Host mutant (tabD)-induced inhibition of bacteriophage T4 late transcription

J. Nob. BioE. (1975) 96, 601-624

Host Mutant (t&D)-induced Inhibition Late Transcription II. Genetic Characterization

of Bacteriophage T4 of Mutants

ANNA COPPO,XNDREAMANZI,JOEIN F. PI-LITZER Idernational

Institute of Genetics and Biophysics, Via Marconi 10: 80125 Naples, Italy

C.N.R.

AND

HIDEO TAKAHASHI Institute of Applied Microbiology University of Tokyo, Bunkyo-ku. Tokyo, Japan (Received

19 December 1974, and in revised form

22 April

1975)

In this paper we show that the MD mutants, selected with ts553 or tsCB53, and tles,cribed in the accompanying paper (Coppo et al.. lYi5): (a) are recessive to tab + ; (b) fail t,o complement each ot)her, and thus map in the samp cistron ; (c) by their linkage to rij and their dominance relationships with well characterized amber mutations in the Escherichia co& RNA polymerase operon, probably all map in the gene controlling the synthesis of the j?’ subunit of the enzyme. We also describe the isolation of a ta + , kD mutant in phage T4 gene 55, used in the selection (a) generates a defective of a new tabD mutant (tabDkZs2 ). This tub mutant: phenotype which differs somewhat from that of the other tabD mutants; (b) complements the other tubD mutants ; (c) by its dominance relationship to amber mutants in the RNA polymerase operon, can be assigned tjo the st’ructural gene coding for the j3 subunit of the enzyme. A new type of interaction between T4 genes 55 and 45 is also described. The P propert,ies of ti553 (gene 55) are suppressed at 3O”C, by a temperature-sensitive tnutation in gene 45. This type of interaction betlvccn missense mutations in genes 35 and 55 apparently occurs even in tub+ strains, since temperature-sensitive mutations in gene 45 accumulate in lysates of two gene 55 mutants (ts553 and tsA81).

1. Introduction In the accompanying paper (Coppo et al.. 1975), we have shown that T4 gene S3 deficiency induced by Escherichia coli tabD mutants reproduces exactly the defective phenotype observed when this gene is inactivated by a viral amber or temperaturesensitive mutation. T4 gene 45-deficiency induced by tabD leads to a block in late transcription but in contrast to classical gene 45defective phenotypes, does not, hinder viral DNA synthesis. In this paper we analyse some genetic properties of the phagc and bacterial components of the tabD conditional lethal system. We will present further evidence fol fnbD-mediated interaction between P45 and P65. whereby mutations in gene 4S

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602

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suppress the phenotypic expression of mutations in gene 55. We will show that tabD5517,5544 and 533 map within the RNA polymerase operon (Errington et al., 1974), possibly in the gene controlling the synthesis of the /3’ subunit ; we will also describe a new mutant, tabDk2g2, that is genetically distinct from the other tabD mutants and maps in the gene coding for the /I subunit.

2. Materials and Methods Media, T4 strains, methods of T4 infection, labelling of proteins with 14C-labelled amino acids, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, labelling of RNA with [5-sH]uridine, hybridization-competition and labelling of T4 DNA with [methyL3H]thymidine are described in Takahashi et al. (1975) and Coppo et al. (1976). Z&D mutants were isolated from nitrosoguanidine-mutagenized CT3 by the general selection procedure described by Takahashi et al. (1975). Other strains are listed in Table 1. General procedures used in constructing the strains for the genetic analysis of f&D mutants are described in Miller (1972).

I.

TABLE

Bacterial

strains

Genotypes

Strains HTC747 HTCBBO

F-, thi-l,purD,,, proA,, h&-4, rif-7, sup48 F-, mm, thy, t&D-533, g&E, guZK,,, ara,,,

HTC272 HTC219 HTC663 HTC308 HTC312 HTC784

F-, thi-1, ilv-1, argH, m&B, r&-l, Zec,T’if-22 P -, &i-l, argE, metB,, proA, his-b, thr-1, Zeu, str F-, thi-l,proA,, hi8-4, TV-7, t&D-633, sup48 F-, metB, recA,c F-, metB, recA,c, rif-31 F-, thi-I, metB,, proA, his-d, thr-1, Zeu, rif-7, t&D-633,8tr-31 F-, azi, str& rel, urgH, riy-Ge, purD Trimethoprim-selected derivative Hfr KL16, recA,, thi, unZA KLFlO, am, d(Zac, pro), naZA, recA, rgr, argE, metB KLFlO derivative carrying rif,&., s non-polar amber mutsnt (Kirschbaum & Scaife, 1974) in the rZf (8) gene KLFlO derivative carrying tif&II,s, a polar amber mutant in the rif (8) gene

k&C, t8%

58182~ 68182c, thy Hlll KLFlO/XAlOO,

rccA

KLFlO, Mii,mz/ XAlOOc, rccA KLFlO, ~~f:mm/al XAlOOc, recA

Origins Tokyo Tokyo Tokyo Tokyo Tokyo Tokyo Tokyo Tokyo J. Miller (Geneva) Naples J. Miller (Geneva) J. Miller (Geneva)

I. Claeys (Geneva) I. Claeys (Geneva)

KLFlO is en episome covering the metB to purD segment of the E. coli chromosome (Fig. 7; Austin et al., 1971). is e polar ember in tif&Dla is a non-polar amber mutation in the rif (8) ge ne, while tif&rls the rif gene which affeots 6 and @’ synthesis (Errington ti al., 1974).

(a) P?uzge PI tramduction The method used is described by Takahashi et al. (1975). The recipients used in the experiments in this paper were: HTC-747, HTC-272, HTC-663 for mapping tiD-533, and thy recom68182~~thy for mapping tabD-5517, 5544 and tabD k202. tabD, arg, rif’ (or +), binants from the transduction experiments were crossed with Hlll to obtain the recA derivatives used in the construction of the merodiploids described below.

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(b) Merodiploid con&u&on (i) KLFlO tab+/arg, tabD, recA (tabD on the chromosome) F-, urgH (or m&B), t&D, tip (or ri,f’), thy, strp recombinsnts were obtained in Pl transduction of t&D to 68182~~thy by selection for Pur+ ; recA was introduced by crossing with streptothese recombinants with Hill, selecting for Thy+ , and counter-selecting mycin. Ret derivatives were detected by their sensitivity to low doses of U.V. The KLFlO episome and its derivatives, carrying t&D (or tab+), tip (or T~F or rijf”) were introduced into the t&D, argH (or m&B), recA strains by crossing with KLFlO/XAlOOc, selecting for Arg + (or Met + ) and counter-selecting with streptomycin. (ii) KLPlO tabD/XAlOOc (tabD on the episome) KLFlO/XAlOOc was mated with the t&D, argH (or metB), ret+ recombinants. Recombination between chromosome and episome was allowed to proceed for 90 min and phenotypically arg + , 2abD‘recombinants were isolated. Some of these had the genetic constitution KLFlO, tiD/argH, t&D, 8trP.The t&D derivatives of KLFlO were transferred and stored in XAlOOc by selecting for Pur+, Arg+ and counter-selecting with nalidixic acid.

3.

