of - Genetics · T4 mutants that overcome this block were isolated by the three groups. All of these mutations map in T4 gene 31 [involved in the ordered assembly of T4 head proteins

A MUTANT OF E. COLI THAT RESTRICTS GROWTH OF BACTERIOPHAGE T4 AT ELEVATED TEMPERATURES

JEAN L. JENSENZ AND MILLARD SUSMAN

Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706

Manuscript received September 2, 1978 Revised copy received August 6,1979

ABSTRACT

After nitrosoguanidine mutagenesis, a Phage Host Defective (phd) mutant of E. coli HfrH was isolated that supported the growth of T4D wild-type bacteriophage at 30", but not at 40" or higher. Eleven independent spontaneous mutants of T4 (go mutants) were isolated that overcame the growth restriction at high temperature. All of these mutants were located within three percent recombination of a gene 39 amber mutation in the clockwise direction on the standard map. In mixed infections, the representative go mutant chosen for further study seems to be recessive to its wild-type allele. Temperature-shift experiments suggested that the mutated host function involved in phage growth is a "late" function, beginning in mid-eclipse.-Electrophoresis of phage proteins labelled early and late in infection showed that under restrictive conditions early protein synthesis was normal, but that certain late proteins were absent. However, measurements of DNA synthesis showed that under restrictive conditions the amount of phage DNA synthesized, and especially the amount of DNA sedimenting as high molecular weight replicative intermediate, was reduced. Pulse-chase experiments showed that the phage DNA made under restrictive conditions was not rapidly degraded.

HE successful infection of E. coZi by its viral parasite T4 involves a shift from Thost-directed to virus-directed metabolism. Some host functions are conserved and can be used directly by T4 (SUEOKA, KANO-SUEOKA and GARTLAND 1966; HASELKORN and SMITH 1969; HASELKORN, VOGEL and BROWN 1969). But, in part, the development of T4 depends on the interactions between phage-coded proteins and pre-existing host functions (FRANKEL 1 966a,b; TRAVERS 1970; MARCHIN, COMER and NEIDHARDT 1972; HORVITZ 1974a,b). The latter kind of interaction is illustrated by the modification of host tRNA's (SUEOKA, KANO- SUEOKA and GARTLAND 1966) and tRNA synthetases (MARCHIN, COMER and NEIDHARDT 1972) following phage infection and by the changes in transcription specificity of the host RNA polymerase resulting from interactions between T4- coded proteins and host core polymerase (TRAVERS 1970; STEVENS 1972; HOR- VITZ 197413). The association of newly synthesized T4 DNA (FRANKEL 1966a,b) and of T4 head (LAEMMLI, BEGUIN and GUJER-KELLENBERGER 1970) and tail

Publication No. 2286 of the Laboratory of Genetics. Research supported in part by Grants AI 05855 and GM 15422 from the Public Health Service and in part by funds from the Graduate School of the University of Wisconsin.

* Present address: Laboratory Safety Products, Janesville, Wisconsin 53545.

Genetics 9-1: 301-325 February, 1980.

302 J. L. JENSEN AND M. SUSMAN

components (SIMON 1969; BROWN and ANDERSON 1969) with a host cell membrane is well documented. It is reasonable to postulate that interactions between host-specified and T4-specified components occur at many stages in T4 development. This sort of specific participation of E. coli in T4 development was proba- bly first postulated by SIMON (1969) and BROWN and ANDERSON (1969). Many investigators (COPPO et al. 1973, 1975; GEORGOPOULOS et al. 1972; TAKANO and KAKEFUDA 1972; PULITZER and YANAGIDA 1971; SIMON, SNOVER and DOERMANN 1974; FRIEDMAN 1971; FRIEDMAN, JOLLY and MURAL 1973; GEORGOPOULOS 1971a,b; SNYDER 1972, 1973, MONTGOMERY and SNYDER 1973; HOMYK and WEIL 1974; TARAHASHI et al. 1975; TAKAHASHI 1978) have attempted to define the stages in phage development that involve specific interactions between phage and host functions.

