactive phage, it is apparent that only %(ant)hyp24 phage grown on E. coli BE at 42°C lack radioactive protein bands pr+ cipihat)ing with antiserum directed against p24 (Fig. 4a and j). There appear to 1~ two vc~ry slightly separated forms of p24 incorporated into phage particles, althougl~ only one hand is observed in capsid formation gene-defective phage-infected cells; both these bands are located under the ~23” band in purified phage (Fig. 4c and 0). Therefore, it appears as though p24 and its processed form ~24” are found in thcb T4 capsid. but the processed form is difficult to distinguish from p24 on standard gels. WC also determined that 24(amE303)byp24 does not incorporate any host protein into its ca.psid in the absence of ~24. E. coli B was prelabeled with 14C-labeled amino acids. The radioactive cells were washed twice and infected with wild type or %3(attt. E303)h;qp24 at a multiplicity of infection of five in the presence of cold medium. At cell Isis the phage were purified by differential centrifugation and their radioactive prot)eins examined by acrylamide gel electrophoresis in the presence of sodium dodec*?;l sulfate. There was no difference between the banding patterns of the wild type ;rntl mutant phage (data not shown). Twc~dimensional isoelectric focusing in urea/acrylamide gradient gels containing sodium dodecyl sulfate was used to confirm our observation that 24(am)byp24 phagc lack 1’24~. The ~24” is apparently more acidic than p23”, so the two proteins can l)e rcsolvcad in the first, isoelectric focusing, dimension even though they have t,he same rnobilit~y in tho second, apparently molecular weight-dependent, urea/acrylamido dimension (Fig. 6). It appears from this and other two-dimensional gels that Z-l(attt NG433)hy~~24 grown on E. coli BE at 42°C (Fig. 6a) are lacking the ~24” band present in ‘l’-tl) + phage (Fig. 6c). The conclusion that ~24 is missing from 2~(cm)bj/p%
~24
BYPASS
MUTATION
276
particles is thus supported by the combined evidence from gels of infected cell lysates (Fig. 3), gels of immunopreeipitat,es of disrupted phage (Fig. 4)) and isoelectric focusing of labeled particle proteins. A very rough estimate can be made of the amount of ~24” in the capsid. There are 144 molecules of ~18 in the T4 particle and the molecular weights of p18 (80,000) and p24 (45,000) have been estimated (Laemmli, 1970; King & Mykolajewycz, 1972). From densitometer tracings of autoradiographs of the p24 bands following immunoprecipitation (Fig. 4d and i) and of the ~18 bands in the phage which were used fo1 the p24 precipitation (Fig. 4a,b and c), it can be estimated that there are 30 molecules of p24 and ~24~ in the phage head. This figure is likely to be an underestimate of the actual amount of ~24” in the phage head, since losses of ~24~ are likely during preparabion of the immunoprecipitates, and it is obvious that such a measurement is subject to considerable variability. We note that only gr0wt.h on E. co& BE at 42°C. does 24(am)byp24 produce phage lacking p24 (Fig. 4j). The 24(am)byp24 phaga grown on E. coli B4OsuI or b;yp24 grown on E. coli BE at 42°C contain p24 in amounts comparable to wild type (Fig. 4d and i). Apparently, the byp24 mutant can utilize ~24 if it is available at any temperature; and when p24 is available, phage assembly is considerably more efficient. (d) Physiology of 24(am)byp24 infection Infection of E. c&i BE with 24(awP)byp24 phage at 42°C leads to the production of phage, polyheads, and T-particles (see section (e), below). The efficiency of phage formation is reduced considerably compared to wild-type infection, perhaps due to this production of aberrant head structures. We examined the time of phage appearance by measuring single-step growth at 42°C (Fig. 7). The 24(amNG433)byp24 progeny appear at a considerably reduced rate and final yield. In the wild-type infection, the first phage is produced at about 15 minutes, and phage production is 50% complete by 20 minutes, whereas in the 24(nwSG433)byp24 infection bhe first phage do not appear until 20 minutes. The reduced