Results

(a) Some genetic Properties of kD mutants Reversion analysis of the kD m&ants We have seen that ts553 and tsA81 in gene 56 and t&B53 and taP73CT in gene 45 fail to grow on tabD host mutants at 3O”C, and thus behave as kD mutants (Coppo et al., 1975). Complementation tests have shown that the kD properties of these mutants are due to the malfunction of the same genes which carry the temperature-sensitive mutations : i.e. gene 55 for ts563 and tsA81, gene 45 for tsCB53 and tsP73CT. A further test is required, however, to determine whether the temperature-sensitive and the kD properties are due to identical or to distinct mutational alterations in genes 55 and 45. If the mutations involved are identical, ts+ revertants of ts553, tsA81, t&B53 and tsP73CYC(obtained by plating about 10” of each mutant on BB (tab+) at 43°C) should all be k+, and thus capable of growth on t&D. Conversely, k+ revertants of the temperature-sensitive mutants (obtained by plating IO6 of each mutant on tobD at 30°C) should be ts + . (1) ts + ts+ reversion. About iO0 ts+ revertants were picked for each mutant. All ts+ revertants of ts553, tsA81, tsCB53 and tsP73CT grow on t&D-5517 and 533 and are thus k+ . (2) il: + k+ reversion. In this case, the results differed from our expectations: 27 out of 30 k+ revertants of tsP73CT and 31 out of 60 k+ revertants of tsCB53 retained the ts phenotype. In the case of ts553,5 k+ revertants out of 100 tested were still ts. Complementation tests showed that the original ts mutations in the ts, k strains were retained in the ts, k+ revertants. We conclude from these results that, since all ts+ revertants are k+ and since at least some k+ revertants are ts + , the k phenotype must be due to the same mutations as the ts phenotypes. To explain the ts, k+ revertants we argue that the k phenotype can be specifically suppressed

by a mutation

(revD) at a site other than that conferring

tem-

perature-sensitivity. We have analysed in some detail one k+ pseudorevertant of tsP73Cl! (tsP73CT,revD-1), one of tsCB53 (t&B53,revD-2) and one of ts553 (ts553,revD-3). The plating properties of the pseudorevertants on t&D mutants are shown in Table 2A.

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(11) Genetic analysis

ET

AT,.

of the k + pseudorecertnnfs

(i) tsP73CT,revD-I We find that tsP73CT is temperature-sensitive at temperatures as low RS 37°C (Table 2B). Complementation tests show that temperature-sensitivity at 37°C is due to an impairment in the function of P45. Consistent with these observations is the fact that T4 DNA synthesis is blocked both at 42.7% (Fig. l(c)) and at 37°C (Fig. l(b)). Notice also that the burst-size and DNA synthesis by tsP73CT are poor even at 30°C (Fig. l(a) : Table 2B).

IO

/ &: IO

;

l

20 30 40 50 Time after infection (mid

h-P73

20 30 40 50 Time after infection(min)

FIG. 1. Replication by tsP73CT and taP73CT, revD-1 on tab+. This experiment was carried out on BB(tnb +) in M9S medium infected at a multiplicity of infnction of 6. [2-‘Wlthymidine was added at 3 min after infection at a specific activity of 0.1 pCi/lS pg per ml. Portions of l-ml were taken at the times indicated and the incorporation of the radioactive precursor, into alkali stable (0.6 N-NaOH overnight at room temperature) trichloroacetic acid-precipitable material, was measured.

As a result of the revD-1 mutation, tsP73CT not only loses its k phenotype, but also its temperature-sensitivity at 37°C; at this temperature the pseudorevertant both replicates and produces phage (Fig. 1(a) and (b) ; Table 2B). The temperature-sensitivity at 42*7”C, however, is retained or possibly enhanced (Fig. l(c) ; Table 2B). Although we have as yet not been able to map revD- 1, the change in the temperaturesensitivity of replication is most simply explained by the assumption that tsP73CT mutant P45-protein is stabilized by an amino acid substitution at a second site within gene 45. A similar phenomenon, referred to as second-site suppression, has been described for a mutant of the E. coli enzyme tryptophan synthetase (Helinski & Yanofsky, 1963).

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TABLE 2

A. Plating eficiencies of ts553 and tsCB53 pseudo-revertants on t&D

l&D-5517

13ecterial strains t&D-5544

0.96 3x 10-c 0.9

1 3x 10-G 0.7

t&B53

0.9

lsCB63, ~evD-2 taP73CT tsP7:%CT, revD-1

0.93 -

3x 10-Z (very turbid plaques) 1.1

Phages

at 30°C

UJD-533

~T4w1 ts663 IS563, reVD-3

Efficiencies top-aga,r.

of pla.ting

are relative

t,o 1nb+(CT3)

and were determined

0.91 0.4x 10-s 2x 10-4-4x 10-l (very turbid plaque*) 8x 10-e 0.94 3.4x 10-4 0.76 using 0.57;

Hershey

B. Effects of revD-I on the temperature-sensitive properties of tsPS3CT Phages

taP73C’T taP73CT, revD-1 Burst-sizes were determined in the legend to Fig. 1.

30°C

Burst-sizes 37°C

7 100 on BB in M9S medium.

0.6 20.5 Conditions

at 42.5OC 0.6 0.02 are t,he same as those tlrscrihed

(ii) tsCR53, revD-2 The suppressing mutations in at least two tsCB53 k + pseudorevertants do not affect the temperature-sensitivity of the mutant noticeably. We have, however, been able to map revD-2 because, as a single mutant (obtained by ts+ reversion of tsCB53, revI)-2), it retains a special property: it grows on some tabC mutants. tabC comprises a class of bacterial mutants which are distinct from tabD and map near the ilv cluster at about 75 minutes on the E. c&i map (Taylor & Trotter, 1972). We know very little about this class, except that it is somehow involved in T4 replicationt. One tabC mutant, tabP-5521, is of interest to us at this point: T4 wild-type fails to grow on this tabC mutant at 42*5”C, while ts + , revD-2 grows almost normally : in other words revD-2 is a cornc mutant (cf. nomenclature in Coppo et al.. 1973 : Takahashi et al., 1975) (Table 3). t l&CL3 is a common mutation; we have classified about 20% to6 mutants selected with T4 wild-type at 42.6”C as t&C. We have also isolated tabC mutants (one of these is t&C?-5521) b!, using ta663 as selector at 30°C. On these mutants, ts653 plates with a very low efficiency, but, is always somewhat leaky, especially in 0.6% top-agar. Usually collzc mutants do not map in gene 4:i and restore phage replication at 42~5°C to almost normal levels. te+,revD-2 plates on tabCts-5521 at 42.S°C, but does not overcome the block on phage development in M9T medium at 42.6OC. to anv We have as yet, been unable to relate the beheviour of ts563 and ts + ,~ewD-2 in t&Y-5621 obvious phenotypio oharacteristic.

606

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TABLE

‘41,.

3

Eficiency of plating of ts + , revD-2 on t&C%5521

Phages

1 1 1

T4wt teCB63, rewD-2 te+,rewD-2 Effiaienoies

Bscterial strains BB (42.b”C) (30°C)

CR63 (42&X!) (30°C)

of plating

1 1.1 1

1 <6x

1O-5 1

were determined

tabcW6621 (42.5’C) (30°C)

1 <4x

10-S 0.96

using 0.6% Hershey

0.89 1.1 0.98

<7x <6x

10-S 1O-5 0.71

top-aggar.

Exploiting this property of revD-2, we have been able to map it with respect to known gene 45 mutants (teCB63, amEl and arnNG18). Consider first the two-factor cross (Fig. 2(a)) : tsCB53, revD-2 x te + , rev + . Neither parental strain plates on tabP-5621 at 426”C, while the ts+ , ana+, rev recombinant does grow. The recombination frequency between tsCB53 and revD-2 can thus be simply determined by plating the issue of the two-factor cross on tabC at 426°C. This frequency turns out to be fairly low (about 2% ; Table 4A) showing that tsCB53 and revD-2 are close. Let us consider now the three-factor crosses: tsCB53, revD-2 x ts + , rev + , amEl tsCB53, revD-2 x ts+, rev+, amNG18. Tw;;Ma;tor

LsscB53 ---~ (a)

I L--

tst

lSCB53 (b)

ts+

Three-factor. crosses

(c

am+ am+

,--revD-2 L ---rev+

isCB53 isi-

rev&2 rev-k

r----

urn+ am+

urn+

revD-2

urn

rev+

(, )

1 MJ353 ts+

Cd1

am+ ---~ om

p--, ---

, L---

am+ revD-2

(2) om rev+

lsC053

ts+ --_

r--I

MD-2 lW+

FIG. 2. Mapping rewD-2: reoombinational events leading to the formation of te+, veuD-2 reoombinmts. (a) Two-factor mosses; (b), (c) and (d) three-factor orosses. For explanation see text.

