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Genetic analysis of T4 tail fiber assembly
VIROLOGY
72, 244-254 (1976)
Genetic
Analysis
I. A Gene 37 Mutation ROBERT Division
of Biology,
of T4 Tail Fiber Assembly
that Allows Bypass of Gene 38 Function
J. BISHOP’ California
AND WILLIAM
Institute Accepted
of Technology, February
B. WOOD2 Pasadena,
California
91125
9,1976
T4 gene 38 function normally is required for assembly of the distal half of the phage tail fiber. From a double mutant with two amber mutations in gene 38, temperaturesensitive revertants have been isolated that produce plaques at 25” on host bacteria nonpermissive for amber mutants but do not produce plaques on any host at 42”. One such revertant, ts3813, has been shown to retain the two original amber mutations, and to produce none of the polypeptide coded by gene 38 (gp38) in nonpermissive host cells. In these cells at 25”, however, ts3813 produces levels of progeny phage and distal half fiber antigens that are 20-408 of those produced in wild-type phage infection, indicating that ts3813 allows a partial bypass of the normal gene 38 requirement. The site of the bypass mutation is near the promoter-distal end of gene 37, which codes for the major structural polypeptide of the distal half fiber, gp37. The properties of ts3813 suggest the possibility that gene 38 product may catalyze the noncovalent dimerization of two gp37 molecules to form the precursor of the distal half fiber. INTRODUCTION
The assembly of tail fibers and their attachment to the tail baseplate during bacteriophage T4 morphogenesis normally require the functions of eight phage genes (Wood and Bishop, 1973; Bishop, Conley, and Wood, 1974). Four of these genes, 34, 35,36, and 37, code for the structural polypeptides of the tail fiber (gp34, gp35, gp36, and gp37j3, whose molecular weights, structural relationships, and sequence of assembly are shown in Fig. 1. The remaining four genes code for the polypeptides gp63, gpwac, gp38, and gp57, which are ’ Present address: Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, California 94143. * Author to whom reprint requests should be sent. 3 Abbreviations used: gp, the polypeptide product of a phage gene (Casjens and King, 1975); P, as in 63P, the active form of a phage gene product (Mason and Haselkorn, 1972); am, amber, referring to suppressor-sensitive mutations; ts, temperature-sensitive; SDS, sodium dodecyl sulfate.
not structural components of the tail fiber or any of its precursors. The functions of these nonstructural proteins are of particular interest, and are not yet well understood. 63P catalyzes the attachment of tail fibers to the tail baseplate (Wood and Henninger, 1969). wacP is a component of the phage whiskers, slender filaments of 20 x 400 A that extend outward from the collar region at the base of the phage head (Dewey, Wiberg, and Frankel, 1974; Follansbee et al., 1974; Conley and Wood, 1975). This protein also is required for efficient attachment of tail fibers, and appears to interact transiently with 36P during the reaction. Since tail fiber attachment occurs readily in vitro, it has been possible to study the functions of 63P and wacP in some detail using isolated components (Wood et al., 1976; Wood and Conley, 1976). By contrast, the functions of 38P and 57P have proven difficult to investigate. As indicated in Fig. 1, 38P is required for the initial step of distal half fiber formation, in which two molecules of gp37 as244
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BYPASS
C
BC
OF
T4 GENE
BC’
FIG. 1. The sequence of steps in T4 tail fiber assembly. Numbers preceded by gp represent proteins coded by the corresponding genes. Dimensions and general appearance of structural intermediates are based on electron microscopy, and antigens A, B, C are determined by serum blocking assay (King and Wood, 1969; Ward et al., 1970). The molecular weights of the four structural proteins are indicated in parentheses (King and Laemmli, 1971; Ward and Dickson, 1971; Dickson, 1973). The molecular weights of the two nonstructural proteins are 26,000 for gp38 (King and Laemmli, 1971) and probably 3000-7000 for gp57 (R. 0. Herrmann, unpublished experiments).
semble into a rod-shaped structure that exhibits a class of antigens designated C antigens (Edgar and Lielausis, 1965; King and Wood, 1969; Beckendorf, 1973). 57P appears to act at three separate points in T4 assembly. In the tail fiber pathway it is required in addition to 38P for the first step in distal half fiber formation, and it is also necessary for the analogous dimerization ofgp34 to form the proximal half fiber precursor which carries the A antigens (Edgar and Lielausis, 1965; King and Laemmli, 1971; Ward and Dickson, 1971). In addition, 57P is required in tail assembly for the conversion of gp 12 to an active form which is the structural component of the short tail fibers that extend downward from the baseplate (Kells and Haselkorn, 1974). Although 38P function has been demonstrated in vitro, the instability of its activity in crude extracts and the low efficiency of the reaction have frustrated our attempts to study it biochemically (Wood and Bishop, 1973; R. J. Bishop, unpublished results). 57P activity has not yet been detected by in vitro complementation, despite many attempts in our laboratory and elsewhere. Consequently, we
245
38 FUNCTION
have taken a genetic approach to obtaining additional information on the functions of 38P and 57P, by isolating and studying mutants in which one of these proteins is no longer required. Analysis of mutations that allow bypass of normally required functions has shed light on the interactions of genetic control elements in both bacteria and bacteriophages (for example, see Butler and Echols, 1970; for reviews, see Reznikoff, 1972, and Herskowitz, 1973). We have taken the same approach in attempting to understand the interactions of proteins in a macromolecular assembly process. In this paper we report the isolation and characterization of phage T4 mutations that allow bypass of the need for 38P function. The accompanying paper (Revel, Herrmann, and Bishop, 1976) describes the isolation and characterization of bacterial host mutations that allow bypass of the need for phage 57P function. MATERIALS
AND
METHODS
Media and buffers. H broth, used for liquid culture of bacteria and bacteriophage, and EHA top and bottom agar, used for plating assays, were prepared as described by Steinberg and Edgar (1962). M9T, a minimal medium supplemented with 50 pg L-tryptophan/ml (Kellenberger and Sechaud, 1957) was used for preparing radioactively labeled infected-cell lysates. Buffers were the dilution medium described by King (1968) and a phosphate buffer used for serum blocking assays (SB buffer), prepared as described by Ward et al. (1970). Strains of bacteriophage and host bacteria. All phage amber (am) and temperature-sensitive (ts) mutants not described
in the text were derivatives of T4D from the Caltech collection and have been described elsewhere (Epstein et al., 1963; Beckendorf, Kim, and Lielausis, 1973; Luftig, Wood, and Okinaka, 1971). Multiple mutants were constructed by phage crosses carried out according to Steinberg and Edgar (1962). The rl mutant r48 was used as the tester phage in serum blocking assays. An rZZ deletion mutant (rdf41) was used in place of wild-type as the reference
246
phage order tester tants gene sults).
BISHOP
AND
in serum blocking experiments in to prevent interference with the phage assay. The host range muhG3 and hG7 carry mutations in 37 (S. Beckendorf, unpublished re-
WOOD
After 100 min of incubation at that temperature, chloroform was added and progeny were assayed on CR63 at 25”. Preparation of ‘Clubeled Zysutes. A saturated culture of B/5 cells in M9T medium was diluted 1:500 into fresh M9T medium and grown for 2.5 hr at 30”, centrifuged, and resuspended at 4 x lo8 cells per ml in the same medium. A 2-ml portion of this suspension was warmed to 30”, infected with phage at a multiplicity of 5 phage per cell, and aerated by agitation on a rotary shaker. After 18 min of incubation at 30”, 2 &i of uniformly 14C-labeled amino acid mixture (Schwarz-Mann) was added. At 26 min, 0.2 ml of 10% casamino acids was added. At 30 min the cells were chilled and centrifuged. The pellet was resuspended in 10 ~1 of 0.05 M Tris (pH 6.8) and lysed by freezing in a dry ice-ethanol bath and thawing at 30”. After addition of 25 pg of DNase and incubation for an additional 15 min at 37”, 100 ~1 of 0.05 M Tris (pH 6.8) containing 2% sodium dodecyl sulfate (SDS) and 2% /3-mercaptoethanol was added. Samples either were used for electrophoresis immediately or were stored at
E. coli host bacteria were used as follows: The K12 strain CR63 and the B strain O-11’ (both permissive for am mutants) for growth of phage stocks and for plating asays; S/6/5 and B/5 (nonpermissive for am mutants) for preparation of defective lysates and for selective plating of urn+ phage; G(h) (nonpermissive for both am and rZZ mutants) for selective plating of the um+rZZ+ tester phage in serum blocking assays; and C31 (resistant to host range mutants hG3 and hG7; S. K. Beckendorf, unpublished experiments) for selective plating of h+ phage. Mapping of phuge mutations. Phage crosses were carried out by the procedure of Steinberg and Edgar (1962) as modified by Beckendorf et al. (1973). All crosses were done at 25” in CR63 host bacteria. Total progeny were assayed by plating on CR63 at 25”. Selective assays for am+, h+, and ts+ progeny were carried out by plat-20”. ing on S/6/5 at 25”, C31 at 25”, and S/6/5 at Electrophoresis on SDS-polyucrylamide 42”, respectively. gels and autoradiography. Samples preTesting for complementution in liquid pared as described in the preceding section culture. Liquid complementation tests were heated in a boiling water bath for 2 were performed by a modification of the min and then electrophoresed in the disstandard phage cross, under conditions re- continuous SDS-buffer system described strictive for both am and ts mutants. The by Laemmli (1970), as modified by Dickson procedure was designed to avoid exposure (1973) and adapted for use with slab gels as of infected cells to KCN at high temperadescribed by Studier (1972). Gels were ture, which results in loss of infective cen- fixed and stained for l-2 hr in an aqueous ters and lowered burst sizes. A saturated solution of 50% (w/v) trichloroacetic acid culture of B/5 was diluted 1:lOOO in H and 0.2% (w/v) Coomassie brilliant blue broth and grown for 2.5 hr at 30”. The cells (Mann). Gels were destained for 12-20 hr were centrifuged and resuspended at 4 x in 10% anhydrous methanol, 10% acetic acid. The gels were dried directly onto lo8 cells per ml in fresh H broth containing 0.004 M KCN at 25”. A sample of the sus- sheets of Whatman No. 3 filter paper and autoradiographed on Kodak no-screen Xpended cells was then mixed with an equal volume of the same H broth containing the ray film by the procedure of Fairbanks et desired phage to give a multiplicity of 7.5 al. (1965) as modified by Dickson (1973). Preparation of defective lysates for deof each phage per cell. After 6 min, anti-T4 antiserum was added to neutralize unadterminations of burst sizes and antigen levels. A saturated culture of either B/5 or sorbed phage, and incubation was continued for an additional 6 min at 25”. The CR63 was diluted 1:lOOO into H broth and cells were then diluted 4 x 104-fold into H grown at either 30 or 41” to 4 x lo6 cells per broth without KCN, prewarmed to 39.5”. ml. Small 5-ml cultures of these cells were