rate of phage formation in the 24(amNG433)byp24 infection is correlated with a greatly reduced rate of head precursor protein cleavage, as is shown in Figure 5. In the 24(amNG433)byp24 infection at 42”C, pulse-labeling of proteins from 12 to 14 minutes followed by a chase with cold amino acids, shows that ~23” appears to a significant extent only in the sample collected at 20.5 minutes (Fig. 5i). The large increase in ~23” concentration at 24.5 and 29.5 minutes (Fig. 5j and k) shows that, p23 cleavage in the 24(amNG433)byp24 infection is continuing at these late times. Thus, following its synthesis, p23 can be normally processed, for an extended period, although the rate of cleavage is slow. In a parallel infection with T4D + , cleavage is already considerable at 14.5 minutes and appears to be essentially complete at 16.5 minutes (Fig. 51 and m). When E. coli BE bacteria are infected with 24(aw&G433)byp24 at non-permissive t’emperature (30”(J), and then shifted to 42°C in the presence of chloramphenicol, we observed a significant increase in the number of phage. However, the yield amounted
1’24
BYPASS
277
MUTATION
t.o only about one phage per cell, suggesting that the 24(am)byp24 or completed inefficiently in the absence of protein synthesis. (e) Electron microscopy
of mutant-infected
heads arc
assembled
cells
We have taken electron micrographs of thin sections from infected cells to det,ermine what structures the mutant phage are able to assemble. When 24(amE303)byp24 is grown under permissive conditions (42°C) the cells contain a mixt’ure of full heads, T-part,icles, and polyheads (Fig. 8). However, at 24°C morphogenesis is blocked and no normal heads are seen (Fig. 9(a) and (b)). The byp24 mutant produces finished heads when grown at either 24°C or 42°C. In agreement with the large amount of uncleaved p23 seen in Figure 3~. the mutant assembles relatively large amounts of polyhead at the higher, partially restrictive temperature (Fig. 10). (f) Osmotic shock resistance of the am24byp24 phage particles WC were interested in the biological consequences of assembling a capsid in the ahscnce of ~24. Since gene 24 has been reported to control the osmotic shock phenotype of T4 capsids (Leibo & Mazur, 1966), we examined the osmotic shock phenotype of the mutant particles. Table 2 lists the survival of various phage following osmotic shock. The 24(amE303)byp24 phage prepared on E. coli B at 42°C are about 15fold more resistant to osmotic shock than were the wild type, suppressed 24(amE303). or 61~~24phage. However, there degree of resistance is not so marked as that of thrh T4BOi mutant, which shows a 96-fold higher survival than wild-type phage. Although t,he 24(amE303)byp24 particles displayed altered osmotic properties. their* ca.psids had normal stability to pH (data not, shown) and to heat inactivatioll D’ig. II). -
I IO
I
20
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30
TIM hn)
FIG. 11.
Heat stability of wild type and 24(amNG433)byp24 phage. A mixture (>f 108 2~(r~mNG433)by~24.B42 and 10 lo 44(umN82)55(amBL292)e(nmH26)~suI42 phage was incuhetl>tl at, 67°C. At. various times samples were plated on BQOsuI at 37°C for total remaining phagc a~itl 011 1%” at. 42°C for remaining 24(amNG433)byp24 phage. - -i ;- I_ ‘-. %J(omNG433)b?yp24: ,’ “, , ~;((~rmN82)55(amHLL’94)e(amH26).
FIG:. 12. Electron micrographs of mgatimly hy ccntrifugation t.hrough a CsCl step gradient (B) T4D + .B42.
stained phagc pwticles. ‘I’ht- phrtgc wwv l)urifkl t+4’L; and cliroct,ly stained. (A) %1( ~lnLE:~(l:l)h!/l”‘4.
1’24 BYPASS
279
MUTATIOK
TABLE
2
Osmotic shock resistance of mutant phuge particles
Host
I’hage
T4D 24(amE303) byp24
B B40suI B
24(antE303)byp24
B
Ratio (mutant survival/ wild-type survival)
Temperature (“C)
2.5(amE303)byp24
B4OsuI
42 42 24 42 42
T4R24(0;)
B
42
(2.38&O+J2) x 10-s (5.03k3.7) x 10-S (2.36-~:0.34) x 1O-2 (3.57kO.65) x 1O-2 (2.35*1.6)x 1O-3
1.0 2.1 0.99 15 3.5
(2.29kO.14)
96
t This value represents the average of 3 independent for thv experimental details.
(g) Electron
microscopic
appearance
x 10-l
experiments.