COPPO et al. (1 973), GEORGOPOULOS et al. (1 972) and TAKANO and KAKEFUDA (1972) independently report the isolation of mutants of E. coli (tab B, gro E, and mop, respectively) in which T4 head assembly is blocked. Compensating T4 mutants that overcome this block were isolated by the three groups. All of these mutations map in T4 gene 31 [involved in the ordered assembly of T4 head proteins (LAEMMLI, BEGUIN and GUJER-KELLENBERGER 1970) 1. The authors independently suggest that the respective bacterial mutations define a host component that interacts with the product of T4 gene 31 during normal T4 head morphogenesis.

In an extension of these studies, TAKAHASHI et al. (1975) report the isolation of additional T4 mutants that overcome the tab B growth restriction. These mutations map in T4 gene 23.

COPPO et al. (1975) report the isolation of E . coli tab D mutants. When infected at 30" with T4 phage carrying temperature-sensitive mutations in genes 45 and 55, these tab D mutants show a block in the transcription of late viral RNA.

TAKAHASHI (1 978) reports the isolation of two temperature-dependent mutants of E. coli, HTC-803 and HTC-619. Infection of these mutants with wild-type T4D under nonpermissive conditions (i.a, 42.5') results in a delay in T4 DNA synthesis and the absence of functional heads and tails. Compen- sating T4 mutants map near T4 gene 39 and in gene 45, respectively.

SIMON, SNOVER and DOERMANN (1974) report the isolation of a strain of E. coli (hd 590) with a possible host membrane mutation that affects T4 head filling and the synthesis of two T4 structural proteins (proteins of the T4 baseplate and tail fibers). Compensating T4 mutants occur in both genes 39 [delayed DNA synthesis ( Y E G I A N ~ ~ ~ ~ . 1971)] and31.

PULITZER and YANAGIDA (1971) report the isolation of an E. coli mutant (W22) in which structurally intact tail fibers are produced, but are not attached to the virion. They postulate the existence of a bacterial protein that is involved in the morphogenesis of functional tail fibers and point to the binding of T4 tail components to the bacterial membrane (LAEMMLI, BEGUIN and GUJER- KELLENBERGER 1970; SIMON 1969) as one possible kind of host function.

RODRIGUEZ (1976) reports the isolation of a host mutant (AR8) that restricts

T4-RESTRICTIVE MUTANTS O F E . Coli 303

the development of T4 mutants carrying a deletion of the region between T4 genes 39 and 56. The restriction appears to occur at the level of T4 DNA synthesis.

SNYDER (1972, 1973), SNYDER and MONTGOMERY (1974) and MONTGOMERY and SNYDER (1973) report the isolation and characterization of rifampicin- resistant mutants of E. coli (@) that result in a delay in the onset of DNA synthesis and the absence of specific late proteins. Compensatory T4 mutants were found to map in T4 genes 45 and 55, the products of which are associated with the host RNA polymerase during normal T4 development (STEVENS 1972; RATNER 1974; HORVITZ 1974b), as well as in the gene for /3-glucosyltransferase.

This paper reports the isolation of a nonlethal host mutant that is conditional for the support of T4 growth. The mutant described in this paper appears to be different from all previously reported mutants.

MATERIALS A N D METHODS

Phage: T4D+ was the wild-type T4 phage strain used in these experiments (DOERMANN and HILL 1953). T4Dra4l is a derivative of T4D+ containing a point in the rII A cistron (EDGAR et al. 1962). The following am mutants kindly provided by R. S. EDGAR and I. LIELAUSIS were used (the gene containing the am mutation is given in parenthesis): amB22(43), amH36 (23), amB25 (34) , amA453 (32), amE51(56), amN116(39), “am”HL626 (60), amB16(7) , amN52(37) and amE18(18). T4D deletion (39-56) #I2 was kindly supplied by A. RODRIGUEZ and J. WEIL.

Bacteria: E. coli strain HfrH was the host strain used in the isolation of phage-restrictive host mutants. B/5 is a derivative of E. coli B. Both Hfr H and B/5 restrict am mutants. CR63/5 is a suf derivative of E. coli K55. CR63(X) is a lysogen that restricts the growth of T4rZZ mutants and permits growth of am mutants (STEINBERG and EDGAR 1962). Shigella was obtained from S . HATTMAN.

Media: H broth (STEINBERG and EDGAR 1962) was the growth medium used in all experiments not involving the use of radioactive tracers. M9 salts and M9 salts f 0.01% casamino acids or M9 salts + 0.1 % casamino acids were prepared as described by RAPPAPORT, RUSSEL and SUSMAN (1974). Tryptone broth was used for all dilutions. All phage and bacterial assays were done on plates of Enriched Hershey Agar ( STEINBERG and EDGAR 1962).