GENETICS

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TABLE 4 Mapping revD-2 A. -

Frequenoy

of recombinants

in two-faotor

oross Recombinant

ta+, arn+, rev

Cross0s

(%I

classes ts+, am+, rev+ (%I

teCB63, revD-2 tsCB63,

TW+

1

x T4 w.t. -

x amEl

tsCB63, rev+ x rcmNGl8

B.

Frequency

of reoombinants

in three-factor

C.

Frequency

crosses selected on taKts-6621

at 42*6”C

Recombinant class fa + , am+, rev (selected on t&C) (%)

C’rosses

taCB63, revD-2 taCB63, revD-2 teCB63, revD-2

1.6 1.3

x amEl x amNG18 x T4 w.t. (control)

of recombinants

in three-factor

0.7 1 1

crosses selected on BB at 42*6Y! Recombinant t8+, am+ (%)

Crosses

taCB63, revD-2

x amEl

tsCB63. revD-2

x avaNGl8

class

1.6 (of which 66% rev) 1.4 (of which SOe/0rev)

Crosses were aarried out at 30°C on CR63 in Hershey broth. Cells were grown to 4 x 1Oe ml at 37% and portions were infeoted at 30°C with equal vohnnas of phage mixes at 4 x lo9 ml (total multiplioity of infection = 10). Infected cells were aerated for 90 min at 30°C and then lyaed by the addition of chloroform. Ratios of parental genomes (1: 1) were oheoked in the input mixes and progeny yields. Plating was in 0.6% Hershey top-agar. A., Two-factor cross; B and C, threefaotor crosses. w.t., wild type.

If the issue of such crosses is plated on CR63 (tab+, su+) at 30°C parental and recombinant classes all grow. If the progeny is plated on BB (to& + , SU- ) at 42*5”C, only ts+, am+, rev and ts+, am+, rev+ recombinants will grow, while if the progeny is plated on tabCY-5521 at 42*5”C, only the ts + , am + , rev recombinant class grows. By comparing the frequency with which the ts + , am + , rev recombinants are produced in the two-factor and three-factor crosses and by determining the fraction of rev among ts + , am+ recombinants selected on BB at 42.5°C in the three-factor cross, we determined the actual order of the markers. Possible orders of the am, ts and rev markers arc shown in Figure 2(b), (c) and (d).

60s

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In Figure 2(b), the ts+, am+, rev recombinant is generated by a single recombinational event between rev and ts at a frequency which, relative to the two-factor cross. should be unaffected by the am mutation in trans. In Figure 2(d), the ts+, am+, rev recombinant is generated by two recombinational events, one between ts and rev and one between ts and am. Its frequency should bc lowered by at least an order of magnitude with respect to the two-factor cross. In Figure 2(c), the ts+, am+, rev recombinant is generated by a single recombinational event between ts and am. If the amber mutation is much closer to ts than to rev, the frequency with which the ts+, am+, rev recombinant is formed will be much lower than in the two-factor cross and it will be easy to distinguish between Figure 2(b) and (c). If, however, the amber is much closer to rev than to ts (Fig. 2(c)(2)), the reduction in the recombination frequency will be imperceptible and it will bc impossible to distinguish between Figure 2(b) and (c)(2). As shown in Table 4B, the frequency with which the ts+, am+, rev recombinant is formed in the three-factor cross is not affected by amEl or amNGl8 in bans, and the actual order must be that outlined in Figure 2(b) or (c)(2). It is possible to distinguish between Figure 2(b) and (c)(2) by determining the frequency of rev as unselected marker among ts + , am + recombinants selected on BB at 42.5%. If am is much closer to rev than to ts, the proportion of rev+ will be much lower than that of rev among the ts + , am + recombinants. As may be seen in Table 4C the actual proportions of rev+ and rev in the two crosses are about the same; this result eliminates the ambiguity in the choice between Figure 2(b) and (c)(2). The true order is shown in Figure 2(b). Since tsCB53, amEl and amNG18 arc known to be in gene 45, and since revD-2 is situated between tsCB53 and the amber mutants, also revD-2 must be in gene 45. (iii) ts553, revD-3 We have seen that, in its reversion properties, ts553 is the most canonical of the kD mutants: reversion from k to k+ without concurrent loss of gene 55 temperaturesensitivity is rare (about 5/100). A clear cut feature characterizes the single ts, k+ pseudorevertant of ts553 (ts553, revD) we have studied: it is more temperature-sensitive than the original ts553 k strain. The original ts553 is rather leaky; when about 10s mutant phages are plated on BB at 43”C, the indicator lawn is “chewed-up”, indicating that some phage growth occurs, while at 41,3”C it grows almost normally. The ts -+ ts + reversion frequency of ts553 at 43.5”C is about 3 x 10m6. On the other hand, tP553, revD-3 is completely inhibited at both 43°C and 4103°C. The reversion frequency is less than low7 at 43~5°C. These data are summarized in Table 5A and suggest that the pseudo-revertant is a double temperature-sensitive mutant, Exploiting the clear cut differences in temperature-sensitivity between ts553 and ts553, revD-3, it is possible to differentially test the gene products involved, by carrying out complementations at the two temperatures. We used amber mutations amBL292 (gene 55), amEl (gene 45) and amN54 (gene 31). The results obtained can be summarized as shown in Table 5B. Since ts55.7, revD-3 fails to complement nmBL292 at 43@C, presumably tllca original temperature-sensitive mutation in gscne 55 is retained. Since it also fails to complement an&El0 either at 43*5”C or 41*3”C, a new temperature-sensitive mutation

GENETICS

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5

Characterization of ts553, revD-3 and tsA81, tsx I\.

B.

Temperature-sensitivity

of ts663, revD-3

Phage mutants

37°C

ts553 ts653, rewD-3

+ +

Complementations

Temperatures 41.3”C

Temperatures 41.3”C 43@C

1.9563 + amBL292 (gene 55) ts563 -1 umEl0 (gene 45) (gene

31)

~553, revD-3 + amBL292 ts563, rewD-3 + amEl ts553, revD-3 +-- amN64

C.

Complementation

+ -

with ts553, revD-3

Phage mixtures

18553 + amN64

43.5”C

-I1.

j.