See Materials
and Methods
of mutant particles
Figure 12 illustrates negatively stained preparations of various mutant phage particles. Tn the absence of ~24, the phage produced by 24(amE303)byp24 on E. co& B at) 42°C have a normal appearance (Fig. 12(A)). Compared to wild-type phage (Fig. 12(B)), the double mutant has a full-size capsid. However, in the presence of normal ~24, the byp24 mutation causes the assembly of abnormally sized heads. Under permissive conditions, E. coli B at 3O”C, byp24 produces a significant fraction of short-headed particles (Fig. 13). Table 3 presents quantitative data from scans of many fields. At 3O”C, 21% of the byp24 phage have an isometric or two-thirds-sized head, and 7’5 have a three-quarter length capsid. In contrast, the wild-type phage preparation showed 2% and the 24(amE303)byp24 grown on E. coli B at 42°C showed 4o/o short,headed phage. When the byp24phage were grown at the more restrictive temperature 42°C. even more particles were abnormal, since 56(x, of the population had isometric heads. From these observations it is clear that the byp24 mutation can affect cap&l lengt’h determination.
TABLE
3
Capsid length of various mutant phage particles Host
Temperat,ure P-3
:h Particles with capsid length?
l/l T4D ktp24 byp 2 4 21(mNG433)byp24 -t ‘L’h~~[)vvrentagtls
B B B B
30 30 42 42
98 72 37 96
3/4 0 7 7 0
are based on tcrtal counts of 118 10 341 total l~avticlcs.
2/3 2 “1 56 4
~24 BYPASS
MUTATION
281
4. Discussion In wild-type T4 infections p24 is essential for normal capsid assembly. It is an integral component of the capsid shell (Aebi et al., 1974) and controls, at least in part’, the shock resistance of the capsid (Leibo & Mazur. 1966). We have osmotic found that both p24 and ~24” are present in the capsid of wild-type T4 phage (Fig. 4a). Although these proteins can be identified on standard gel electrophoresis of infected cell extracts, they are difficult to detect in phage particles, where they are obscured by large amounts of ~23” (see Results, section (c)). Either clect,rophoresis of immunoprecipitation or two-dimensional isoelectric focusing, urea/ acrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate is required for the separation of ~24~ and ~23”. In addition, there seems to be a requirement, for p24 to prevent aberrant capsid development, since gene 24 missense mutants cause formation of polyheads and maturation of giant phages (Paietta et al., 1976). However, p24 does not seem to play an essential role in shape determination, since 24 defective mutants also accumulate s-particles, which can be directly converted to mature phage when 2d function is restored (Bijlenga et al., 1973). The capsid protein cleavage reactions which occur during normal T4 morphogenesis are blocked in 24- infect’ions, although p24 is not directly implicated in the protein processing reactions (Onorato & Showe, 1975). Our results establish the nature of a specific suppressor of the requirement for ~24 in the morphogenesia of T4 capsids: a particular mutation within gene 23. called 117~~24,permits the production and maturation of T4 heads in the absence of p24 (Figs 3: 4> and 6). The map position of this mutation near 23(amHll) (Fig. 2) strongly suggests that it alters a portion of p23 which is removed during capsid morphogenesis. A pept’ide corresponding to this portion of p23 which is excised has not been found. and it is not known to have a function following cleavage. Therefore, the hyp24 supprcssiort of gene 2d mutants should precede the stage of head assembly at which p23 is cleaved. It is surprising that a single mutation in gene 23 can overcome the requirements for ~24 in capsid assembly. Mutations bypassing normally essential gene products of phage T4 have recently been described, but these gene products are not structural components of the phage particle (Bishop $ Wood, 1976; Revel el al., 1976). Existence of the h;qp24 mutation is an additional exampIe of the great plasticity of the T4 phage head. Elimination of major structural components of the assembly core (p.lPI, pIPI1, and pLP[II) and of the capsid shell (hoc and sot) has litt,lr or no effect upon active head formation (Showe & Black, 1973; Ishii & Yanagida, 1975). What is the mechanism of the byp24 suppression ? It is clear that the mut’ation leads t,o a structural change in ~23, which is most extreme at high temperature. In the absence of normal ~24, the byp24 protein allows capsid production at 42”C, but not at lower temperatures (Table 1). When p24 is present, the byp24 mutation is tompc,rature-sensitive and produces a high frequency of short-headed particles, rspccially at 42°C (Tables I and 3). Thus the byp24 mutation clearly affects hcbnd lcbngth determination, although it maps at a site different from the other known gene 23 hnad length mutations (Aebi et al., 1974; Doermann, personal communication). ‘L’hc* hyp24 suppression of various amber and temperature-sensitive mutants in gem: 24, and the normally stoichiometric requirement for p24 in head assembly (Aebi et d.. 1974) make it ext,remely unlikely that small amount,s or fragments of 1’24 arc: used for phage product’ion. Therefort~, an import,ant unanswered question is
282
I,.