Hepes buffer was purchased from Calbiochem and used at 0.1 M.

Chemicals: N-methyl-”nitro-N-nitrosoguanidine (NMNG) was a gift from R. DEMARS. NMNG solutions were made at 0.5 mg per ml in sterile distilled water before each mutagenesis experiment. Thymidine (A grade) was obtained from Calbiochem. 2’-deoxyadenosine hydrate was obtained from Aldrich Chemical Co. and stored at 4” until use. Scintifluor, a POP-PPOPOP counting cocktail, was obtained from Isolab. It was diluted 10-fold into toluene before being used.

Sodium dodecyl sulfate (SDS), acrylamide, methylene-bis-acrylamide and Coomassie bril- liant blue were obtained from Bio-Rad. All were electrophoresis purity grade chemicals.

Thymidine-methy1-W (0.05 mCi/0.22 mg dissolved in 0.5 ml sterile distilled water) and thymidine-methyl-3H (5mCi/0.06 mg dissolved in 5 ml sterile distilled water) were obtained from New England Nuclear.

Mixed 14C-amino acids (57 mCi/m atom dissolved at 250 pCi/5 ml) were obtained from Amersham.

BacteriologicaL techniques: Preparation of host and indicator bacteria, phage stocks and plating techniques for phage and bacterial assays are as described by STEINBERG and EDGAR (1 962).

One-step growth and premature lysis experiments were done as generally described by STEINBERG and EDGAR (1962). Unadsorbed phage and cell survivors were measured in all ex-


periments. The multiplicity of infection and burst size were calculated on the basis of the actual number of bacteria infected (infective centers present after the treatment with antiserum to inactivate free phage).

Tempersatures of incubation: The phage-restrictive bacterial mutant described in this paper is temperature dependent. The temperatures of incubation required for phage restriction in liquid culture were lower than those required in solid media, presumably because of the time required to warm the agar in a petri dish. We used 40" as the restrictive temperature for liquid cultures and 42" for plates.

Isolation of phdBH b y mutagenesis with nitrosogrcanidine: Nitrosoguanidine mutagenesis was performed using the following procedure modified from ADELBERG, MANDEL and CHEIN- CHING-CHEN (1965). Exponential-phase cultures of HfrH grown in H broth at 30" were sedimented three times by low-speed centrifugation and resuspended in Hepes buffer (pH7.4) after each centrifugation. The titer of the bacteria after the final resuspension was 1 x l o 9 to 2 X IO9 cells per ml. 0.8 ml nitrosoguanidine was added to 3.2 ml bacteria (to give a final concentration of mutagen of 0.1 mg per ml) and the mixture was incubated at 37" for 30 min without aeration. The mutagenized stock was diluted 10-fold with Hepes, sedimented twice, resuspended in H broth after each centrifugation and grown for three hr in H broth at 30" with aeration. In these experiments, cell survival after mutagenesis varied from 20% to 50%; the frequency of strr mutants among cell survivors after mutagenesis was about 10-4.

Screening for phd mutants: Stocks of HfrH, mutagenized with nitrosoguanidine, were plated and grown overnight at 30". Individual colonies were picked, resuspended in H broth and grown overnight at 30". (Growth at 30" prevents selection against slightly detrimental temperature-sensitive mutations). Each overnight culture was spotted onto two sets of plates. One set of plates was incubated overnight at 30" and the second at 42". Colonies that grew at both temperatures were then tested in microtiter plates for their ability to support growth of wild- type T4D. Four microtiter plates were prepared; tw3 contained 5 x 107 to 5 x 10s bacteria and 0.i ml soft agar per well and two contained 5 x 107 to 5 x 10' bacteria, 5 x 106 T4D+ and 0.1 ml soft agar per well. One member of each pair of plates was incubated at 30", the other at 42". A colony was scored as a potential temperature-dependent phage restrictive mutant if it produced turbid bacterial growth at 42", but not at 30", in the presence of phage, and produced turbid growth at both temperatures in the absence of phage. One of these mutants, designated phd-SH, is the subject of this report.