+ + I-

+ +

with taAS1, tsx

Phage mixtures tsA81, tsx + amBL292 (gene 55) tsA81, tsx + amEl (gene 45) tsA81, tax + amN66 (gene 24)

Temperatures 42.5”C 43.6”C i_ +

i-

Complement&ion was performed by mixing the appropriate viral strains (2 x lo* phage/ml) and spotting on tab+ (BB) in 0.7% Hershey top-agar overlayered on Tryptone-agar plates. After spotting, plates to be tested at 43.5“C were preincubated in a 60°C incubator for 10 min. Preincubation is necessary to avoid leakiness in t&63.

gene 45 must be present in the pseudo-revertant. Furthermore, the temperaturesensitive mutation in gene 45 (ts45) and rewD-3 coincide, since at least some ts+ revertants picked at 41.3% (which have lost the P45 temperature-sensitivity while retaining P55 temperature-sensitivity) regain the original kD gene 55-deficiency (of 96 ts+ revertants in gene 45 which retained ts553, eight were kn on tubD-5517 at 30°C). In the 88 other revertants that retained their ability to plate on tabD-5617, in spite of t’he loss of ts45, the latter probably did not revert to the wild-type allele but to a ts+ mutant allelic form which conserved the revD-3 mutant property. As an additional check of the identity of ts45 and rewD-3 we tried to separate the two by forcing recombination between ts45 and amber mutations in genes 45 (amE10) and 46 (amN130) as described in Figure 3. Since we were unable to separate the two phenotypes, .we conclude that revD-3 is either very close to ts45 or, as strongly indicated by t,he reversion analysis, is identical to ts45.

in

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. cross

cross A

B I 13553 7 .

ts553 ; I $@! l

645

I

r----J t ; o(I)

am NE0 (gene 46) : I ’

umElO(gene45)

i r--- .1 ; (2)

.

I

FIN. 3. An attempt to separate revD-3 from te45. (a) Possible position of rewD-3, if reuD-3 + t846. teEi alone does not grow ct 43*6”C!, but does grow at 41*3”C!; ts46 does not grow at 41.3% ta+. urn+ recombirmnts at 41.3% were selected by plating the issues of crosses A and I3 on BB(au-) 8t the non-permissive tempereture. (1) Represents the selected recombinational events, while (2) represents non-selected recombination81 event~s. 136 of In oross A, 2% of the progeny w&s represented by nm +, ta+ (at 41*3’C) recombinants. suoh recombirmnts were tested, and 120 were found to be k+ on l&D-6617 and ts+ on BB at 43*6”C, while 16 were kD and temperature-sensitive at 43.6”C. None were simultaneously t,empertlture-sensitive at 43.6% and k + . In oross B, 6% of the progeny WBS represented by am+, la+ (at 41.3OC) recombinents. 128 of such were tested and 77 were found to be k+ on t&D-6517 at 30°C and t.~+ on BB at 43.6”C, while 47 were kD 8nd tempereture-sensitive at 43.6’C. None were simultaneously temperaturesensitive at 43*6”C and k+ , If revD-3 were distinct end loosely linked to t846, some of the recombinents, temperature-sensitive at 43~6°C but not at 41*3’C, should have been k+.

(c) Effect of revD-2 and revD-3 on replication Since revI)-2 and revD-3 map in gene 45, it was of interest to see what their effects would be on replication. At 30°C revD-2 depresses replication when coupled with t&B63 (Fig. 4(e)), but not as a single mutant (Fig. 4(b)); similarly, VevD-3 depresses replication in the double mutant ts553, revD-3 (Fig. 4(c)) (we have not isolated the single mutant ts+, revD-3). Similar patterns of replication are observed on tub+ (data not shown); the double mutant ts553,revD-3, as expected, does not replicate at 42*7”C (data not shown)?. (d) f&cod-site mutdio?~9 in

of gene 55 temperature-sensitive mutants (ts553 and tsA81)

.!y8a&.s

As mentioned in Materials and Methods of the accompanying paper (Coppo et al., 1975), a stock derived from strain ts553 x 4 described in Pulitzer (1970) and in Pulitzer & Geiduschek (1970) which originally replicated at 43°C in M9S medium (Diggelman et al., 1970), acquired a secondary temperature-sensitive mutation which blocks replication at the non-permissive temperature (Beguin, 1973; Bolund, 1973). We will refer to this double mutant as ts553, tsx. t It is not possible to relate the effeots of revD-2 and revD-3 in tiD to the observed depression in replioetion at 30%. tsP73CT replioetion is also depressed at 30°C (Fig. l), however it behaves 8s 8 kD mutant.

GENETICS

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6-

20

40

6( Time after infection (min)

1

FIG. 4. Repliaation of k + pseudo-revertants on tabDkaaa at 30°C. Infeotion was in MQT medium et 30°C at e multiplioity of infeotion of 7 by the prooedure desoribed in Takahashi et al. (1976). Lsbelling wes with [m&y&%]thymidine at a apeoiflo motivity of 2 @i/30 erg per ml. Portions of 0.2-ml were taken at the times indiaatad and the inoorporation of the radioeative precursor in triohloroecetio said-preoipitable material WSB determined. (0) T4wt; (A) WB63; (A) tiB63. revD-2; (0) f&63; (0) ta663, reuD-3; (m) te+, WUD-2.

The appearance of a secondary temperature-sensitive mutation (tsx) affecting replication in t&53 was puzzling, especially in view of the fact that a secondary temperature-sensitive replication mutation had also been observed early in the characterization of another gene 55 temperature-sensitive mutant, t8A81 (Pulitzer, unpublished observation). Since in t&3, revD3 a mutation in gene 45 somehow suppresses a phenotypic trait of t&3, it was of interest to reinvestigate the t&63 double mutant (t8563, tsx). t8663, t8x behaves like t8553 at 3O”C, since it fails to plate on tubD bacterial mutants. In both cases we established by complement&ion tests, that the kn phenotype was due to an impairment in the function of gene 55. When t&i63 and t&53, t8x were complemented on tab + (BB) at 43.5°C with umBL292 (gene 55), amEl (gene 45) and umN65 (gene 24), t8553 complemented the amber mutants in genes 45 and 24, but not that in gene 55, while tsb53, t8x complemented the amber in gene 24, but not those in genes 55 or 45. Apparently t8x is in gene 45. We also tested the other temperature-sensitive mutant in gene 55, teA81, derived from a stook used by Pulitzer (1970) and Pulitzer & Geiduschek (1970), whioh also replioated in 11698medium at 43°C after having been freed of seoondary mutations. We found that this viral strain, plated on BB at 42*5”C, gave a reasonable number of ta+ revertants (0.8 x 10ee). At 43.5”C, however, t.s+ revertants were less than lo-‘, suggesting that taA8l was a double mutant (i.e. tsA81, tsx). Indeed, DNA synthesis is blocked in

612

A. C’OYPO

E:L1’ &4L .