BYPASS
OF
T4 GENE
then infected at a multiplicity of 4 phage per cell. At 5 and 40 min after infection for 30” cultures, and 3 and 22 min after infection for 41” cultures, samples were withdrawn into screw-cap vials and shaken with chloroform. Viable phage were assayed by plating on O-11’ host cells, and A and BC antigens were determined by the endpoint serum blocking assay of Ward et al!. (1970). RESULTS
Rationale
and Plan
of Experiments
In order to determine whether the normal requirement for gene 38 function in tail -fiber assembly can be bypassed as a result of compensating mutation in another gene, we have isolated and characterized apparent revertants (pseudorevertants) of mutant T4 that carry two am mutations in gene 38. First, the gene 38 double mutant was plated on S/6/5, a restrictive host for am mutants. Apparent revertants were isolated, and one was selected for intensive study. Second, genetic and biochemical tests were performed to establish that the revertant was a pseudorevertant; that is, it still carried the original gene 38 defect in nonpermissive hosts. Third, backcrossed phage carrying the second-site mutation, both alone and together with a gene 38 am mutation, were examined for their production of phage progeny and tail fiber antigen on both permissive and nonpermissive hosts. Fourth, the genetic location of the second-site mutation was determined. Isolation of Pseudorevertants amB262:C290
from
When T4 carrying two am mutations in gene 38 (amB262:C290) were plated on S/ 6/5 bacteria at a level of about 10s phage per plate at 25”, plaques appeared at a frequency of about 10e7. The plaques were generally turbid and heterogeneous in size. Their frequency was too high to be explained either by double reversion of the two am mutations or by mutation to a phage-coded am suppressor (Wilson and Kells, 1972). This result suggested the occurrence of second-site mutations that allowed either restoration or bypass of 38P
247
38 FUNCTION
function. In order to have a marker for genetic analysis of such a second-site mutation, a large number of the pseudorevertant plaques were screened for temperature sensitivity. The frequency of ts clones among the pseudorevertants varied from 0.2-0.4% depending on the amB262:C290 stock from which the revertants were isolated. Since most of the pseudorevertants grew very poorly on the nonpermissive host S/6/5, ts isolates were screened for ability to grow well enough at 25” for further study. One revertant, ts3813, was chosen for the characterization described below. Several other ts pseudorevertants, although not studied in detail, have been tested for degree of linkage of the secondsite mutation to ts3813. Pseudorevertant ts3813 Still Carries Original Gene 38 am Mutations
To
establish
genotype as the pseudorevertant was crossed to wild-type phage and the progeny were screened for am phenotype. The appearance of am phage among such progeny requires recombination between the ts site and one of the am mutations. Since amB262 and amC290 are closely linked (about 1 map unit), the frequency of such recombinants provides an estimate of the genetic distance between the ts and the am mutations. About 0.5% of the total progeny exhibited the am phenotype and were shown by additional crosses to carry both amB262 and amC290. This result established the genotype of the pseudorevertant as ts3813: amB262:C290 and showed that the ts mutation is closely linked to the am sites. The reciprocal recombinant, ts3813:am+, occurred less frequently but was isolated also. To establish that ts3813 rather than some other mutation was responsible for bypass of the gene 38 mutation, ts:am double mutants were reconstructed by crossing ts3813 with both amB262 and amC290. The ts3813:am double mutants isolated from these crosses both plate on S/ 6/5 with high efficiency. The reconstructed double mutant ts3813:amB262 was used in the characterization of phenotype described in the following sections. ts3813:amB262:C290,
its
the
248
BISHOP AND WOOD
The Pseudorevertant ts3813:amB262:C290 Remains 38-Defective in Nonpermissive Hosts An am mutation in gene 38 could be suppressed in either of two general ways by a second-site mutation. Mutation of a T4 tRNA gene to a phage-coded suppressor could allow gp38 synthesis in nonpermissive hosts. Alternatively, the second-site mutation could somehow obviate the necessity for 38P function in tail fiber assembly. Both genetic and biochemical evidence support the latter alternative as the explanation for ts3813 pseudoreversion. Several observations argue against mutation to a phage-coded am suppressor. First, the frequency of pseudoreversion is higher by a factor of about lo4 than the previously observed frequency of T4 am suppressor mutations (Wilson and Kells, 1972). Second, the ts3813 mutation is closely linked to gene 38, whereas all the known T4 tRNA genes are separated from gene 38 by ‘half of the genome (Wilson, Kim and Abelson, 1972; Wood, 1974a). Third, construction and analysis of various ts3813:am mutants showed that ts3813 does not suppress am mutations in several genes other than 38. None of the following double mutants, constructed by crossing ts3813 with the indicated am mutants, showed better growth in nonpermissive hosts than the am mutants alone (data not shown): ts3813:amB25(gene 34), ts3813: amA (gene 34), ts3813:amB252 (gene 35), ts3813:amXl(gene 361, and ts3813: amE198cgene 57). Direct biochemical evidence against suppression of the am mutations in gene 38 was obtained by electrophoretic analysis of phage-coded polypeptides radioactively labeled following infection of the nonpermissive host B/5 (Fig. 2). 14C-labeled lysates of cells infected with T4 wildtype (+ +), amB262:C290 (3%), ts3813: amB262:C290 (ts:38-), and amN52 (37-) were electrophoresed on polyacrylamide gels in the presence of SDS, and phage polypeptides were visualized by autoradiography of the dried gels. As shown by previous investigators (King and Laemmli, 1973; Dickson, 1973; Vanderslice and Yegian, 19741, gp38 can be identified as a
FIG. 2. Autoradiogram of YJ-labeled lysates of cells infected with T4D wild-type, amB.262, ts3813:amB262, and amX52. ‘YXabeled lysates were prepared as described in Materials and Methods, and electrophoresed on a slab gel of 10% polyacrylamide containing 1% SDS. Each sample contained approx lo5 disintegrations per minute. The gel was fixed, stained, destained, dried, and autoradiographed as described in Materials and Methods. The additional bands seen in the 38- lysate are unexplained; they were not found in other similar experiments.
26,000 dalton polypeptide that is present in lysates of cells infected with wild-type phage but is missing from lysates of cells infected with gene 38 am mutants. In several comparisons of wild-type with amB262:C290, the presence or absence of this band was found to be the only repro-
BYPASS
OF
T4
GENE
ducible difference between the two lysates. The gel pattern (Fig. 2) indicates that ts3813:amB262:C290-infected cells contain no detectable gp38 under these conditions, although infectious phage are being assembled. The amN52 (37-)-infected cells lack gp37 (120,000 daltons) and produce a reduced amount of gp38 relative to wildtype. This reduction is known to be due to a polar effect of the gene 37 am mutation on expression of the neighboring gene 38 (King and Laemmli, 1971). These results indicate that the ts3813 mutation somehow permits a bypass of the normal requirement for 38P in tail fiber assembly. Production of Tail Fiber Antigens and Progeny Phage by a Gene 38 am Mutant That Carries ts3813 Conceivably, ts3813 could allow bypass of 38P function in either of two ways. It could specifically affect the 38P-mediated step in tail fiber assembly, allowing it to proceed in the absence of 38P, or it could render T4 particles infectious in the absence of tail fibers. In view of earlier results, the latter possibility seemed unlikely. Mutants of T4 able to infect without tail fibers were not found in an earlier search that would have detected such phage at a frequency of lo-lo (Wilson and Kells, 19721, and these mutants would be unexpected due to the necessity of tail fibers not only for attaching phage to host cells, but probably also for triggering subsequent baseplate attachment and sheath contraction (Yamamoto and Uchida, 1975). Moreover, ts3813 bypasses only am mutations in gene 38, and not am mutations in other tail fiber genes. To determine how ts3813 affects tail fiber assembly in the presence and absence of 38P, production of tail fiber antigens and phage progeny was measured under various conditions. Lysates were prepared using permissive (CR63) and nonpermissive (B/5) host cells infected at 30 and 41” with wildtype amB262, ts3813, and ts3813:amB262 phage. The lysates were assayed for levels of A and BC tail fiber antigens as well as for viable phage titer. Results obtained at 30 and 41” are summarized in Table la and b, respectively.
249
38 FUNCTION TABLE
1
PRODUCTION OF TAIL FIBER ANTIGENS AND INFECTIOUS PI-IAGE IN WILD TYPE- AND MUTANTINFECTED CELLS AT (a) 30” AND (b) 41% Phage
Host
Burst size IPha!&
(phageequivalents/cell)’
(a) 30 ++ amB262 ts3813 ts3813: amB262 (b) 41” ++ amB262 ts3813 ts3813: amB26i
,
!
Percentage of
CR63 B/5 CR63 B/5 CR63 B/5 CR63 B/5
100 48 111 0.004 136 41 44 21
188 137 248 228 238 283 148 249
390 314 473 11 344 601 138 109
CR63 B/5 CR63 B/5 CR63 B/5 CR63 B/5
26 17 16 0.01 0.01 0.26 co.7 CO.28
71 69 88 116 104 140 91 133
75 160 124 5.6 4.2 11.9 2.4 2.0
1BC+a+ntigenb
100 100 93 1.9 78 91 41 18
100 100 141 2.1 3.8 3.5 2.4 0.6
a Preparation of lysates and measurements of A and BC antigens were carried out as described in Materials and Methods. All values are averages of two independent experiments. The phage designated ++ in these experiments is the rZZ deletion mutant rdf41. b BC antigen levels produced by mutants were normalized to those of wild-type by using A antigen as an internal standard, since none of the mutants employed here are defective in A antigen production. Values for BC antigen as percent of wild-type level were calculated using the formula: BC/A (mutant)/BC/A (+ +) x 100%. The mean standard deviation for the duplicate results averaged in this column was 226% of the values shown. c Antigen levels are expressed in phage.equivalents as defined by Ward et al. (1970).