Isolation of go-9H phage: An unmutagenized stock of T4D+ (titer = 2 x l o l l particles per ml) was diluted 1000-fold and plated on phd-QH host bacteria. The plates were incubated overnight at 42". Plaques appearing after this time were picked, resuspended in 1 ml H broth with chloroform and plated on B/5 at 30". Plaques were picked from the B/5 plates after four hr incubation and stocks were prepared from the four hr plaques.

TABLE 1

Mapping go; two-factor crosses to locate go-9H

Parental s t r a i n s

1, go-9H am+ X go+ am56 2. go-9H am+ X go+ am32 3. go9Ham+ x go+ am39 4. go-9H am+ X go+ am60 5. go-9H r+ X go+ ra41 6. go+ am39 r+ X go+ am+ rad1

Cross 1 Frequency of recombination

W% 43 %

5% 8%

12%, 12% 6%

Cross 2 Frequency of recombination

26% 48% 6% 9%

12%, 12% 6%

Crosses were done as described in MATERIALS AND METHODS.

T4-RESTRICTIVE MUTANTS OF E . Coli 305

Mapping experiments: All mapping experiments were done at 30" using B/5 as the host bacterium. CR63/5 was used as the permissive bacterium for assays of T4 am mutants. B/5 was used as the permissive bacterium for assays of T4ra41. CR63(X) was used as the selective indicator for detection of r+ in crosses involving T4ra41.

Spot tests of individual progeny plaques were performed by resuspending single plaques in 1 ml H broth containing 0.1 ml CHC1, and spotting a sample of the resuspended phage onto the appropriate indicator bacterium.

For the two-factor crosses (Table l ) , host cultures of B/5 were prepared and infected with the parental phage strains a t a multiplicity of infection (MOI) of seven for each parent phage. Free phage were inactivated with antiserum. Progeny phage were taken at 55 min after infection at 30" and the recombination percentages determined. Each cross was done twice. Lines 1 through 4 show the results of two-factor crosses using go-9H and an am mutant as the parental phage. Equal input crosses of am f x $- g0-9H were scored in two steps as follows: First, the progeny were plated on E. coli B/5 to select am+ offspring. Of these, 200 were then spotted on a phd-9H lawn and incubated at 42". A replicate phd-9H plate was incubated at 30" The percent recombination i s reported as

no. of negative spots a t 42" total number of spots at 30"

x 100. ____

The recombination values in line 5 were determined by picking r o r r+ plaques (200 of each) from the platings on B/5 and spot testing each on phd-9H at 30" or 42", as described previously. The first number of each experiment is the percent recombination among r+ plaques picked and the second number is the percent recombination among r plaques picked. For crosses involving mutations in genes 39 and 60, which produce very small plaques on lawns of SIC bacteria, the spot tests were done only with large, am+ plaques picked from the B/5 plates. In line 6, Table 1, the distance between ra41 and amNl16 was determined by plating the lysate from an equal input cross on E. coli B/5 and scoring r versus r+ plaque morphology among the selected am f offspring that grew on the plate. Percent recombination is

r+ plaques on B/5 total plaques on B/5

XIOO.

For the three-factor crosses (Table 2), a host culture of B/5 was prepared and infected at 30" with the parental phage at an MO1 N 7 each. The culture was lysed at 55 min after infection, and the genotypes of progeny phages were scored by a combination of selective plating and spot testing as described above. Only the am+ offspring were scored. It was assumed that am offspring would give symmetrical proportions.

Measurement of DNA synthesis: Host cells were grown to 1 x I O 7 cells per ml in M9 + 0.1% casamino acids a t 30", spun at 2000 x g for 15 min at 0" and resuspended at 5 X 108 cells per ml in M 9 + 0.01% casamino acids containing deoxyadenosine (200 pg/ml) and cold thymidine ( 2 pg/ml). The host cells were aerated for ten minutes before infection and then infected with phage at an MO1 of 10. Two minutes after infection, the infected cells were diluted two-fold in M9 f 0.01% casamino acids containing deoxyadenosine (200 pg/ml), thymidine (2 pg/ml) and methyl-3H-thymidine (20 pCi/ml; final specific activity 10 pCi/pg). At intervals, 100 pl aliquots were removed and diluted into ice-cold 50 p l aliquots of 0.1 M NaN,. 100 pl samples of these mixtures were spotted onto filter discs (Whatman 3MM). The discs were washed four times with ice-cold 7.5% trichloroacetic acid (TCA), dried and allowed to sit overnight in cold TCA. The discs were washed with 95% ethanol, dried, put into vials containing 15 ml Scintifluor solution and counted in a Packard scintillation counter.