tsA81 in M9T medium at 42*7”C. When &A81 was complementrcl witJi nnrBLZ9:! (gene 55), amEl (gene 45) and amN65 (gene 24), the pattern shown in Tahlc SC was observed. The secondary temperature-sensitive mutation in tsA81, tsx is agaiu in gene 45. We suggest that the appearance of temperature-sensitive mut’ations in gene 45 in lysates of gene 55 temperature-sensitive mutants is not fortuitous, but reflects a selective advantage of the double mutant at 30”Ct. The idea is reinforced by the results of reversion analysis. We have seen that when k+ revertants of ts553 are picked on t&D-5517, most (about 95%) are also ts +. When, however, we picked k+ revertams of ts553,tsx or tsA81,tsx on t&D-5517 the results were quite different. Of 15 k+ revertants of ts553, tsx all, of course, retained the gene 45 temperature-sensitivity, but, unexpectedly, only two lost the gene 55 temperature-sensitivity. Similarly of 11 k+ revertants of tsA81, tsx, only one lost its gene 55 temperature-sensitivity, while ten were temperature-sensitive in both genes 55 and 45. Thus, while the single mutants tend to lose the kn property by reverting to ts+, the double mutants appear to preferentially loose the F’ property by gaining an additional mutation. Presumably. tsx is already a partial suppressor of the gene 55 kD phenotype (indeed, in plating experiments on t&D-5517 at 3O”C, ts553, tsx is clearly leakier than ts553). (e) Behuviour of suppressed phage T4 amber m&ants on tabD-533 (su + ) Suppression of amber mutants occurs by the translation of the polypeptide termination codon UAG as an amino acid. Three well studied suppressors, szl-1, su-2 and a-3, translate UAG respectively into the amino acids serine, glutamine and tyrosine. It is. thus, likely that suppression of an amber mutation in a given gene, by at least one of the suppressors, will lead to the synthesis of a protein with mutant properties. Is the kn phenotype a mutant property conferred by the suppression of amber mutations in genes 55 and 4511 To determine this, su-1,526-2 and 526-3derivatives of tabD-533 and the tab + parental strain were constructed. The plating efficiencies of amEl (gene 45), amBL292 and am552 (gene 55) and amB22 (gene 43) on these a + strains are shown in Table 6. amBL292 suppressed by su-2 shows a clear cut kD phenotype. The kD phenotype of am552 suppressed by m-2 at 30°C is correlated with the previously described (Pulitzer, 1970) suppression-induced temperature-sensitivity of this mutant. am552 behaves as a kD mutant at 42*5”C also when it is suppressed by su-1. The gene 45 mutant, amEl0. behaves as a more or less leaky kD on all suppressor tabD-533 strains; the kD phenotype is, however, most clear-cut on tabD-533 (~-2). Complementation tests carried out on tabD-533 (m-2) establish that the kD phenotype of amBL292 is due to a malfunction of gene 55, while the kD phenotype of amEl is due to a malfunction of gene 45 (Table 7). (f) Isolation of a non-temperdwre-sensitive kD mutant (kD-292) and a new ta,bD mwtand with unusual properties (tabDkzg2) Having ascertained the kD properties of suppressed amber mutants in genes 55 and 45, we isolated am+ revertants of amBL292 and amEl in the hope of picking up t The effectiveness of selection may have been enhanced by the fact that, for a time, lsA81 and k9653 lysates were grown serially from single plaques (i.e. a new lysete was grown from a single plaque of the older lysete).

Plating

eficiencieu

Bacterial strains

of suppressed awher mutantv OWL t&D-533,

Temperature (“C)

T4wt

nmEl0

Viral strains nmBL292

su + derivatives

am552

//rnB32

tub , SIC-1

30 42.5

1.0 1.0

I.0 1 .o

14 0.9

1.0 1.0

1.0 I.”

trrh ‘-, 8 I, - 2

30 42.5

0.9 1.0

0.9 0.8

0.9 0.9

1.0 2 >: 10-G

I.(1 1.0

td +, su-3

30 42.5

I.0 I.0

I.0 1.0

I.1 I.2

t&D-533,

su-1

30 42.5

I.0 1.0

10-Z 4x 10-7

t&D-533,

SW-L’

30 42.5

I.0 I.1

10-B 10-7

30 42.5

I.0 I.0

10-S 10-e

l&ID-537 * , W-3

Efficiencies platns.

of plating

were determined

Complementation Phage mixtures amBL292 (gene 55) amEl (gene 45) a7nB22 (gene 43) t8653 (gene 55) tsCB63 (gene 45) amBL292 + amEl amBL292 + amB22 amBL292 + t8663 amBL292 + t&B53 anaEl0 + amB22 amEl + ts563 amEl + taCB63 amB22 + t8553 amB22 + t8CB53 t8553 + t&B53

in tabD-533,

0.3 0.2

0.4 10-s

I.1 1%I

5x IO-4 G x 10-s

10-s 10-e

0.9 1.1

1.2 I.0

in 0.5 9/, Hershey

TABLE

I.0 1.1

top-agar

_-

overlayered

I.1 I.0 on Tryptone-agar

7

su- an& tabD-533, m-2 at 35.577

in l&D-533, 1.3 2.3 0.6 0.3 0.6 25.6 117.0 2.1 23.0 82.0 32.0 2.0 73.0 79.0 31.0

Burst-sizes BUin t&D-533,8u-2

0.6 0.7 0.02 0.65 50~0 0.2 34.5 43.1 0.4 -31.1

Complementation tests were carried out by growing bacteria to 4 x IO* cells/ml in M9T medium (Takahashi et 0.2.. 1975) and infecting a portion with an equal volume of phage mixes at a concentration of 4 x lo9 phage/ml (multiplicity of infection 5+5). After 5 min adsorption, T4 antiserum was added at K w 1 and incubated for an additional 4 min; at this time, the infected cells were diluted lO,OOO-fold into M9T medium aerated at 35*5”C. At 60 min after infection, t,he cell* were lysed by the addition of chloroform. Plating was on CR63(8u+ ).

.I.

014

COPPO

E!L’ AL.

TABLE

of tabl)k2Q2 and kD-292

Growth properties A.

Plating

efficiencies

on tabDkZs2

Viral strains

Bacterial strain t&D”292

T4 w.t. tab63 tsbb3, revD-3 taCBb3 taCBb3, revD-2 kD-292

0.43 0.6 x 1O-B 0.73 0.6 x 10-B 0.66 2.6~ 1O-3 (small plaques) 3.4 x 10-s (leaky background) 0.9

tsP73CT taP73CT, revD-I

B.

Plating

efficiency

Viral strain

of kD-292

t&D-6617

k=-292 (very

C.

Burst-sizes

8

Bacterial strains t&D-6644 t&D-633

0.6 0.9 0.3 small plaques on all 3 strains)

on t~bD~*~~

Viral strains

Bacterial tabDk292

T4 w.t. l.8653 18663, revD-3 t&B63 taCBb3, revD-2 t8+, revD-2 k=‘-292

18.5 0.09 lb.8 0.7 5.3 21.2 0.51

strains tab + 34.7 37 43 9.5 14.5 25 21

A and B. Plating efficiencies were determined in O.b% Hershey top-agar overlayered on Tryptone plates; C. Burst-sizes were determined in M9T medium at 30°C (multiplicity of infeotion = 7). Conditions of infection were standard (Takahashi et al., 1976). w.t., wild type.

among these, non-temperature-sensitive am + , kD mutants of the type described in the tabB system (Coppo et al., 1973; Takahashi et al., 1975). While none of about 100 am+ revertants of amEl retained the kn phenotype, we were successful with amBL292; out of 15 am+ revertants of this mutant, one retained the kD phenotype. This am+, ts + ,h+’revertant of amBL292 (IP-292) is somewhat leaky on MD-533 as judged by the chewed-up bacterial lawn observed when about lo6 kD-292 particles were plated on MD-533. For this reason, kD-292 was used as selector in the isolation of a new bacterial mutant, tabDk2Q2. The plating properties and burst-sizes of the new viral and bacterial

GENETICS

OF LATE

RNA-DEFECTIVE

MUTANTS

615

mutants are shown in Table 8, where they are compared to the other members of the tabD system. The specificity of the permissive and non-permissive combinations generated by the interaction of tabDk2e2 with the various T4 mutant members of the tabD system is intermediate between that observed with MD-5517 on one hand and t&D-6544 and 533 on the other; ts553, rewD-3 plates well on tabDkaQ2and t&D-5517, while tsCB53 is more strongly restricted on tabDk2Q2and 5544 than on t&D-5517. (g)i Phage T4-specijic protein synthesis and nucleic acid synthesis in tabDk2 Q2 Phage proteins synthesized in tabDk2Q2 infected by various T4 mutants were labelled by the addition of 14C-labelled amino acids either from 12 to 60 minutes after infection at 30°C (late labelling), or from 3 to 15 minutes followed by a second portion of r4C-labelled amino acids from 15 to 60 minutes after infection (early + late labelling) . In both cases, late protein synthesis in tabDkzg2 infected by T4 wild-type is depressed relative to tab+ infected by T4 wild-type (the variability in burst-size of TP wild-type on ta6D k292 5 to 30 progeny phages/cell, is probably related to this depression) and P32 overproduced. However, when the non-permissive conditions are imposed by infecting with ts553, t&B53 or kD-292, the additional inhibition of late protein synthesis is unimpressive (Plate I). When rest,riction due to ts553 is relieved by the secondary mutation in gene 45, revD-3 , late protein synthesis is restored to the level o’bserved when tabDk2Q2is infected with T4 wild-type, and stimulated above this level when the bacterial mutant is infected with ts+ , revD-2. Observations on late transcription in tabDk292 are consistent with the *protein synthesis experiments: on the one hand, the relative proportion of late RNA is reduced (compared to tub + ) upon infection with T4 wild-type and, about to the same extent, by infection with tsCB53; on the other hand, late transcription is even more strongly inhibited when infection is by ts553 or kD-292 (Table 9; Fig. 5). Moreover, late transcription is restored to the wild-type level when infection is by ts553, revD-3 and stimulated over the wild-type level when infection is by ts+ : rewD-2. TABLE 9 Late tramwiptim