At 30” (Table la) on the nonpermissive host, the amB262 mutation greatly reduces the production of both BC antigens and viable phage relative to wild-type infected cells. The ts3813 mutation at least partially overcomes the gene 38 defect as measured by production of both antigen and viable phage, although the levels are lower than in wild-type infected cells. In the permissive host, ts3823:amB262 also
250
BISHOP
produces somewhat lower antigen and viable phage levels than does wild-type. At 30”, the single mutant ts3813 produces normal levels of BC antigen and viable phage on both CR63 and B/5 hosts. At 41” (Table lb) amB262 displays the same defective phenotype as at 30”: both BC antigen and viable phage production are drastically reduced in the nonpermissive host. At 41”, however, the ts3813 mutation does not restore either antigen or Mutants that carry phage production. ts3813 produce less than 4 and 1% of the wild-type levels of BC antigen and phage progeny, respectively, on either CR63 or B/ 5 host cells. Therefore, the bypass mutation also leads to temperature sensitivity of gp37 assembly. The ts3813 mutation does not significantly increase the heat lability of phage produced at low temperature. Both ts3813 and ts3813:amB262 phage incubated at 39.5 and 45” in dilution medium containing 1% SDS and 1% P-mercaptoethanol were inactivated at about the same rate as wildtype phage (data not shown).
AND
WOOD TABLE
2
RESULTS OF LIQUID COMPLEMENTATION TESTS WITH t.s3813:amB262:C290 AND am MUTANTS DEFECTIVE IN GENES 27, 37, AND 38a Infecting ++ amB262 (38-j amN52 (ST-) amNl20 (27-) ts3813:amB262:C290 amB262 + amNl20 amN52 + amNl20 ts3813:amB262:C290 ts3813:amB262:C290 ts3813:amB262:C290
phage
+ amNl20 + amB262 + amN52
Burst size (phagekell) 2.3b 0.02 0.01 0.01 0.09 23.8 18.1 43.3 0.04 0.15
a Complementation tests were carried out at 39.5” with B/5 host cells in liquid culture as described in Materials and Methods. b Burst sizes obtained with wild-type phage at this temperature were variable, ranging from 2-50 phage per cell.
can be complemented by a&120(27-1, but not by amB262(38-) or amN52(37-). An additional control experiment (not shown) under comparable conditions The ts3813 Mutation Maps near the Proshowed that despite the polar effect of moter-Distal End of Gene 37 amN52 on gp38 production (Fig. 21, The genetic location of ts3813 was deteramN52 and single gene 38 am mutants mined in two steps. First, the mutation complement to give burst sizes in the was shown to be in gene 37 by complemenrange of wild-type. Therefore, the ts3813 tation tests at nonpermissive temperature defect is in gene 37, which codes for the with the ts3813 mutant and appropriate major structural polypeptides of the distal am mutants. Second, the map position of half tail fiber (Fig. 1). ts3813 within gene 37 was determined by a To determine the map position of ts3813 series of 3-factor crosses. within gene 37, ts3813:amB262 and The previously observed linkage of ts3813:amC290 were crossed with phage ts3813 to am mutations in gene 38 and the carrying various ts mutations in genes 37 and 38 and/or host range mutations in lack of BC antigen production at high temperature in mutants carrying ts3813 sug- gene 37 (hG3 and hG7). Examples of three gested that this mutation might be in gene such crosses are diagrammed in Fig. 3. 37, which is adjacent to gene 38 on the T4 Progeny of ts:am x ts crosses were plated on CR63 bacteria at high temperature to genetic map. Accordingly, ts3813:amB262: select ts+ recombinants, which then were C29O was tested for genetic complementation of amN52(37-1, amB262(38-1, and as scored for the unselected am marker to a control amNl20(27-), at 39.5” in B/5 determine the position of ts sites relative host cells. The results of this test are to amB262. Progeny of ts:am x h crosses shown in Table 2. Control cultures in- were plated on C31 bacteria at high temfected with each of the mutants alone gave perature to select ts+h+ recombinants, which were then scored for the unselected burst sizes less than 5% of the wild-type control. The results of the pair-wise in- am marker as above. The results of these crosses, tabulated in Table 3, show that fections show that ts3813:amB262:C290
BYPASS
OF
T4
GENE
ts3813 maps close to the host range mutations hG3 and hG7, in the region of gene
C290. In these isolates, the second-site ts mutations were unlinked to gene 38 and did not appear to be in any of the genes required for tail fiber assembly. Although of considerable interest, these mutations were more difficult to work with, in that they bypassed 38 function less effectively than ts3813, and in some cases produced
37 that codes for the C-terminal portion of the gp37 molecule (Fig. 4). We conclude that gp37 can be mutationally altered in such a way that it no longer requires 38P for assembly into a functional tail fiber. Mapping experiments also were carried out on a number of other independently isolated ts pseudorevertants of amB262: A
ts3ei3
A
lS38/3
.
d
251
38 FUNCTION
4 4 \5
!‘JB
(I
ts38I3
I
IIl,l p +
:-------L
__.
,__--- .__..
.
-
tsN36
(a)
lb1
FIG. 3. Diagrammatic factor crosses involving
___..-A -
Gene 37
tsA3l
hG3
representation ts3813.
OF ts3813:amB262
Cross
CC)
of three-
AND ts3813:amC290 Recombinants lected
x x x x x x x
tsCT32(38-I tsN36(37-) tsB78(37-I tsN2(37-) t&20(37-) tsA31(37-) tsC9(49-)
ts+ ts+ ts+ ts+ ts+ ts+ ts+
ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262
x tsCZ’32(38-1 x tsN36(37-) x tsB78(37-) x tsN2(37-) x t&20(37-) x tsA31(37-) x tsC9(49-1
ts+ ts+ ts+ ts+ ts+ ts+ ts+
ts3813:amB262 ts3813:amB262
x hG3
ts+h+ ts+h+
hG3
x hG7
h+
hG3 hG3 hG7 hG7
x x x x
ts+h+ ts+h+ ts+h+ ts+h+
tsN2(37-)” tsCT32(38-I” tsN2(37-)” tsCT32(38-)”
II
6% 9’ i
II Gene 38
4. Map of ts and h markers in genes 37 and 38. Relative distances are estimated from the data in Table 3 and earlier mapping experiments (Beckendorfet al., 1973; Beckendorf, unpublished results).
ts3813:amC290 ts3813:amC290 ts3813:amC290 ts3813tamC290 ts3813:amC290 ts3813:amC290 ts3813:amC290
x hG7
4 (3% &$ ,+ \S‘ 0 0 fi (f
FIG.
TABLE RESULTS
,$y@~
3 CROSSES
se-
WITH ts AND h MUTANT@ am/am+’ Percentage recombinatiox? ___---2.0 0.6 1.4 4.3 2.6 1.6 31.9
3.1 1.0 1.4 4.4 2.7 1.6 39.0
2.61 0.08 0.13 0.11 0.05 2.12
1.7 2.2
0.13 0.21
eo.1 10.3 5.2 9.8 4.7
0 Crosses were carried out as described in Materials and Methods, using CR63 bacteria SO that am markers were unselected among the progeny in all crosses. * Percentage recombination values are calculated as 200% x recombinants/total progeny. I’ The am/am+ ratio was determined by stabbing at least 100 recombinant plaques with sterile pins, which then were stabbed into a lawn of S/6/5 bacteria and then into a second lawn of CR63 bacteria. Plaques that produced a clear spot on CR63 but not on S/6/5 were scored as am. Plaques that failed to produce a spot on CR63 were not scored, and plaques that produced spots on both hosts were scored as am+. ” ts x h crosses gave higher frequencies of recombinants than expected from ts X ts crosses.
252
BISHOP
AND
labile phage particles that rapidly lost viability during storage. These pseudorevertants have not yet been investigated further.