For rate measurements, initial infections were done as above. At intervals, samples were removed from the nonradioactive adsorption tubes, diluted two-fold into the labelling medium and aerated for two min. Labelling was then terminated by removing an aliquot (100 pl) and diluting into NaN, as described above. (Preliminary experiments were done to show that NaN, is effsctive in stopping DNA synthesis.)

306 J. L. J E N S E N AND M. SUSMAN

TABLE 2

Three-factor crosses involving go-PH, am NI16 and ra41

Genotypes Percent of scored genotypes

Interpretations of the crossover classes for each of the three possible orders of the markers used in the cross

among among Order 1 Order 2. order 3 progeny progeny go'amIlr g o W a m rIgdIam

Cross I go am+r+

X go+ am r

Cross I1 go am r+

x go+am+r

am+r go+am+r go+ am + r + go+am+

5.1 0.7 5.7 coinIonly 6.0

CO in I1 zk CO in I CO in I and 11*

CO in I & CO in I1

coin I1 t coin I CO in I1 only CO' in I only co in I or I1

am+r+ go am+?-+ go am+r go+am+r+

6.1 CO in I1 +- CO in I 1.2f CO in I and I1 6.6 CO in I only 4.9 CO in I1 only

co in I1 -f coin I CO in I1 only CO in I only CO in I and I1

co in I or I1 CO in I1 only CO in I and I1 coin11 k c o i n 1

CO in I or I1 CO in I1 only CO in I and 11 CO in I only

Cross I11 go am+ 5.2 CO I -+ CO in I1 CO in I or I1 coin I1 t co in I go am r f go am+r+ 1.2+ CO in I and I1 CO in I1 only CO in I1 only

X go+ am+r

* Coincidence coefficient for order I is 2 to 3. j- HET'S for go/go+ will be scored as go. For r/r+ will be scored as r+ . Consequently, the

double crossaver class in crosses I1 and I11 is slightly overestimated. All crosses were equal input crosses with MOI's of approximately seven for each parent phage.

The am+ offspring were selected and the recombinant classes shown were scored by testing for go and ra4I as described in MATERIALS AND METHODS. Parent genotypes are shown in Column 1. The genotypes of progeny phages and the frequencies of these genotypes are shown in Columns 2 and 3. Not all recombinant classes were scored in each cross. Columns 4,5 and 6 show the three possible orders of the markers used in these crosses and the crossover pattern for each order that would lead to the recombinant class observed. Only order I (shown in column 4) is consistent with the frequencies observed.

For the pulse-chase experiments, infection was done at 40". After two min adsorption, the infected cells were diluted two-fold into the labelling medium (growth tube medium) and incubated at 40" with aeration for 13 min. Then, an aliquot was removed from the radioactive growth tube and diluted five-fold into chase medium containing 200 pg/ml deoxyadenosine and 3 mg/ml thymidine. This is a 1000-fold isotopic dilution of thymidine, A control aliquot was also taken from the radioactive growth tube and diluted five-fold into radioactive growth medium. After initiation of the chase, samples were taken at intervals from both the chase tube and the diluted growth tube and then mixed with NaN, as described above.

For all labelling experiments the data are plotted as cpni per sample (per disc). Sucrose gradient centrifugation of DNA: The method used is as described by ALTMAN and

LERMAN (1970). Replicating DNA was continuously labelled from two min after infection as described above. Samples were taken 18 min after infection at 40" and treated as described by ALTMAN and LERMAN (1970). Gradients consisted of 4.8 ml 5-20% (w/w) sucrose in 0.005 M tris, pH 7.4, 0.001 M MgSO, and 0.06 M NaCl, beneath which was a 0.2 ml cushion of 55% CsCl (w/w-) in 20% sucrose. Gradients were spun in a SW 50.1 rotor (Beckman model L Spinco ultracentrifuge) at room temperature for 6.5 minutes at 3.2 x lO*rpm. Forty-eight 0.1 ml fractions were collected from each gradient.