Viral

strains

T4 w.t. t&53 t&63, revD-3 kD-292 tsCB63

ts+, revD-2

Time of labelling (min) 20-22 20-22 20-22 20-22 20-22 29-22

in tabDkZg2 “,(, label not competed by 2 mg cold RNA/ml (L) (El 23 11 “3 13 20 27

10 11 12 I2 11 10

Conditions of i&&ion at 30°C were standard (Takaha.shi et al., 1976). RNA was labelled with 20 pCi [sH]uridine/ml and wea extracted at the end of the pulse as described in Coppo et al. (1976). Hybridization-competition was in DNA excess and followed the procedure of Belle et al. (1968). [3H]RNA, 3 to 4 pg/ml; DNA, 8 pg/ml. E. early competitor prepared 6 min after infection at 30°C; L, late competitor prepared 20 min after infection at 30°C. w.t., wild type.

616 __.

IO /~553---tabD~~~~ 0.0 0.6

1

.-I ---1~-.--..105 IO 15 Cold competitor(mg/ml)

20

FIG. 5. Detailed anelysk of T4 RNA synthesized in k~blP~~. This is a mixed-comp&itor experiment (Bolle et ccl., 1968). Conditions are those described in the legend to Table 9. (0) 5.min unlebelled RNA from T4 wild-type infected f//b+ ; (a) XI-min RNA.

We feel that the low burst-sizes observed under non-permissive conditions may not be wholly accounted for by the unimpressive overall reduction in late synthesis. A closer examination of the gels shown in Plate I reveals two interesting details: the cleavage of T4 head precursors (Laemmli, 1970) appears to be preferentially affected. P23 modification to P23* is inhibited even in t&B53 infected cells. in which the reduction in late protein synthesis and transcription is only slight; also noticeable is the inhibition of the cleavage of internal protein IPIII to IPIII*. These effects are likely to be an important cause of the reduced burst-sizes. On the other hand, the synthesis of some proteins seems to be almost completely abolished (for instance P9). We tentatively propose that the tabDkzB2mutation affects the amount or kinetics of synthesis of some late gene products more than others, and that at least one of the preferentially inhibited proteins is involved in T4 head maturat,ion. Further work will be necessary to check this idea. (h) The bwterial wmtantv According to the hybrid complex model defined in the preceding paper (Takahashi et al., 1975), a tab mutant identifies a host gene coding for a protein which is directed to the performance of viral functions by interaction with specific phage proteins; the genes coding for these viral proteins are identified by corn and/or k mutants. In the tabD conditional-lethal system, we have seen that k mutants map in gene 5s and 45. Since P55 has been shown to copurify with the (a),/3,53’ (core enzyme) subunits

P34 P37

P23 P23* P32

PLATE 1. Late protein synthrds in tr~bl)~~~~ at XJ’C’. tloct~~cyl ~r~lphittc~-I)~)l~~rt~\-lwmidr: Xutoradiogram of T4 proteins separat,cct on a 12.5? o sodium dab gel (cf. Takahashi et cd., 1975). amino acitls/ml (NPW England Nuclear) from 12 to 60 La,belling was with 1 PLci “C~-labelled Ilnin after infection in M9T medium, at 30°C’. (‘ontlitions of infwtion aw itlantical tc) thaw tlcwribed ill ‘Ltkahashi et d. (1975). w.t., wild typo, IJOC;II~ p, tilli

GENETIC8

OF

LATE

RNA-DEFECTIVE

615

JLUT.-\BTS

of E. CO& RNA polymerase (Stevens, 1972), and since at least the p subunit is used throughout infection for t,he transcription of T4 genes. we expect that the f&D mutat’ions identify one or more of the genes coding for the subunits of the host RN.4 polymerase. TABLE

Mappi/rg .I.

tabD-533

10

by phuge PI tmnsductio~l:

three-factor wo6se.s

Dorm-, HTC650 (pw+, TifS’, t&D-633) Recipient, HTC747 (purD, rif-7, lab+) Selected

So. of transductants

Unselected

IZi

tub + , rij’

0.337

I) 3

Inb’ ) rif s tab, rif p trrb,r(ffs (Total

Yrequency

‘: 0~00:~ O~OOX () 6 5 5

247 377)

13. Donor, HTCGKO (met+, art+, rqs, tabD-633) Ilecipimt, 272 (metB, argH, rv-22, tab+) SPleatetl

TJnselectetl

trrg +

tub+, rif’ ttcll+, rif tub, Tif 1 tatI, rifS

So. of transtluctants

61 4 1 06 (Total

c’.

I’recpenq 0,376 0+?*5

0.006 0.593

162)

Donor, HTC663 (met+, arg+, rif-7, tabD-533) I?ecipient, HTC219 (metB, argE, tip, tab+) Unselected

i-if’

No. of tra,nsductants

met, m-g, tab+

0.033 047CJ
(Total

3 61 II “0 0 0 0 7 91)

0.032 0.596 0.372 -co.011

(Total

3 56 35 0 94)

met, arg, tab met, m-g+, tab+

met, m-g + , tab met + , arg, tab+ met+, arg, tab met+, arg+, tab+ met + , m-g+, tab

wg +

tub+, rif’ tub+, rif 6 tab, rif’ tab, rif s

Phage Pl transduction was carried in Takahashi et ul. (1975).

Frequenq-

out using PlCMclrlOO

following

the procedure

desoribetl

This map w&s constructed

FIG. 6. Map position of tabD-633. from the Pl transduction data shown in Table 10.

Preliminary crosses with tabD-5617 showed that indeed, this mutant maps in the region (79 min; Taylor t Trotter, 1972) between argE-H and purD (data not shown). This region is of particular interest because it is where all the known RNA polymerase mutations map (Tocchini-Valentini et al., 1968; Yura t Igarashi, 1968; Khesin et al., 1962; Austin et al., 1971; Iwakura et al., 1973; Miller, manuscript in preparation). More accurate mapping data by Pl transduction in two and three-factor crosses show that t&D-533 maps just to the right of a rifr mutant (rif-7) (Table 10; Fig. 6). Similarly tabDkzQ2 maps close to a rif’ mutant (rif-Ge) and tabD-5Fi44 and 5617 map close and to the right of r$‘-Ge (Table 11). TABLE

11

tabD three-factor-crosse,v freqolency of unselected recomb&ants among pur + phuge PI-mediated

transdudants Unselected

tab mutants rijif’

tnbD’a9a

t&D-6644 t&D-6617 MD-633

markers

tab +

tab rip

rif

Colonies Frequency

66 0.36

2 0.012

6 0.03

92 0.69

Colonies

Frequency

73 0.4

6 0.03

13 0.07

92 0.6

Colonies Frequency

37 0.34

0
10 0.09

62 0.67

Colonies Frequency

27 0.34

0 < 0.002

3 o*oos

247 0.66

Phage Pl transduction w&s carried out using PlCAPolrlOO as described in Tekehaehi et al. (1976). Recipient for experiments with tabD has2, tabD-6644 and m6D-6617 w&8 68182c, thy (argH, @&I, tab+, purD). Selection w&s for pm-+. The experiment with tabD-633 is the cleme ELSthet shown in Table 10A.