WOOD
showed that gp38 is not a structural component of either the completed tail fiber or any of its precursors (King and Laemmli, 1971; Dickson, 1973). These results suggested that 38P acts catalytically in tail DISCUSSION fiber assembly, in agreement with earlier A number of earlier genetic studies sug- evidence from gene dosage experiments gested an interaction between 38P and the (Snustad, 1968). If so, what could be the C-terminal portion of gp37 during tail fi- mechanism of 38P action? Two general ber assembly. Both proteins are required possibilities can be suggested. for tail fiber formation (Epstein et al., (1) 38P could catalyze a covalent modifi1963; Eiserling et al., 1967) and for normal cation ofgp37 tliat promotes dimerization. tail fiber antigen production (Edgar and The molecular weights of gp37 before and Lielausis, 1965). In mixed infections of the after dimerization are the same as judged closely related phages T2 and T4, these by SDS polyacrylamide gel electrophoretwo proteins are functional only in the sis, but the uncertainty in these experihomologous combinations, i.e., both pro- ments is about *3%. Therefore, they canteins from T2 or both from T4. Such mixed not rule out either proteolytic cleavage of a infections produce no viable progeny few amino acid residues from one end of phage if each of the parental phage strains gp37 or modification of amino acid side carries a defect in one of the two genes 37 chains by 38P. A possibly analogous example of the latter process is the enzymic and 38 (Russell, 1967, 1974); for example, of proline residues in procolnonpermissive cells infected with a T2 hydroxylation gene 38 am mutant and a T4 gene 37 am lagen to increase its helix-coil transition mutant produce no progeny. In subsequent temperature and thereby allow triple-hestudies Beckendorf (1973) showed that this lix formation (Berg and Prockop, 1973; RoT2-T4 incompatibility is limited to gene 38 senbloom et al., 1973). However, the propand the promoter-distal end of gene 37, erties of the ts3813 mutant suggest that corresponding to the C-terminal portion of drastic 38P-catalyzed covalent modificagp37. Within the incompatibility region, tion of gp37 is not necessary for dimerizarecombination between T2 and T4 does not tion, since the requirement for 38P can be occur, and there is no detectable homology bypassed by what is probably a single between T2 and T4 DNA as shown by ex- amino acid substitution near one end of the gp37 monomer. If 38P does catalyze a amination of T2-T4 heteroduplex DNA covalent change, this change must promolecules in the electron microscope dimerization quite specifically, (Beckendorf, Kim, and Lielausis, 1973). mote The C-terminal end ofgp37 carries the tail rather than generally as in procollagen fiber recognition site for bacterial cell sur- hydroxylation. (2) An alternative possibility is that 38P face components and is largely responsible association of for determining the host range of the catalyzes the noncovalent phage. Phages T2 and T4, although closely two gp37 molecules, perhaps by tranrelated otherwise, have diverged considersiently stabilizing an intermediate comably in host range, and their tail fibers plex in the dimerization reaction. Gene 37 recognize quite different cell surface com- am mutants produce no C antigen, even is near the proponents (Jesaitis and Goebel, 1953). The when the am mutation moter-distal end of the gene so that long apparent codivergence of gene 38 function fragments of gp37 are synthesized. This with the region of gene 37 that determines host range suggests that 38P interacts observation suggests that dimerization must be initiated by interaction of the Cwith the C-terminus of gp37 during tail fiber assembly, to promote the dimerizaterminal ends of two gp37 molecules. If the resulting initial complex is either unstable tion of gp37 into the rod-shaped precursor (e.g., conformationally strained) or imhalf fiber that carries the C antigens. The question of how 38P acts became probable (e.g., one of many isoenergetic configurations), then 38P could increase more intriguing when additional studies
BYPASS
OF
T4 GE NE
the rate of dimerization by transiently combining with and specifically stabilizing the complex. According to this model, the amino acid substitution in ts3813 would likewise stabilize the initial complex. Recent experiments of Kikuchi and King (1975a, 197513, 1975c) have demonstrated elegantly that almost all of the thermodynamically favorable subunit associations in T4 tail assembly fail to occur in the absence of specific structural components, formed in previous assembly steps, which presumably provide nucleation sites that help to overcome activation energy barriers to subunit assembly. In tail fiber assembly, a similar function may be served by the nonstructural protein 38P. Catalysis of noncovalent association by a nonstructural protein also has been suggested as the mechanism of action for 63P in tail fiber attachment to the baseplate (Wood and Bishop, 1973; Wood et al., 1976), and as a possible general phenomenon in macromolecular assembly (Wood, 197433). Whatever the mechanism of 38P function, it can be partially bypassed by a mutational alteration in the structural protein gp37. As a consequence of the bypass mutation, however, assembly of the distal half of the tail fiber is blocked at higher growth temperatures. Most of the other bypass mutants isolated, although not studied in detail, appeared to grow poorly under all conditions, and some of them produced unstable phage particles as well. These observations suggest that gene 38 function cannot be bypassed without considerable cost in fitness to the phage; consequently, it is not surprising that this gene has been retained in the course of evolution. ACKNOWLEDGMENTS These studies were supported by a research grant from the U.S. Public Health Service (AI092381 to W.B. W., and a postdoctoral fellowship from the U.S. Public Health Service (GM40392) to R.J.B. REFERENCES BECKENWRF, S. K. (1973). Structure of the distal half of the bacteriophage T4 tail fiber. J. Mol. Biol. 73, 37-53. BECKENDORF, S. K., KIM, J.-S., and LIELAUSIS, I. (1973). Structure of bacteriophage T4 genes 37 and
38 FUNCTION
253
38. J. Mol. Biol. 73, 17-35. BERG, R. A., and PROCKOP, D. J. (19731. Purification of [i4C]protocollagen and its hydroxylation by prolyl-hydroxylase. Biochemistry 12, 3395-3401. BISHOP, R. J., CONLEY, M. P., and WOOD, W. B. (1974). Assembly and attachment of bacteriophage T4 tail fibers. J. Supramolec. Structure 2, 196-201. BUTLER, B., and ECHOLS, H. (1970). Regulation of bacteriophage A development by gene N; properties of a mutation that bypasses N control of late protein synthesis. Virology 40, 212-222. CAEJENS, S., and KING, J. (1975). Virus assembly. Anna. Rev. Biochem. 44, 555-611. CONLEY, M. P., and WOOD, W. B. (1975). Bacteriophage T4 whiskers; A rudimentary environmentsensing device. Proc. Nat. Acad. Sci. USA 72, 3701-3705. DEWEY, M. J., WIBERG, J. S., and FRANKEL, F. R. (1974). Genetic control of whisker antigen of bacteriophage T4D. J. Mol. Biol. 84, 625-634. DICKSON, R. C. (1973). Assembly ofbacteriophage T4 tail fibers IV. Subunit composition of tail fibers and fiber precursors. J. Mol. Biol. 79, 633-647. EDGAR, R. S., and LIELAUSIS, I. (1965). Serological studies with mutants of phage T4D defective in genes determining tail fiber structure. Genetics 52, 1187-1200. EISERLING, F. A., BOLLE, A., and EPSTEIN, R. H. (1967). Electron microscopic studies of the structure of mutants of bacteriophage T4D defective in tail fiber genes. Virology 33, 405-412. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KEI~ LENBERGER, E., BOY DE LA TOUR, E., CHEVALLEY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375394. FAIRBANKS, G., LEVINTHAL, C., and REEDER, R. H. (1965). Analysis of Ci4-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20, 393-399. FOLLANSBEE, S. E., VANDERSLICE, R. W., CHAVEZ, L. G., and YEGIAN, C. D. (1974). A new set of absorption mutants of bacteriophage T4D: Identification of a new gene. Virology 58, 180-199. HERSKOWITZ, I. (1973). Control of gene expression in bacteriophage lambda. Annu. Rev. Genetics 7, 289-324. JESAITIS, M. A., and GOEBEL, W. F. (1953). The interaction between T4 phage and the specific lipocarbohydrate of phase II Sh. sonnei. Cold Spring Harbor Symp. Quant. Biol. l&205-208. KELLENBERGER, E., and SECHAUD, J. (1957). Electron microscopical studies of phage multiplication. II. Production of phage-related structures during multiplication of phages T2 and T4. Virology 3, 256-274.
254
BISHOP
AND WOOD
S., and HASELKORN, R. (1974). Bacteriophage T4 short tail fibers are the product of gene 12. J.
KELLS,
Mol. Biol. 83, 473-485. KIKUCHI, Y., and KING,
J. (1975a). Genetic control of T4 baseplate morphogenesis. I. Sequential assembly of the major precursor, in uiuo and in uitro.
J. Mol. KIKUCHI,
Biol.
99, 645-672. and KING, J.
Y., (1975b). Genetic control of T4 baseplate morphogenesis. II. Mutants unable to form the central part of the baseplate. J.
Mol. Biol. 99, 673-694. KIKUCHI, Y., and KING,
J. (1975c). Genetic control of T4 baseplate morphogenesis. III. Formation of the central plug and overall assembly pathway. J. Mol. Biol. 99, 675-716. KING, J. (1968). Assembly of the tail of bacteriophage T4. J. Mol. Biol. 32, 231-262. KING, J., and LAEMMLI, U. K. (1971). Polypeptides of the tail fibers of bacteriophage T4. J. Mol. Biol. 62, 465-477. KING, J., and LAEMMLI, U. K. (1973). Bacteriophage T4 tail assembly: Structural proteins and their genetic identification. J. Mol. Biol. 75, 315-337. KING, J., and WOOD, W. B. (1969). Assembly of bacteriophage T4 tail fibers: The sequence of gene product interaction. J. Mol. Biol. 39, 583-601. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LUFTIG, R. B., WOOD, W. B., and OKINAKA, R. (1971). Bacteriophage T4 morphogenesis: On the nature of gene Is-defective heads and their role as intermediates, J. Mol. Biol. 57, 555-573. MASON, W. S., and HASELKORN, R. (1972). Product of T4 gene 12. J. Mol. Biol. 66, 445-469. REVEL, H. R., HERRMANN, R., and BISHOP, R. J. (1976). Genetic analysis of T4 tail fiber assembly. II. Bacterial host mutations that bypass the requirement for gene 57 function. VirologY 72, 255-265. REZNIKOFF, W. S. (1972). The operon revisited. Annu. Rev. Genetics 6, 133-156. ROSENBLOOM, J., HARSCH, M., and JIMENEZ, S. (1973). Hydroxyproline content determines the denaturation temperature of chick tendon collagen. Arch. Biochim. Biophys. 158, 478-484. RUSSELL, R. L. (1967). Speciation among the T-even bacteriophages. Ph.D. dissertation, California Institute of Technology, Pasadena, Calif. RIJ~~ELL, R. L. (1974). Comparative genetics of the T-even bacteriophages. Genetics 78, 967-988. SNUSTAD, D. P. (1968). Dominance interactions in Escherichia coli cells mixedly infected with bacteriophage T4D wild-type and amber mutants and their possible implications as to type of gene product function: Catalytic vs. stoichiometric. Virol-
ogy 35, 550-563. C. M., and EDGAR, R. S. (1962). A critical test of a current theory of genetic recombination in bacteriophage. Genetics 47, 187-208. STUDIER, F. W. (1972). Bacteriophage T7. Science STEINBERG,
176, 367-376. VANDERSLICE,
R. W., and YEGIAN, C. D. (1974). The identification of late bacteriophage T4 proteins on sodium dodecyl sulfate polyacrylamide gels. Virology 60, 265-275. WARD, S., LUFTIG, R. B., WILBON, J. H., EDDLEMAN, H., LYLE, H., and WOOD, W. B. (1970). Assembly of bacteriophage T4 tail fibers. II. Isolation and characterization of tail fiber precursors. J. Mol. Biol. 54, 15-31. WARD, S., and DICKSON, R. C. (1971). Assembly of bacteriophage T4 tail fibers. III. Genetic control of the major tail fiber polypeptides. J. Mol. Biol. 62, 479-492. WILSON,
J. H., and KELLS, S. (1972). Bacteriophage T4 transfer RNA. I. Isolation and characterization of two phage-coded nonsense suppressors. J. Mol.
Biol. 69, 39-56. WILSON, J. H., KIM,
J.-S., and ABELSON, J. N. (1972). Bacteriophage T4 transfer RNA: III. Clustering of the genes for the T4 transfer RNA%. J.
Mol. WOOD,
Biol.
Mol.
Biol.
71, 547-556.
W. B. (1974a). Bacteriophage T4. In “Handbook of Genetics” (R. C. King, ed.), Vol. I, pp. 327331. Plenum Press, New York. WOOD, W. B. (1974b). Undelivered summary remarks for the 1974 Squaw Valley Meeting on Assembly Mechanisms. J. Supramolec. Structure 2, 512-514. WOOD, W. B., and BISHOP, R. J. (1973). Bacteriophage tail fibers: Structure and assembly of a viral organelle. In “Virus Research” (C. F. Fox and W. S. Robinson, eds.), pp. 303-324. Academic Press, New York. WOOD, W. B., and CONLEY, M. P. (1976). Attachment of tail fibers in bacteriophage T4 assembly. III. Role of the phage whiskers. Submitted for publication. WOOD, W. B., and HENNINGER, M. (1969). Attachment of tail fibers in bacteriophage T4 assembly: Some properties of the reaction in vitro and its genetic control. J. Mol. Biol. 39, 603-618. WOOD, W. B., CONLEY, M. P., LYLE, H. L., and DICKSON, R. C. (1976). Attachment of tail fibers in bacteriophage T4 assembly. II. Purification, properties, and site of action of the catalytic protein 63P. Submitted for publication. YAMAMOTO, M., and UCHIDA, H. (1975). Organization and function of the tail of bacteriophage T4. II. Structural control of the tail contraction. J. 92, 207-223.