Preparation of labelled T4 DNA: The procedure is as described above except that methyl. I%-thymidine (4 pCi/ml, final specific activity 2 pCi/pg) was used. Cells were lysed with CHC1, at 90 min after infection, the lysate was spun at 0" for 10 minutes at 6000 rpm in a GSA rotor (Sorvall), and the supernatant was spun for 90 minutes a t 0" at 13,000 rpm in stainless steel tubes in the SS34 (Sorvall) rotor. The phage pellet was resuspended in 0.1 M tris, pH 7.9

T4-RESTRICTIVE M U T A N T S O F E . Coli 307

and 0.3 M NaC1. The stock was extracted three times with phenol and dialyzed against 0.1 M

tris, pH 7.9 and 0.01 M NaCl for eight hr at 4". Radioactive labelling of proteins in phage-infected cells: Host cells were grown at 30" to a

titer of 1 x I O 7 per ml in M9 + 0.1% casamino acids, centrifuged at 2006 X g for 15 min and resuspended at 4 x 108 cells per ml in M9 + 0.01% casamino acids. An equal volume of phage was added to give an MO1 of 10 to 15. At different times after infection, 2 ml aliquots were removed and added to 0.1 ml of a mixture of 14C-labelled amino acids (57 mCi/m atom; final concentration = 2 pCi per ml). The incorporation of radioactive amino acids was terminated by the addition of an equal volume of M9 + 0.1% casamino acids and the immediate removal of the mixture to an ice bath. The cells were centrifuged at 0" and resuspended in SDS sample buffer (LAEMMLI 1970) at 1/10 the original volume. The cells were immersed in boiling water for I to 2 minutes and stored at -70". Thawed samples were immersed in boiling water for one minute before they were dispensed into the gel and 0.005 ml of 0.1% bromophenol blue dye was added IO them. Although no chase time was allowed for completion of nascent proteins, no background of incomplete polypeptides obscured our gels.

Gel electrophoresis and autoradiography: Electrophoresis of radioactive proteins was done on a Hoefer vertical slab apparatus (0.75 mm gels). The SDS-gel and buffer system used was as described by LAEMMLI (1970). A 10% separating gel and a 3% stacking gel were used. Samples (15 pl) of the dissociated radioactive proteins were dispensed into the wells. Gels were run at room temperature at a constant current of 2 mA until the bromophenol blue tracking dye reached bottom (approximately two hr). After electrophoresis, proteins were stained in 0.2% Coomassie blue solution in 7.5% acetic acid for 60 min and diffusion-destained overnight in 7.5% acetic acid. The gels were dried and put under Kodak No Screen X-ray film for 3 to 4 days. The films were developed in Kodak D19 and fixed in Rapid Fix.

RESULTS

Isolation and characterization of phd-9H and go-9H: E. coli HfrH was mutagenized as described in MATERIALS AND METHODS, and several thousand single- colony isolates were screened for the presence of temperature-dependent, phage- host defective (phd) mutants. Among these, five mutants were found that could form colonies at 30" and 42" and support phage growth at 30°, but not at 42", as determined by the microtiter plate technique. One of these, designated phd-SH, was chosen for further study.

Strain phd-9H is conditionally restrictive for the growth of T4D+ at higher temperatures as shown by the plating efficiency and burst-size data in Table 3.

TABLE 3

Efficiency of plating and burst size of T4D+ on phd-9H and phd+

Host

30" EfIiciency Burst of plating* size+

420/4QO Efficiency Burst of plating' SiZet

phd-SH$ 0.99 165 -10-6 0.02 phd+ 0.97 150 0.95 135

* For measurements of plating efficiencies, the number of T4D+ added was determined by assaying the T4D+ stock on B/5 indicator bacteria. t The burst-size data are the results of one-step growth experiments. For experiments done at 30", burst sizes are calculated at 60 min after infection. For experiments done at 40", burst sizes are calculated at 50 min after infection. Measurements of efficiency of plating were done at 30" and 42".

$ The MOI's are: for phd+ + T4D+, 0.5; for phd-9H + T4D+, 0.6.


T4D+ plates at 42" with an efficiency 1.3 X 10-6 of that measured at 30". No significant differences in plating efficiency at the two temperatures are seen when phd+ is used as host bacterium. In one-step growth experiments at 40°, the burst size of T4D+ in phd-9H is 0.02, roughly a 7000-fold reduction from that seen with phd-9H at 30" or with phd+ at 30" or 40". The burst size of T4D+ in phd-9H at 40" was not increased by extending the incubation time to 180 minutes after infection or by using exogenously added lysozyme to effect cell lysis. Efficiencies of phage adsorption and host-cell killing were measured in these experiments. The results were essentially the same with phd-9H and phd+ at high and low temperatures.