A.
McNICOL,
L.
I).
SIMON
ANI)
L.
LV. BLAC’K
whether in Z-l(nm)byp24 phage p24 is substituted or is simply missing. WC havcl apparent’ly eliminated new phage or host proteins as substitutes for 1124. However, an obvious replacement’ for ~24” is p23”, the molecular weights of t,he prot8eins art’ very similar, and the 0~~24 mutation maps in gene 23. UnfortunatIely, it, is very difficult to resolve experimentally whether the amount of ~23” increases very slightly in phage lacking p24”, because ~23” is present in such excess (1000 copies per head compared with an est’imated 30 copies per head of ~24”; Results, section (c)). Two arguments can be made in favor of a ~23” substitution for ~24” : (1) the 24(am)byp24 head has wild-type stability toward heat and pH (Fig. ll), and (2) the 24(am)byp24 phage head shows an osmotic shock resistance which is greater than wild type, but less than 24(0:) (Table 2). One reasonable model for the osmotic shock phenotype is that in the 0: mutant, p24 structure is altered so that the capsid becomes more porous to small ions, and bherefore less subject to osmotic shock. If, (1) this is indeed the basis for osmotic shock resistance, and since, (2) a p24 “hole” should be more porous than any type of structure containing an altered p24 protein, it can be argued that an intermediate porosity requires a p24 substitute, perhaps ~23”. Neither of these arguments is especially persuasive, since we lack certain knowledge of the location or structural role of p24 in the capsid and of the physical basis for the osmotic shock resistance. However, these considerations lead to an interest,ing speculation about the relationship between genes 23 and 24. This question arises naturally from t,he observations that mutation in gene 23 can make gene 24 non-essential, that the products of the two genes found in the capsid are very similar in molecular weight,, and that the two genes are adjacent to each other on the genetic map. It is possible that gene 24 arose as a duplication of gene 23, whose structure was eventually altered to allow more efficient assembly. Greater knowledge of the structure and interaction betlveen t,hese genes and proteins could provide an answer to this speculation. Tllis work was supported in part by grant AI11863 from the National Institutes of Allergy and Infectious Diseases t,o one of ns (L. D. S.) ; by a National Institutes of Health Postdoctoral Fellowship to another author (L. A. M.); and by grant ,4111676 from the Pu’ational Institutes of Allergy and Infectious Diseases to the third author (L. W. B.). We tllank Dr William Mason for his extremely helpful criticism of the manuscript, Dr Michael K. Showo for the generous donation of anti-p24 serum, and Pat Coon for excellent assistance with some of these experiments, REFERENCES Adams, M. H. (1959). Bacteriophages, Interscionce, New York. F., KellenAebi, U., Bijlenga, R. K. L., van den Broek, R., van den Broek, J., Eiserling, berger, C., Kellenberger, E., Mesyanzhinov, V., Muller, L., Showe, M., Smith, R. & Steven, A. (1974). J. Suprarnol. Stmct. 2, 253-275. Anderson, T. F., Rappaport, C. & Muscatine, N. A. (1953). An%. Inst. Pmt. 84, 5-10. Bijlenga, R. K. L., Scraba, D. & Kellenberger, E. (1973). Virology, 56, 250-267. Bijlenga, R. K. L., van den Broek, R. & Kellenberger, E. (1974). Nature (London), 249, 825-827. Bijlenga, R. K. L., Aebi, U. & Kellenberger, E. (1976). J. Mol. Biol. 103, 469-498. Bishop, R. J. & Wood, W. B. (1976). ?‘i’iroZogy, 72, 244-254. Black, L. W. & Ahmed-Zadeh, C. (1971). J. Mol. Biol. 57, 71-92. Black, L. W. & Brown, D. T. (1976). J. Vi7iroZ. 17, 894-905. Castillo, C. J., Hsiao, C.-L., Coon, P. & Black, L. W. (1977). J. Mol. BioZ. 110, 585-601. Celis, J. E., Smith, J. D. & Brenner, S. (1973). Nature New BioZ. 241, 130-132. Coppo, A., Manzi, A., Pulitizer, J. F. & Takahashi, H. (1973). J. Mol. BioZ. 76. 61-87.
p24
BYPASS
MUTATION
Xl
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