72, 244-254 (1976)
Genetic
Analysis
I. A Gene 37 Mutation ROBERT Division
of Biology,
of T4 Tail Fiber Assembly
that Allows Bypass of Gene 38 Function
J. BISHOP’ California
AND WILLIAM
Institute Accepted
of Technology, February
B. WOOD2 Pasadena,
California
91125
9,1976
T4 gene 38 function normally is required for assembly of the distal half of the phage tail fiber. From a double mutant with two amber mutations in gene 38, temperaturesensitive revertants have been isolated that produce plaques at 25” on host bacteria nonpermissive for amber mutants but do not produce plaques on any host at 42”. One such revertant, ts3813, has been shown to retain the two original amber mutations, and to produce none of the polypeptide coded by gene 38 (gp38) in nonpermissive host cells. In these cells at 25”, however, ts3813 produces levels of progeny phage and distal half fiber antigens that are 20-408 of those produced in wild-type phage infection, indicating that ts3813 allows a partial bypass of the normal gene 38 requirement. The site of the bypass mutation is near the promoter-distal end of gene 37, which codes for the major structural polypeptide of the distal half fiber, gp37. The properties of ts3813 suggest the possibility that gene 38 product may catalyze the noncovalent dimerization of two gp37 molecules to form the precursor of the distal half fiber. INTRODUCTION
The assembly of tail fibers and their attachment to the tail baseplate during bacteriophage T4 morphogenesis normally require the functions of eight phage genes (Wood and Bishop, 1973; Bishop, Conley, and Wood, 1974). Four of these genes, 34, 35,36, and 37, code for the structural polypeptides of the tail fiber (gp34, gp35, gp36, and gp37j3, whose molecular weights, structural relationships, and sequence of assembly are shown in Fig. 1. The remaining four genes code for the polypeptides gp63, gpwac, gp38, and gp57, which are ’ Present address: Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, California 94143. * Author to whom reprint requests should be sent. 3 Abbreviations used: gp, the polypeptide product of a phage gene (Casjens and King, 1975); P, as in 63P, the active form of a phage gene product (Mason and Haselkorn, 1972); am, amber, referring to suppressor-sensitive mutations; ts, temperature-sensitive; SDS, sodium dodecyl sulfate.
not structural components of the tail fiber or any of its precursors. The functions of these nonstructural proteins are of particular interest, and are not yet well understood. 63P catalyzes the attachment of tail fibers to the tail baseplate (Wood and Henninger, 1969). wacP is a component of the phage whiskers, slender filaments of 20 x 400 A that extend outward from the collar region at the base of the phage head (Dewey, Wiberg, and Frankel, 1974; Follansbee et al., 1974; Conley and Wood, 1975). This protein also is required for efficient attachment of tail fibers, and appears to interact transiently with 36P during the reaction. Since tail fiber attachment occurs readily in vitro, it has been possible to study the functions of 63P and wacP in some detail using isolated components (Wood et al., 1976; Wood and Conley, 1976). By contrast, the functions of 38P and 57P have proven difficult to investigate. As indicated in Fig. 1, 38P is required for the initial step of distal half fiber formation, in which two molecules of gp37 as244
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
BYPASS
C
BC
OF
T4 GENE
BC’
FIG. 1. The sequence of steps in T4 tail fiber assembly. Numbers preceded by gp represent proteins coded by the corresponding genes. Dimensions and general appearance of structural intermediates are based on electron microscopy, and antigens A, B, C are determined by serum blocking assay (King and Wood, 1969; Ward et al., 1970). The molecular weights of the four structural proteins are indicated in parentheses (King and Laemmli, 1971; Ward and Dickson, 1971; Dickson, 1973). The molecular weights of the two nonstructural proteins are 26,000 for gp38 (King and Laemmli, 1971) and probably 3000-7000 for gp57 (R. 0. Herrmann, unpublished experiments).
semble into a rod-shaped structure that exhibits a class of antigens designated C antigens (Edgar and Lielausis, 1965; King and Wood, 1969; Beckendorf, 1973). 57P appears to act at three separate points in T4 assembly. In the tail fiber pathway it is required in addition to 38P for the first step in distal half fiber formation, and it is also necessary for the analogous dimerization ofgp34 to form the proximal half fiber precursor which carries the A antigens (Edgar and Lielausis, 1965; King and Laemmli, 1971; Ward and Dickson, 1971). In addition, 57P is required in tail assembly for the conversion of gp 12 to an active form which is the structural component of the short tail fibers that extend downward from the baseplate (Kells and Haselkorn, 1974). Although 38P function has been demonstrated in vitro, the instability of its activity in crude extracts and the low efficiency of the reaction have frustrated our attempts to study it biochemically (Wood and Bishop, 1973; R. J. Bishop, unpublished results). 57P activity has not yet been detected by in vitro complementation, despite many attempts in our laboratory and elsewhere. Consequently, we
245
38 FUNCTION
have taken a genetic approach to obtaining additional information on the functions of 38P and 57P, by isolating and studying mutants in which one of these proteins is no longer required. Analysis of mutations that allow bypass of normally required functions has shed light on the interactions of genetic control elements in both bacteria and bacteriophages (for example, see Butler and Echols, 1970; for reviews, see Reznikoff, 1972, and Herskowitz, 1973). We have taken the same approach in attempting to understand the interactions of proteins in a macromolecular assembly process. In this paper we report the isolation and characterization of phage T4 mutations that allow bypass of the need for 38P function. The accompanying paper (Revel, Herrmann, and Bishop, 1976) describes the isolation and characterization of bacterial host mutations that allow bypass of the need for phage 57P function. MATERIALS
AND
METHODS
Media and buffers. H broth, used for liquid culture of bacteria and bacteriophage, and EHA top and bottom agar, used for plating assays, were prepared as described by Steinberg and Edgar (1962). M9T, a minimal medium supplemented with 50 pg L-tryptophan/ml (Kellenberger and Sechaud, 1957) was used for preparing radioactively labeled infected-cell lysates. Buffers were the dilution medium described by King (1968) and a phosphate buffer used for serum blocking assays (SB buffer), prepared as described by Ward et al. (1970). Strains of bacteriophage and host bacteria. All phage amber (am) and temperature-sensitive (ts) mutants not described
in the text were derivatives of T4D from the Caltech collection and have been described elsewhere (Epstein et al., 1963; Beckendorf, Kim, and Lielausis, 1973; Luftig, Wood, and Okinaka, 1971). Multiple mutants were constructed by phage crosses carried out according to Steinberg and Edgar (1962). The rl mutant r48 was used as the tester phage in serum blocking assays. An rZZ deletion mutant (rdf41) was used in place of wild-type as the reference
246
phage order tester tants gene sults).
BISHOP
AND
in serum blocking experiments in to prevent interference with the phage assay. The host range muhG3 and hG7 carry mutations in 37 (S. Beckendorf, unpublished re-
WOOD
After 100 min of incubation at that temperature, chloroform was added and progeny were assayed on CR63 at 25”. Preparation of ‘Clubeled Zysutes. A saturated culture of B/5 cells in M9T medium was diluted 1:500 into fresh M9T medium and grown for 2.5 hr at 30”, centrifuged, and resuspended at 4 x lo8 cells per ml in the same medium. A 2-ml portion of this suspension was warmed to 30”, infected with phage at a multiplicity of 5 phage per cell, and aerated by agitation on a rotary shaker. After 18 min of incubation at 30”, 2 &i of uniformly 14C-labeled amino acid mixture (Schwarz-Mann) was added. At 26 min, 0.2 ml of 10% casamino acids was added. At 30 min the cells were chilled and centrifuged. The pellet was resuspended in 10 ~1 of 0.05 M Tris (pH 6.8) and lysed by freezing in a dry ice-ethanol bath and thawing at 30”. After addition of 25 pg of DNase and incubation for an additional 15 min at 37”, 100 ~1 of 0.05 M Tris (pH 6.8) containing 2% sodium dodecyl sulfate (SDS) and 2% /3-mercaptoethanol was added. Samples either were used for electrophoresis immediately or were stored at
E. coli host bacteria were used as follows: The K12 strain CR63 and the B strain O-11’ (both permissive for am mutants) for growth of phage stocks and for plating asays; S/6/5 and B/5 (nonpermissive for am mutants) for preparation of defective lysates and for selective plating of urn+ phage; G(h) (nonpermissive for both am and rZZ mutants) for selective plating of the um+rZZ+ tester phage in serum blocking assays; and C31 (resistant to host range mutants hG3 and hG7; S. K. Beckendorf, unpublished experiments) for selective plating of h+ phage. Mapping of phuge mutations. Phage crosses were carried out by the procedure of Steinberg and Edgar (1962) as modified by Beckendorf et al. (1973). All crosses were done at 25” in CR63 host bacteria. Total progeny were assayed by plating on CR63 at 25”. Selective assays for am+, h+, and ts+ progeny were carried out by plat-20”. ing on S/6/5 at 25”, C31 at 25”, and S/6/5 at Electrophoresis on SDS-polyucrylamide 42”, respectively. gels and autoradiography. Samples preTesting for complementution in liquid pared as described in the preceding section culture. Liquid complementation tests were heated in a boiling water bath for 2 were performed by a modification of the min and then electrophoresed in the disstandard phage cross, under conditions re- continuous SDS-buffer system described strictive for both am and ts mutants. The by Laemmli (1970), as modified by Dickson procedure was designed to avoid exposure (1973) and adapted for use with slab gels as of infected cells to KCN at high temperadescribed by Studier (1972). Gels were ture, which results in loss of infective cen- fixed and stained for l-2 hr in an aqueous ters and lowered burst sizes. A saturated solution of 50% (w/v) trichloroacetic acid culture of B/5 was diluted 1:lOOO in H and 0.2% (w/v) Coomassie brilliant blue broth and grown for 2.5 hr at 30”. The cells (Mann). Gels were destained for 12-20 hr were centrifuged and resuspended at 4 x in 10% anhydrous methanol, 10% acetic acid. The gels were dried directly onto lo8 cells per ml in fresh H broth containing 0.004 M KCN at 25”. A sample of the sus- sheets of Whatman No. 3 filter paper and autoradiographed on Kodak no-screen Xpended cells was then mixed with an equal volume of the same H broth containing the ray film by the procedure of Fairbanks et desired phage to give a multiplicity of 7.5 al. (1965) as modified by Dickson (1973). Preparation of defective lysates for deof each phage per cell. After 6 min, anti-T4 antiserum was added to neutralize unadterminations of burst sizes and antigen levels. A saturated culture of either B/5 or sorbed phage, and incubation was continued for an additional 6 min at 25”. The CR63 was diluted 1:lOOO into H broth and cells were then diluted 4 x 104-fold into H grown at either 30 or 41” to 4 x lo6 cells per broth without KCN, prewarmed to 39.5”. ml. Small 5-ml cultures of these cells were