Although phd-9H cannot support T4D+ growth at high temperatures, the mutant bacterium grows at both low and high temperature. The ratio of the doubling time for phd+ to that of phd-9H in H broth at 30" is 1.04; at 40" it is 1.8.

Since E. coli hosts normally restrict the growth of T4 phage containing nonglucosylated DNA ( SYMONDS et al. 1963) , it was conceivable that phd-9H was temperature sensitive for a bacterial enzyme required for glucosylation of phage DNA. If infections at 42" yielded a normal number of progeny phage with nonglucosylated DNA, these phages would not form plaques on the strains of E. coli used as plating bacteria in these experiments. To test for the presence of such phage particles, a strain of Shigella that does not restrict T4 with nonglucosylated DNA (LURIA and HUMAN 1952) was used as the plating bacterium for lysates made on phd-9H at 42". The results showed no difference in phage titer, regardless of the bacterial strain used as the indicator.

It is clear that phage growth in phd-9H is restricted at some stage after adsorption and DNA injection have occurred. Temperature-shift experiments were used in an attempt to determine the time at which the restriction occurs. The results of temperature up-shift experiments (Figure 1 ) show that, when phd-9H is infected with T4D+ at 30" and upshifted to 40" at various times after infection, no phage appear if the up-shift is made before 10 minutes. i.e., about half- way through the eclipse period at 30". If the up-shift is made after 10 minutes, phage are produced, the number depending on the time of the upshift. The results of the temperature down-shift experiment in Figure 2 show that if phd-9H is infected with T4D+ at 40" and down-shifted to 30" at various times after infection, no drop in phage titer appears if the down-shift is made during the first three minutes of the eclipse period at 40". For shifts six minutes or later, the phage titer decreases as the time before the down-shift increases. The temperature shifts have virtually no effect on phage production in phd+ host cells. These two temperature-shift experiments are in reasonable agreement with each other, and are consistent with the model that the host function affected by the phd-9H mutation is not needed for phage development until approximately the mid-eclipse period, but is then needed until the end of the latent period.

To help define the function supplied by phd-9H for T4D+ growth, mutants of T4D+ that overcome the growth restriction at high temperature were isolated

T4-RESTRICTIVE MUTANTS O F E. C o l i 309

40- z W

[L W 30- n

&-A T4Dtphage+pN+bacteria, premature lysis at 30"

0 IO 20 30 40 50 60 M I N U T E S D E V E L O P M E N T , 30"

I I

~ T 4 D ' p h a g e + phd-9H bacteria,

-T4D4phage+ pfi'bacterio,

1 O--OT4D4phage+@-9H bacteria,

I temperature upshift

temperature upshift

premature lysis at 30° 2o t &-A T4Dtphage+pN+bacteria,

premature lysis at 30"

0 IO 20 30 40 50 60 M I N U T E S D E V E L O P M E N T , 30"

FIGURE 1 .-Temperature upshift experiment with phd-9H and phdf hosts. Host cultures of both strains were grown in H-broth and infected with T4Df in H-broth at 30". At various times after infection (these times are indicated along the abscissa) aliquots were transferred to 40" and incubation was continued for an additional 50 min. Phage yields are expressed as percent of the yield of phage after 50 min in the control (premature lysis) tube maintained at 30". Thr MOI's in this experiment were: for phdf, 0.7; far phd-SH, 0.8. The 30" burst sizes were: for phd+, 201; for phd-SH, 183.

by plating a stock of T4D+ on phd-SH, incubating at 42" and looking for plaques. Ten such mutant plaques were isolated from 10 independent wild-type stocks of T4D. All 10 mutants showed less than 0.5 percent recombination with each other, suggesting that they may be within the same gene. Phage that grew on phd-9H at the nonpermissive temperature occurred at a frequency of 1 X in each of the stocks. One of these mutants, designated go-SH, was chosen for further study.