GENETICS

OF

LATE

RNA-DEFECTIVE

MUTANTS

619

There is now good evidence that the gene coding for the /3 subunit of RNA polymerase (identified by at lea& some of the rip mutations) and the gene coding for the ,3’ subunit are situated in the same operon and are transcribed from left to right in the order B to /?’ (Errington et al., 1974) ; there is, also, genetic evidence that an additional member of the operon is situated to the right of /?’ (J. B. Kirschbaum, personal communication). We have not been able to prove that the tabD mutants map within the rif cluster (i.e. between two different riF mutations). Since the crosses establish only that the tabD mutants are clustered close to the rif cistron, it was important to establish the relationship between t&D alleles and rif alleles in merodiploicls. (i) Behaviour of tabD mutants in merodiploids In E. wli it is possible to construct stable merodiploicls: in such strains each cell carries two sets of a given portion of the bacterial genome, one on the chromosome and one on a substituted episome (F’). The stability of this arrangement is ensured if the chromosomal segment carries a gene defective in some metabolic function, while the episomd segment carries the functional allele: if, in addition, recombination between episome and chromosome is prevented by a mutation on the chromosome in the recombination gene recA (Clowes & Moody, 1966), growth in minimal media lacking the appropriate metabolite will only be possible if the episome is retained. We have used merodiploids constructed with an episome KLFlO which is known to span the rijregion (Austin et al., 1971) (Fig. 7), and have screened for Tab+ and Tab phenotypes by cross-streaking merodiploid isolates with appropriate phage mutants.

(78.0) I met

From Austin

, I urgE-H

(7?0)

(81-O)

\

I

I

I

bfe

n’f

purD

I 1 ma10

FIG. 7. The KLFlO episome. et al. (1971) and Taylor & Trotter (1972).

(ii) tabD is recmsive to tab+ F- , tabD, arg, SW, recA recombinanta were constructed and the arg+, tab + , rifs, KLFlO episome was introduced into the tabD recombinant by crossing the latter with KLFlO/XAlOOc (KLFlO/arg, pzbr, pro, etrs) (see Material and Methods), and selecting for arg+ (by omitting a&nine in the minimal selective media), and counter-selecting with streptomycin. Such merodiploids carrying t&D-5544, -533, -5517 and tabDkzg2 are all phenotypically tab + , showing that these t&D alleles are recessive. (iii) Compkmentahm between tabD mutants To determine the cistron assignations for the tabD mutants, we have isolated a derivative of KLFlO carrying the tabD-533 mutation, and a derivative of KLFlO carrying tabDkaea. The episome KLFlO-rif-7-t&D-533 was introduced into t&D, recA recipients; we found that, while tabD-533 does not complement tabD-5544, it complements weakly tabDkaea (Table 12). Similarly, when KLFIO, tu&Dk202 was

.\.

(.‘OI’l’O

TABLE

Summary

Episomes

dominance

tnb +

tabD-5544

tab +

KLFlO, l-if-7 KLFlO, KLFlO, KLFlO,

tabD-533,

12

tabDkzg2 r$;$

+ +

tif&,Dla

+

+

K--lo,

rif&/s

i-

-

tests with tabD

Chromosomes tabD-533, tabD-533, T’if rif-Ge

trrbD-6617,

rifS

-t

(TV“)

WfY

(rif “)

T

t

i

+

+ (+-P)

+

-

t (rim -

ttrbJFg2

+

1-

(Tif “)

‘ 4/. 2

and compleme~atdion

KLFlO,

I-

ET

-

+

-t

&A+

-

@if 7 + Indicates a tab + phenotype; - , indicates a tab phenotype. tab phenotype was determined for each strain by cross-streaking at least 8 individual colonies, picked from minimal citrate selective plates and resuspended in 10 ~1 droplets of minimal citrate buffer, against streaks of t8653, &CBS3 and T4 wild-type (lo* phage/ml in T broth) applied to Tryptone-agar plates. Plates were incubated overnight at 36 to 37°C; both t&63 and t&B53 grow well at this temperature and adsorption to the bacterial strains is better than at 30°C. Strains were classified as tab when bacterial growth w&s not inhibited at the intersection of the bacterial streak and the ts663 (and/or tsCB53) phage streak, but was inhibited at the intersection with the T4 wild-type streak; classification was tab+ when the bacterial growth was inhibited both by the ts and the T4 wild-type streak. The notation + indicates that inhibition of bacterial growth, at the intersection between phage ts mutant and bacterium, is incomplete (presumably phage production is less than normal). t This diploid, as well as a diploid oonstructed with KLFlO, rg’, appears to be partially resistant to rif at a concentration of <60 pg/ml in minimal citrate plates. $ rifz is representative of 7 independently isolated and unoharacterized rip mutants.

introduced into t&D, recA recipients, it failed to complement itself, but weakly complemented t&D-5644, -5517 and -533. These observations allow us to assign t&D-5544 and t&D-533 to the same cistron (we have not established whether t&D-6517 also belongs to this cistron). The weak complementation between tabDkZe2 and the other three tabD mutants is in itself difficult to interpret since tabD mutants are missense mutations which might permit intracistronic complementation. However, in view of the result of the next experiments, it probably does reflect the fact that tubDk2Q2 and tabD-5544, 533 and 5517 are in different cistrons. (iv) Dominance relationships between tabD and rif” mutants rip/@ merodiploids are phenotypically rifampicin-sensitive; however, as shown by Austin & Scaife (1970), it is possible to isolate rif’ derivatives of such diploids which have acquired resistance to rifampicin because of mutations which abolish the function of the wild-type rzp allele. Such mutants, called rip, can be either due to m&sense (temperature-sensitive) or nonsense (amber) mutations or, presumably, deletions. They are distinguishable from rip mutants which, of course, would render the

GENETICS

01” LATE

RNA-J)EYECTIVE

BlUTAKTS

621

merodiploid rifampicin-resistant, by their inability to give rif” recombinants when introduced into a rip, ret + recipient. Seven such r;f” mutants were isolated and characterized by the above criteria. KLFlO-rife episomes do not abolish rifampicin-resistance when introduced into rp, recA recipients and fail to generate rip recombinants when introduced into rip, ret + recipients. MD-533, t&D-5544 or tabDk2s2 recipients carrying any one of the seven KLFlO-rip episomes retain the t&D phenotype. This finding strongly suggests that all three t&D mutants affect RNA polymerase; however, since the genes coding for the p and ,8’ subunits are part of the same transcription unit, and since we cannot exclude that any of the rife mutants are small deletions or point mutations in the ,f?gene which might exert polar effects on the synthesis of fl’ and other members of thcb transcription unit, this experiment, while strongly suggesting that tabD mutations are in RNA polymerase genes, does not necessarily assign the three tahD mutations to the same cistron. (v) Dominance relationships between tabD and amber mutations in the rif gene In a further attempt to determine the cistron assignation of the tabD mutants, we investigated the relationships between tabD and two amber mutations in the which maps in the rif gene and only affects the synthesis of rif (8) gene: f-if&~ the /3 subunit; ri&,rIIls a polar mutant which maps in the rif gene, but affects thr synthesis of both the /? and /Y subunits (Errington et u.l., 1974). Each of these two mutations was introduced into the KLFlO episome by I. Claeys. thus providing an ideal genetic tool for determining the cistron assignations of RNA polymerase mutants. KLFlO-rij&,,, (non-polar, affecting /3) and KLF1O-rij&lll,, (polar, affecting ,9 and ,3’) were introduced into tabD-5544, recA, tabD533, recA, tabD-5517, recA and tabDk2s2, recA; the characterization of the resulting merodiploids is shown in Table 12. t&D-5544, MD-5517 and t&D-533 are recessive in merodiploids carrying KLFlOriflmn12, and dominant in merodiploids carrying KLFlO-tijf~,,,,,, showing that tabD-5544, -5517 and 533 are not in the /I cistron, but in the /?’ cistron or in another cistron of the RNA polymerase operon situated to the right of /3’ (the existence of this cistron has been inferred indirectly by deletion analysis : J. B. Kirschbaum, personal communication). On the other hand, tabDk2s2 is dominant in merodiploids carrying ; it is thus situated in the rif (/?) cistron. The both KLFlO-$&,,, and KLFlO-rij&,,,, localization of tabDk2s2 in a different cistron from the other t&D mutants is consistent with the complementation observed between tabDk2s2 and tabD-5544, -5517 and -533. The dominance relationships observed in merodiploids are summarized in Table 12t.