BYPASS
OF
T4 GENE
then infected at a multiplicity of 4 phage per cell. At 5 and 40 min after infection for 30” cultures, and 3 and 22 min after infection for 41” cultures, samples were withdrawn into screw-cap vials and shaken with chloroform. Viable phage were assayed by plating on O-11’ host cells, and A and BC antigens were determined by the endpoint serum blocking assay of Ward et al!. (1970). RESULTS
Rationale
and Plan
of Experiments
In order to determine whether the normal requirement for gene 38 function in tail -fiber assembly can be bypassed as a result of compensating mutation in another gene, we have isolated and characterized apparent revertants (pseudorevertants) of mutant T4 that carry two am mutations in gene 38. First, the gene 38 double mutant was plated on S/6/5, a restrictive host for am mutants. Apparent revertants were isolated, and one was selected for intensive study. Second, genetic and biochemical tests were performed to establish that the revertant was a pseudorevertant; that is, it still carried the original gene 38 defect in nonpermissive hosts. Third, backcrossed phage carrying the second-site mutation, both alone and together with a gene 38 am mutation, were examined for their production of phage progeny and tail fiber antigen on both permissive and nonpermissive hosts. Fourth, the genetic location of the second-site mutation was determined. Isolation of Pseudorevertants amB262:C290
from
When T4 carrying two am mutations in gene 38 (amB262:C290) were plated on S/ 6/5 bacteria at a level of about 10s phage per plate at 25”, plaques appeared at a frequency of about 10e7. The plaques were generally turbid and heterogeneous in size. Their frequency was too high to be explained either by double reversion of the two am mutations or by mutation to a phage-coded am suppressor (Wilson and Kells, 1972). This result suggested the occurrence of second-site mutations that allowed either restoration or bypass of 38P
247
38 FUNCTION
function. In order to have a marker for genetic analysis of such a second-site mutation, a large number of the pseudorevertant plaques were screened for temperature sensitivity. The frequency of ts clones among the pseudorevertants varied from 0.2-0.4% depending on the amB262:C290 stock from which the revertants were isolated. Since most of the pseudorevertants grew very poorly on the nonpermissive host S/6/5, ts isolates were screened for ability to grow well enough at 25” for further study. One revertant, ts3813, was chosen for the characterization described below. Several other ts pseudorevertants, although not studied in detail, have been tested for degree of linkage of the secondsite mutation to ts3813. Pseudorevertant ts3813 Still Carries Original Gene 38 am Mutations
To
establish
genotype as the pseudorevertant was crossed to wild-type phage and the progeny were screened for am phenotype. The appearance of am phage among such progeny requires recombination between the ts site and one of the am mutations. Since amB262 and amC290 are closely linked (about 1 map unit), the frequency of such recombinants provides an estimate of the genetic distance between the ts and the am mutations. About 0.5% of the total progeny exhibited the am phenotype and were shown by additional crosses to carry both amB262 and amC290. This result established the genotype of the pseudorevertant as ts3813: amB262:C290 and showed that the ts mutation is closely linked to the am sites. The reciprocal recombinant, ts3813:am+, occurred less frequently but was isolated also. To establish that ts3813 rather than some other mutation was responsible for bypass of the gene 38 mutation, ts:am double mutants were reconstructed by crossing ts3813 with both amB262 and amC290. The ts3813:am double mutants isolated from these crosses both plate on S/ 6/5 with high efficiency. The reconstructed double mutant ts3813:amB262 was used in the characterization of phenotype described in the following sections. ts3813:amB262:C290,
its
the
248
BISHOP AND WOOD
The Pseudorevertant ts3813:amB262:C290 Remains 38-Defective in Nonpermissive Hosts An am mutation in gene 38 could be suppressed in either of two general ways by a second-site mutation. Mutation of a T4 tRNA gene to a phage-coded suppressor could allow gp38 synthesis in nonpermissive hosts. Alternatively, the second-site mutation could somehow obviate the necessity for 38P function in tail fiber assembly. Both genetic and biochemical evidence support the latter alternative as the explanation for ts3813 pseudoreversion. Several observations argue against mutation to a phage-coded am suppressor. First, the frequency of pseudoreversion is higher by a factor of about lo4 than the previously observed frequency of T4 am suppressor mutations (Wilson and Kells, 1972). Second, the ts3813 mutation is closely linked to gene 38, whereas all the known T4 tRNA genes are separated from gene 38 by ‘half of the genome (Wilson, Kim and Abelson, 1972; Wood, 1974a). Third, construction and analysis of various ts3813:am mutants showed that ts3813 does not suppress am mutations in several genes other than 38. None of the following double mutants, constructed by crossing ts3813 with the indicated am mutants, showed better growth in nonpermissive hosts than the am mutants alone (data not shown): ts3813:amB25(gene 34), ts3813: amA (gene 34), ts3813:amB252 (gene 35), ts3813:amXl(gene 361, and ts3813: amE198cgene 57). Direct biochemical evidence against suppression of the am mutations in gene 38 was obtained by electrophoretic analysis of phage-coded polypeptides radioactively labeled following infection of the nonpermissive host B/5 (Fig. 2). 14C-labeled lysates of cells infected with T4 wildtype (+ +), amB262:C290 (3%), ts3813: amB262:C290 (ts:38-), and amN52 (37-) were electrophoresed on polyacrylamide gels in the presence of SDS, and phage polypeptides were visualized by autoradiography of the dried gels. As shown by previous investigators (King and Laemmli, 1973; Dickson, 1973; Vanderslice and Yegian, 19741, gp38 can be identified as a
FIG. 2. Autoradiogram of YJ-labeled lysates of cells infected with T4D wild-type, amB.262, ts3813:amB262, and amX52. ‘YXabeled lysates were prepared as described in Materials and Methods, and electrophoresed on a slab gel of 10% polyacrylamide containing 1% SDS. Each sample contained approx lo5 disintegrations per minute. The gel was fixed, stained, destained, dried, and autoradiographed as described in Materials and Methods. The additional bands seen in the 38- lysate are unexplained; they were not found in other similar experiments.
26,000 dalton polypeptide that is present in lysates of cells infected with wild-type phage but is missing from lysates of cells infected with gene 38 am mutants. In several comparisons of wild-type with amB262:C290, the presence or absence of this band was found to be the only repro-
BYPASS
OF
T4
GENE
ducible difference between the two lysates. The gel pattern (Fig. 2) indicates that ts3813:amB262:C290-infected cells contain no detectable gp38 under these conditions, although infectious phage are being assembled. The amN52 (37-)-infected cells lack gp37 (120,000 daltons) and produce a reduced amount of gp38 relative to wildtype. This reduction is known to be due to a polar effect of the gene 37 am mutation on expression of the neighboring gene 38 (King and Laemmli, 1971). These results indicate that the ts3813 mutation somehow permits a bypass of the normal requirement for 38P in tail fiber assembly. Production of Tail Fiber Antigens and Progeny Phage by a Gene 38 am Mutant That Carries ts3813 Conceivably, ts3813 could allow bypass of 38P function in either of two ways. It could specifically affect the 38P-mediated step in tail fiber assembly, allowing it to proceed in the absence of 38P, or it could render T4 particles infectious in the absence of tail fibers. In view of earlier results, the latter possibility seemed unlikely. Mutants of T4 able to infect without tail fibers were not found in an earlier search that would have detected such phage at a frequency of lo-lo (Wilson and Kells, 19721, and these mutants would be unexpected due to the necessity of tail fibers not only for attaching phage to host cells, but probably also for triggering subsequent baseplate attachment and sheath contraction (Yamamoto and Uchida, 1975). Moreover, ts3813 bypasses only am mutations in gene 38, and not am mutations in other tail fiber genes. To determine how ts3813 affects tail fiber assembly in the presence and absence of 38P, production of tail fiber antigens and phage progeny was measured under various conditions. Lysates were prepared using permissive (CR63) and nonpermissive (B/5) host cells infected at 30 and 41” with wildtype amB262, ts3813, and ts3813:amB262 phage. The lysates were assayed for levels of A and BC tail fiber antigens as well as for viable phage titer. Results obtained at 30 and 41” are summarized in Table la and b, respectively.
249
38 FUNCTION TABLE
1
PRODUCTION OF TAIL FIBER ANTIGENS AND INFECTIOUS PI-IAGE IN WILD TYPE- AND MUTANTINFECTED CELLS AT (a) 30” AND (b) 41% Phage
Host
Burst size IPha!&
(phageequivalents/cell)’
(a) 30 ++ amB262 ts3813 ts3813: amB262 (b) 41” ++ amB262 ts3813 ts3813: amB26i
,
!
Percentage of
CR63 B/5 CR63 B/5 CR63 B/5 CR63 B/5
100 48 111 0.004 136 41 44 21
188 137 248 228 238 283 148 249
390 314 473 11 344 601 138 109
CR63 B/5 CR63 B/5 CR63 B/5 CR63 B/5
26 17 16 0.01 0.01 0.26 co.7 CO.28
71 69 88 116 104 140 91 133
75 160 124 5.6 4.2 11.9 2.4 2.0
1BC+a+ntigenb
100 100 93 1.9 78 91 41 18
100 100 141 2.1 3.8 3.5 2.4 0.6
a Preparation of lysates and measurements of A and BC antigens were carried out as described in Materials and Methods. All values are averages of two independent experiments. The phage designated ++ in these experiments is the rZZ deletion mutant rdf41. b BC antigen levels produced by mutants were normalized to those of wild-type by using A antigen as an internal standard, since none of the mutants employed here are defective in A antigen production. Values for BC antigen as percent of wild-type level were calculated using the formula: BC/A (mutant)/BC/A (+ +) x 100%. The mean standard deviation for the duplicate results averaged in this column was 226% of the values shown. c Antigen levels are expressed in phage.equivalents as defined by Ward et al. (1970).
At 30” (Table la) on the nonpermissive host, the amB262 mutation greatly reduces the production of both BC antigens and viable phage relative to wild-type infected cells. The ts3813 mutation at least partially overcomes the gene 38 defect as measured by production of both antigen and viable phage, although the levels are lower than in wild-type infected cells. In the permissive host, ts3823:amB262 also
250
BISHOP
produces somewhat lower antigen and viable phage levels than does wild-type. At 30”, the single mutant ts3813 produces normal levels of BC antigen and viable phage on both CR63 and B/5 hosts. At 41” (Table lb) amB262 displays the same defective phenotype as at 30”: both BC antigen and viable phage production are drastically reduced in the nonpermissive host. At 41”, however, the ts3813 mutation does not restore either antigen or Mutants that carry phage production. ts3813 produce less than 4 and 1% of the wild-type levels of BC antigen and phage progeny, respectively, on either CR63 or B/ 5 host cells. Therefore, the bypass mutation also leads to temperature sensitivity of gp37 assembly. The ts3813 mutation does not significantly increase the heat lability of phage produced at low temperature. Both ts3813 and ts3813:amB262 phage incubated at 39.5 and 45” in dilution medium containing 1% SDS and 1% P-mercaptoethanol were inactivated at about the same rate as wildtype phage (data not shown).
AND
WOOD TABLE
2
RESULTS OF LIQUID COMPLEMENTATION TESTS WITH t.s3813:amB262:C290 AND am MUTANTS DEFECTIVE IN GENES 27, 37, AND 38a Infecting ++ amB262 (38-j amN52 (ST-) amNl20 (27-) ts3813:amB262:C290 amB262 + amNl20 amN52 + amNl20 ts3813:amB262:C290 ts3813:amB262:C290 ts3813:amB262:C290
phage
+ amNl20 + amB262 + amN52
Burst size (phagekell) 2.3b 0.02 0.01 0.01 0.09 23.8 18.1 43.3 0.04 0.15
a Complementation tests were carried out at 39.5” with B/5 host cells in liquid culture as described in Materials and Methods. b Burst sizes obtained with wild-type phage at this temperature were variable, ranging from 2-50 phage per cell.
can be complemented by a&120(27-1, but not by amB262(38-) or amN52(37-). An additional control experiment (not shown) under comparable conditions The ts3813 Mutation Maps near the Proshowed that despite the polar effect of moter-Distal End of Gene 37 amN52 on gp38 production (Fig. 21, The genetic location of ts3813 was deteramN52 and single gene 38 am mutants mined in two steps. First, the mutation complement to give burst sizes in the was shown to be in gene 37 by complemenrange of wild-type. Therefore, the ts3813 tation tests at nonpermissive temperature defect is in gene 37, which codes for the with the ts3813 mutant and appropriate major structural polypeptides of the distal am mutants. Second, the map position of half tail fiber (Fig. 1). ts3813 within gene 37 was determined by a To determine the map position of ts3813 series of 3-factor crosses. within gene 37, ts3813:amB262 and The previously observed linkage of ts3813:amC290 were crossed with phage ts3813 to am mutations in gene 38 and the carrying various ts mutations in genes 37 and 38 and/or host range mutations in lack of BC antigen production at high temperature in mutants carrying ts3813 sug- gene 37 (hG3 and hG7). Examples of three gested that this mutation might be in gene such crosses are diagrammed in Fig. 3. 37, which is adjacent to gene 38 on the T4 Progeny of ts:am x ts crosses were plated on CR63 bacteria at high temperature to genetic map. Accordingly, ts3813:amB262: select ts+ recombinants, which then were C29O was tested for genetic complementation of amN52(37-1, amB262(38-1, and as scored for the unselected am marker to a control amNl20(27-), at 39.5” in B/5 determine the position of ts sites relative host cells. The results of this test are to amB262. Progeny of ts:am x h crosses shown in Table 2. Control cultures in- were plated on C31 bacteria at high temfected with each of the mutants alone gave perature to select ts+h+ recombinants, which were then scored for the unselected burst sizes less than 5% of the wild-type control. The results of the pair-wise in- am marker as above. The results of these crosses, tabulated in Table 3, show that fections show that ts3813:amB262:C290
BYPASS
OF
T4
GENE
ts3813 maps close to the host range mutations hG3 and hG7, in the region of gene
C290. In these isolates, the second-site ts mutations were unlinked to gene 38 and did not appear to be in any of the genes required for tail fiber assembly. Although of considerable interest, these mutations were more difficult to work with, in that they bypassed 38 function less effectively than ts3813, and in some cases produced
37 that codes for the C-terminal portion of the gp37 molecule (Fig. 4). We conclude that gp37 can be mutationally altered in such a way that it no longer requires 38P for assembly into a functional tail fiber. Mapping experiments also were carried out on a number of other independently isolated ts pseudorevertants of amB262: A
ts3ei3
A
lS38/3
.
d
251
38 FUNCTION
4 4 \5
!‘JB
(I
ts38I3
I
IIl,l p +
:-------L
__.
,__--- .__..
.
-
tsN36
(a)
lb1
FIG. 3. Diagrammatic factor crosses involving
___..-A -
Gene 37
tsA3l
hG3
representation ts3813.
OF ts3813:amB262
Cross
CC)
of three-
AND ts3813:amC290 Recombinants lected
x x x x x x x
tsCT32(38-I tsN36(37-) tsB78(37-I tsN2(37-) t&20(37-) tsA31(37-) tsC9(49-)
ts+ ts+ ts+ ts+ ts+ ts+ ts+
ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262 ts3813:amB262
x tsCZ’32(38-1 x tsN36(37-) x tsB78(37-) x tsN2(37-) x t&20(37-) x tsA31(37-) x tsC9(49-1
ts+ ts+ ts+ ts+ ts+ ts+ ts+
ts3813:amB262 ts3813:amB262
x hG3
ts+h+ ts+h+
hG3
x hG7
h+
hG3 hG3 hG7 hG7
x x x x
ts+h+ ts+h+ ts+h+ ts+h+
tsN2(37-)” tsCT32(38-I” tsN2(37-)” tsCT32(38-)”
II
6% 9’ i
II Gene 38
4. Map of ts and h markers in genes 37 and 38. Relative distances are estimated from the data in Table 3 and earlier mapping experiments (Beckendorfet al., 1973; Beckendorf, unpublished results).
ts3813:amC290 ts3813:amC290 ts3813:amC290 ts3813tamC290 ts3813:amC290 ts3813:amC290 ts3813:amC290
x hG7
4 (3% &$ ,+ \S‘ 0 0 fi (f
FIG.
TABLE RESULTS
,$y@~
3 CROSSES
se-
WITH ts AND h MUTANT@ am/am+’ Percentage recombinatiox? ___---2.0 0.6 1.4 4.3 2.6 1.6 31.9
3.1 1.0 1.4 4.4 2.7 1.6 39.0
2.61 0.08 0.13 0.11 0.05 2.12
1.7 2.2
0.13 0.21
eo.1 10.3 5.2 9.8 4.7
0 Crosses were carried out as described in Materials and Methods, using CR63 bacteria SO that am markers were unselected among the progeny in all crosses. * Percentage recombination values are calculated as 200% x recombinants/total progeny. I’ The am/am+ ratio was determined by stabbing at least 100 recombinant plaques with sterile pins, which then were stabbed into a lawn of S/6/5 bacteria and then into a second lawn of CR63 bacteria. Plaques that produced a clear spot on CR63 but not on S/6/5 were scored as am. Plaques that failed to produce a spot on CR63 were not scored, and plaques that produced spots on both hosts were scored as am+. ” ts x h crosses gave higher frequencies of recombinants than expected from ts X ts crosses.
252
BISHOP
AND
labile phage particles that rapidly lost viability during storage. These pseudorevertants have not yet been investigated further.