4. Discussion We have performed the experiments described in this and in the accompanying paper (Coppo et aZ., 1975) in pursuit of two aims: t The observation that complement&ion between tabDk292 and the other t&D mutants is inoomplete (Table 12), although all four tab mutants are clearly recessive when tested by crossstreaking with phage, remains to be explained. Possibly tiDkasa, or the other tiD mutants, contain one weak mutation in the rif gene and one in another member of the polymerase operon; combination of the two mutations is necessary for a Rtringent TabD phenotype.

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(1) to establish whether the tab/k/ corn system of conditional lethal mutants defined in the previous paper (Takahashi et nl., 1975) is generally applicable to the analysis of host-virus interactions, and implicitly to test the validity of the hybrid complex model which provides a conceptual framework for interpreting the interactions between viral and host mutants that define a given tab system. (2) To explore further the mechanisms involved in the control of bacteriophage T4 late transcription. In this section we will discuss our results as they relate to these general goals. (a) Post-infective RNA pdynaera.se a.s a hybrid cona$ex As shown in this and in the preceding manuscript (Coppo et al., 1975), exploiting the potential k” properties of a known mutation in gene 55 (ts553), we have been able to construct a new tab/k system of conditional lethal mutants (tabD/kD) comparable to the previously described tabB/k* system (Coppo et al., 1973; Takahashi et al., 1975). The elements of the tabD/kD system identify host and viral proteins that, upon interaction, control the synthesis of T4 late RNA. These proteins are, the viral products P55 and P45, identified by kD mutations in genes 55 and 457; the host RNA polymeraae p subunit, identified by the bacterial mutant tabDkzg2, and the fi’ subunit (or another subunit ; see below) identified by tubD-5544, -5517 and -553. In other words, by our interpretation of the mutant interactions in the tubD system, the minimal structure of T4 post-infective RNA polymerase is : P55 (13/u p45 Indeed P55 is known to copurify with RNA polymerase from T4 infected cells (Stevens, 1972) and to be a positive control element in late transcription (Pulitzer $ Geidusohek, 1970) ; Ratner (1974) has recently shown that P45 also binds to T4 post-infective RNA polymerase, and Wu et al. (1975) have shown that it is a control element in late transcription. The interpretation of k mutants as identifying phage coded structural components of hybrid complex is thus corroborated, in the case of tabD, by direct biochemical evidence. Moreover, Haselkorn et al. (1969) and di Mauro et al. (1969) have shown that the host p subunit is active in T4 transcription. That three mutants, to&D-6544, tabD-5517 and tabD-533, which block late transcription map in the polymerase operon but not in the ri,f gene is a strong indication that a subunit other than p also plays an important role in the in vivo transcription of T4 RNA. The assignation of tabD-5544, t&D-5517 and tabD-533 to the /3’ cistron by their dominance relationship with polar and non-polar amber mutations in the rif cistron, is not unequivocal. There is indirect evidence for an additional RNA polymerase cistron, to the right of the /I’ cistron, which is blocked by the polar amber mutation in rif (J. B. Kirschbaum, personal communication). However, since the tabD mutants (especially tabD-533; Table 1OC) are very closely linked to rif (Table ll), it seems t The product of viral gene 33 also hae been shown to copurify with post-infective RNA polymerase (Horvitz, 1973). However, since miasense mutationa are not available in gene 33, we did not attempt to determine the interaction of this protein in the t&D system. This may, however, be possible by using suppressed Bmber mutants in gene 33, following the procedure described in this paper.

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MUTANTS

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unlikely, barring strong marker effects, that they (with the possible exception of tabD-5517) are situated beyond the F gene. (b) Interactions

between P55, P45 and RNA

polymerme

Since RNA polymerase is a hetero-multimer, alterations in one subunit are apt to induce configurational alterations in other subunits. For this reason the cistron assignations of the tabD mutants do not necessarily establish with which host subunit P55 and P45 interact. Mutually induced distortions in /3 and p’ might explain why tabD mutants in different genes affect the interactions of P55 and P45 with RNA polymerase. On the other hand, we have no idea why the defective phenotype generated by tabDkzQ2is somewhat different from that generated by the other mutants, and seems to affect the expression of some late genes more than others; nor do we know whether this difference is significant or whether the properties of tabDkzQ2are typical of tab mutants in the j? gene. Mutually induced configurational alterations may also explain the suppressing effect of some mutations in gene 45 on kD gene 55 deficiency (for instance, in the case of ts.553, revD-3). If the conformation of each component contributes to the overall stability (function) of the RNA polymerase complex, P45 altered by mutation revD-3 might, upon binding to RNA polymerase, change the P55 binding site, so that it will accept P55 in its kD mutant form. Alternat)ively, P55 and P45 might form a complex (P55-P45) which in its turn interacts with RNA polymerase. In this case a kD mutation in gene 55 would also affect the interaction of P45 and RNA polymerase and z&e versa; a kD mutant in gene 55 might, on tho other hand, be compensated by a secondary mutation in gene 45. In the preceding paper (Coppo et al., 1975) we have also proposed a model in which individual RNA polymerase molecules interact either with P55 or with P45 ; in the former case, RNA polymerase is competent for late transcription, in the latter case it is competent for a step required for the replication of T4 DNA. fabD-induced P56 and P45 deficiencies, which affect transcription but not replication (in contrast, classical gene 45 mutations block both replication and transcription), were imagined to be due to RNA polymerase depletion by P45, resulting from an increased aEinity between RNA polymerase and P45, and a decreased affinity between RNA polymerase and P55. By this model the lower affinity of P55 for tabD RNA polymerase might be compensated for by a mutation in gene 45 (revD-3) that decreases the affinity of P45 for RNA. polymerase. The method of affinity chromatography on Sepharose RNA-polymerase columns developed by Ratner (1974), for investigating the interaction of viral proteins with RNA polymerase, should be an extremely useful tool in testing some of these ideas. We thank A. Caacino, G. Martire and F. Visco for help in the initial characterization of some of the bacterial and viral mutants. We are especially grateful to I. Claeys, J. Kirschba,um, J. Miller and S. Nasi for their advice and generous gifts of strains, which were both essential in the genetic characterization of the t&D mutants. REFERENCES .4ust,in, S. J. & Scaife, J. G. (1970). J. Mol. Austin, S. .1., Titt,awella, I. P. B., Hayward, 232, 133. 136.

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