WOOD
showed that gp38 is not a structural component of either the completed tail fiber or any of its precursors (King and Laemmli, 1971; Dickson, 1973). These results suggested that 38P acts catalytically in tail DISCUSSION fiber assembly, in agreement with earlier A number of earlier genetic studies sug- evidence from gene dosage experiments gested an interaction between 38P and the (Snustad, 1968). If so, what could be the C-terminal portion of gp37 during tail fi- mechanism of 38P action? Two general ber assembly. Both proteins are required possibilities can be suggested. for tail fiber formation (Epstein et al., (1) 38P could catalyze a covalent modifi1963; Eiserling et al., 1967) and for normal cation ofgp37 tliat promotes dimerization. tail fiber antigen production (Edgar and The molecular weights of gp37 before and Lielausis, 1965). In mixed infections of the after dimerization are the same as judged closely related phages T2 and T4, these by SDS polyacrylamide gel electrophoretwo proteins are functional only in the sis, but the uncertainty in these experihomologous combinations, i.e., both pro- ments is about *3%. Therefore, they canteins from T2 or both from T4. Such mixed not rule out either proteolytic cleavage of a infections produce no viable progeny few amino acid residues from one end of phage if each of the parental phage strains gp37 or modification of amino acid side carries a defect in one of the two genes 37 chains by 38P. A possibly analogous example of the latter process is the enzymic and 38 (Russell, 1967, 1974); for example, of proline residues in procolnonpermissive cells infected with a T2 hydroxylation gene 38 am mutant and a T4 gene 37 am lagen to increase its helix-coil transition mutant produce no progeny. In subsequent temperature and thereby allow triple-hestudies Beckendorf (1973) showed that this lix formation (Berg and Prockop, 1973; RoT2-T4 incompatibility is limited to gene 38 senbloom et al., 1973). However, the propand the promoter-distal end of gene 37, erties of the ts3813 mutant suggest that corresponding to the C-terminal portion of drastic 38P-catalyzed covalent modificagp37. Within the incompatibility region, tion of gp37 is not necessary for dimerizarecombination between T2 and T4 does not tion, since the requirement for 38P can be occur, and there is no detectable homology bypassed by what is probably a single between T2 and T4 DNA as shown by ex- amino acid substitution near one end of the gp37 monomer. If 38P does catalyze a amination of T2-T4 heteroduplex DNA covalent change, this change must promolecules in the electron microscope dimerization quite specifically, (Beckendorf, Kim, and Lielausis, 1973). mote The C-terminal end ofgp37 carries the tail rather than generally as in procollagen fiber recognition site for bacterial cell sur- hydroxylation. (2) An alternative possibility is that 38P face components and is largely responsible association of for determining the host range of the catalyzes the noncovalent phage. Phages T2 and T4, although closely two gp37 molecules, perhaps by tranrelated otherwise, have diverged considersiently stabilizing an intermediate comably in host range, and their tail fibers plex in the dimerization reaction. Gene 37 recognize quite different cell surface com- am mutants produce no C antigen, even is near the proponents (Jesaitis and Goebel, 1953). The when the am mutation moter-distal end of the gene so that long apparent codivergence of gene 38 function fragments of gp37 are synthesized. This with the region of gene 37 that determines host range suggests that 38P interacts observation suggests that dimerization must be initiated by interaction of the Cwith the C-terminus of gp37 during tail fiber assembly, to promote the dimerizaterminal ends of two gp37 molecules. If the resulting initial complex is either unstable tion of gp37 into the rod-shaped precursor (e.g., conformationally strained) or imhalf fiber that carries the C antigens. The question of how 38P acts became probable (e.g., one of many isoenergetic configurations), then 38P could increase more intriguing when additional studies
BYPASS
OF
T4 GE NE
the rate of dimerization by transiently combining with and specifically stabilizing the complex. According to this model, the amino acid substitution in ts3813 would likewise stabilize the initial complex. Recent experiments of Kikuchi and King (1975a, 197513, 1975c) have demonstrated elegantly that almost all of the thermodynamically favorable subunit associations in T4 tail assembly fail to occur in the absence of specific structural components, formed in previous assembly steps, which presumably provide nucleation sites that help to overcome activation energy barriers to subunit assembly. In tail fiber assembly, a similar function may be served by the nonstructural protein 38P. Catalysis of noncovalent association by a nonstructural protein also has been suggested as the mechanism of action for 63P in tail fiber attachment to the baseplate (Wood and Bishop, 1973; Wood et al., 1976), and as a possible general phenomenon in macromolecular assembly (Wood, 197433). Whatever the mechanism of 38P function, it can be partially bypassed by a mutational alteration in the structural protein gp37. As a consequence of the bypass mutation, however, assembly of the distal half of the tail fiber is blocked at higher growth temperatures. Most of the other bypass mutants isolated, although not studied in detail, appeared to grow poorly under all conditions, and some of them produced unstable phage particles as well. These observations suggest that gene 38 function cannot be bypassed without considerable cost in fitness to the phage; consequently, it is not surprising that this gene has been retained in the course of evolution. ACKNOWLEDGMENTS These studies were supported by a research grant from the U.S. Public Health Service (AI092381 to W.B. W., and a postdoctoral fellowship from the U.S. Public Health Service (GM40392) to R.J.B. REFERENCES BECKENWRF, S. K. (1973). Structure of the distal half of the bacteriophage T4 tail fiber. J. Mol. Biol. 73, 37-53. BECKENDORF, S. K., KIM, J.-S., and LIELAUSIS, I. (1973). Structure of bacteriophage T4 genes 37 and
38 FUNCTION
253
38. J. Mol. Biol. 73, 17-35. BERG, R. A., and PROCKOP, D. J. (19731. Purification of [i4C]protocollagen and its hydroxylation by prolyl-hydroxylase. Biochemistry 12, 3395-3401. BISHOP, R. J., CONLEY, M. P., and WOOD, W. B. (1974). Assembly and attachment of bacteriophage T4 tail fibers. J. Supramolec. Structure 2, 196-201. BUTLER, B., and ECHOLS, H. (1970). Regulation of bacteriophage A development by gene N; properties of a mutation that bypasses N control of late protein synthesis. Virology 40, 212-222. CAEJENS, S., and KING, J. (1975). Virus assembly. Anna. Rev. Biochem. 44, 555-611. CONLEY, M. P., and WOOD, W. B. (1975). Bacteriophage T4 whiskers; A rudimentary environmentsensing device. Proc. Nat. Acad. Sci. USA 72, 3701-3705. DEWEY, M. J., WIBERG, J. S., and FRANKEL, F. R. (1974). Genetic control of whisker antigen of bacteriophage T4D. J. Mol. Biol. 84, 625-634. DICKSON, R. C. (1973). Assembly ofbacteriophage T4 tail fibers IV. Subunit composition of tail fibers and fiber precursors. J. Mol. Biol. 79, 633-647. EDGAR, R. S., and LIELAUSIS, I. (1965). Serological studies with mutants of phage T4D defective in genes determining tail fiber structure. Genetics 52, 1187-1200. EISERLING, F. A., BOLLE, A., and EPSTEIN, R. H. (1967). Electron microscopic studies of the structure of mutants of bacteriophage T4D defective in tail fiber genes. Virology 33, 405-412. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KEI~ LENBERGER, E., BOY DE LA TOUR, E., CHEVALLEY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375394. FAIRBANKS, G., LEVINTHAL, C., and REEDER, R. H. (1965). Analysis of Ci4-labeled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 20, 393-399. FOLLANSBEE, S. E., VANDERSLICE, R. W., CHAVEZ, L. G., and YEGIAN, C. D. (1974). A new set of absorption mutants of bacteriophage T4D: Identification of a new gene. Virology 58, 180-199. HERSKOWITZ, I. (1973). Control of gene expression in bacteriophage lambda. Annu. Rev. Genetics 7, 289-324. JESAITIS, M. A., and GOEBEL, W. F. (1953). The interaction between T4 phage and the specific lipocarbohydrate of phase II Sh. sonnei. Cold Spring Harbor Symp. Quant. Biol. l&205-208. KELLENBERGER, E., and SECHAUD, J. (1957). Electron microscopical studies of phage multiplication. II. Production of phage-related structures during multiplication of phages T2 and T4. Virology 3, 256-274.
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S., and HASELKORN, R. (1974). Bacteriophage T4 short tail fibers are the product of gene 12. J.
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J. (1975c). Genetic control of T4 baseplate morphogenesis. III. Formation of the central plug and overall assembly pathway. J. Mol. Biol. 99, 675-716. KING, J. (1968). Assembly of the tail of bacteriophage T4. J. Mol. Biol. 32, 231-262. KING, J., and LAEMMLI, U. K. (1971). Polypeptides of the tail fibers of bacteriophage T4. J. Mol. Biol. 62, 465-477. KING, J., and LAEMMLI, U. K. (1973). Bacteriophage T4 tail assembly: Structural proteins and their genetic identification. J. Mol. Biol. 75, 315-337. KING, J., and WOOD, W. B. (1969). Assembly of bacteriophage T4 tail fibers: The sequence of gene product interaction. J. Mol. Biol. 39, 583-601. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LUFTIG, R. B., WOOD, W. B., and OKINAKA, R. (1971). Bacteriophage T4 morphogenesis: On the nature of gene Is-defective heads and their role as intermediates, J. Mol. Biol. 57, 555-573. MASON, W. S., and HASELKORN, R. (1972). Product of T4 gene 12. J. Mol. Biol. 66, 445-469. REVEL, H. R., HERRMANN, R., and BISHOP, R. J. (1976). Genetic analysis of T4 tail fiber assembly. II. Bacterial host mutations that bypass the requirement for gene 57 function. VirologY 72, 255-265. REZNIKOFF, W. S. (1972). The operon revisited. Annu. Rev. Genetics 6, 133-156. ROSENBLOOM, J., HARSCH, M., and JIMENEZ, S. (1973). Hydroxyproline content determines the denaturation temperature of chick tendon collagen. Arch. Biochim. Biophys. 158, 478-484. RUSSELL, R. L. (1967). Speciation among the T-even bacteriophages. Ph.D. dissertation, California Institute of Technology, Pasadena, Calif. RIJ~~ELL, R. L. (1974). Comparative genetics of the T-even bacteriophages. Genetics 78, 967-988. SNUSTAD, D. P. (1968). Dominance interactions in Escherichia coli cells mixedly infected with bacteriophage T4D wild-type and amber mutants and their possible implications as to type of gene product function: Catalytic vs. stoichiometric. Virol-
ogy 35, 550-563. C. M., and EDGAR, R. S. (1962). A critical test of a current theory of genetic recombination in bacteriophage. Genetics 47, 187-208. STUDIER, F. W. (1972). Bacteriophage T7. Science STEINBERG,
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R. W., and YEGIAN, C. D. (1974). The identification of late bacteriophage T4 proteins on sodium dodecyl sulfate polyacrylamide gels. Virology 60, 265-275. WARD, S., LUFTIG, R. B., WILBON, J. H., EDDLEMAN, H., LYLE, H., and WOOD, W. B. (1970). Assembly of bacteriophage T4 tail fibers. II. Isolation and characterization of tail fiber precursors. J. Mol. Biol. 54, 15-31. WARD, S., and DICKSON, R. C. (1971). Assembly of bacteriophage T4 tail fibers. III. Genetic control of the major tail fiber polypeptides. J. Mol. Biol. 62, 479-492. WILSON,
J. H., and KELLS, S. (1972). Bacteriophage T4 transfer RNA. I. Isolation and characterization of two phage-coded nonsense suppressors. J. Mol.
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W. B. (1974a). Bacteriophage T4. In “Handbook of Genetics” (R. C. King, ed.), Vol. I, pp. 327331. Plenum Press, New York. WOOD, W. B. (1974b). Undelivered summary remarks for the 1974 Squaw Valley Meeting on Assembly Mechanisms. J. Supramolec. Structure 2, 512-514. WOOD, W. B., and BISHOP, R. J. (1973). Bacteriophage tail fibers: Structure and assembly of a viral organelle. In “Virus Research” (C. F. Fox and W. S. Robinson, eds.), pp. 303-324. Academic Press, New York. WOOD, W. B., and CONLEY, M. P. (1976). Attachment of tail fibers in bacteriophage T4 assembly. III. Role of the phage whiskers. Submitted for publication. WOOD, W. B., and HENNINGER, M. (1969). Attachment of tail fibers in bacteriophage T4 assembly: Some properties of the reaction in vitro and its genetic control. J. Mol. Biol. 39, 603-618. WOOD, W. B., CONLEY, M. P., LYLE, H. L., and DICKSON, R. C. (1976). Attachment of tail fibers in bacteriophage T4 assembly. II. Purification, properties, and site of action of the catalytic protein 63P. Submitted for publication. YAMAMOTO, M., and UCHIDA, H. (1975). Organization and function of the tail of bacteriophage T4. II. Structural control of the tail contraction. J. 92, 207-223.