Plating efficiency and average burst-size data for go-9H in phd-9H and phd+ are given in Table 4. T4 go-9H plates on E. coli phd-9H with equal efficiency at 30" and 42". The burst size of go-9H on phd-9H at the restrictive temperature in this experiment is 10. In subsequent experiments, the burst size of go-9H on phd-9H at high temperature was found to be variable, ranging from 5-25. The


gOl \

a -I w

0 T4Dtphage+ phd-9H bacieria, a temperature downshift

50 A T4D'phoge + @'bacteria, L L temperoture downshift I-

MINUTES DEVELOPMENT, 40"

FIGURE 2.-Temperature down-shift experiment with phd-9H and phd+ hosts. Host cultures of phd-9H and p h d f were grown in H-broth and infected with T4D+ in H-broth at 40". At various times after infection (these times are shown on the abscissa) aliquots were removed from 40" to 30" and the infection was continued until t = 90. Phage yields are expressed as percents of the average of the yield of phage obtained after 50 min in the 30" control (data not shown). The MOI's in this experiment were 0.9 for both infections. The burst sizes were: for phd+, downshifted at t = 0, 183; for phd+ incubated at 30", 185; for phd-SH, down-shifted at t = 0, 187; for phd-SH, incubated at 30°, 187. The end of the eclipse period in T4D-infected phd+ cells at 40" is shown by the arrow.

burst size of go-9H at 30" in phd-9H in this experiment is 152. Although the burst size for go-9H on phd-9H at 40" is 500 to 1000-fold larger than the burst size for go+, go-9H gives a reproducibly smaller burst (a reduction of 10- to 30-fold) in phd-9H than in phd+ at 40", suggesting that the go-9H mutation does not completely compensate for the growth restriction imposed by phd-9H. Pre- mature lysis data measuring eclipse and latent periods for go+ or go-9H infections of phd-9H at 30" were identical (data not shown).

The burst-size data for go-9H in phd+ at 30" and 40" are essentially the same

T4-RESTRICTIVE MUTANTS O F E . Coli

TABLE 4

Efficiency of plating and burst size of T4D go-9H compared to go+

31 1

30' 42'/W0 Phage-host Efficiency Burst Efficiency Burst

combinationt of plating* size+ of plating' SiZ*

go+-phd-gH 0.99 132 -10-6 0.01 go-9H-phd-9H 1.02 152 0.96 1o.m

go-9H-phdf - 151 - 121 go+-phd+ - 14.5 - 135

* Plating experiments done at 30" and 42". Measurements of plating efficiency are determined by dividing the number of plaques on a lawn of E. coli phd-SH by the number of plaques produced by an equal volume of the same phage stock plated on E. coli B/5. + Burst sizes are the result of one-step growth experiments at 30" and 40". For experiments done at 30°, burst sizes are calculated at 60 min after infection. For experiments done at No, burst sizes are calculated at 50 min after infection.

$The MOI's are: for go+ + phd-9H at 30", 0.7; for go+ + phd-9H at a", 0.8; for go + phd-SH at 30", 0.5; for go + phd-SH at N", 0.5; for go+ + phd+ at 30", 0.5; for go+ + phd+ at 40", 0.5; for go + phd+ at 30°, 0.7, for go + phd+ at 40", 0.8.

as seen with go+ in phd+ at both temperatures. Premature lysis data measuring eclipse and latent periods for go+ or go-SI-I infected phd+ at either 30" or 40" were identical at both temperatures (data not shown).

The go-9H mutation was mapped by a series of 2- and 3-factor crosses. The 2-factor cross data are shown in Table 1. The go-9H mutation shows linkage to the am mutations in genes 39 and 60 and to the rZZA mutation. The 3-factor cross data given in Table 2 distinguish between the orders go-9H-gene 39-rZZ and gene 39-go-9H-rZZ. The order indicated by these data is go-gene 39-rZZ.

The mapping data place go-9H near a so-called "dispensable region" of the T4 genome. Recently, a series of overlapping deletions in this region, between genes 56 and 39, has been isolated by HOMYK and WEIL (1974). The longest of these deletions, called deletion (39-56) #12, was obtained from WEIL'S laboratory. When plated on phd-9H at 42", deletion (39-56) #12 acts like T4D+. One reasonable interpretation of this observation is that go-9H lies outside the region deleted in deletion (39-56) # 12.

Dominance tests on go+ and go-9H: In order to determine whether go-9H is dominant or recessive to its wild type allele, a long series of single-burst experiments was done. It was hard to get reproducible results because of two trouble- some properties of phd-9H: it adsorbs T4 poorly so that it is hard to control the multiplicity of infection, and it reverts to phd+ at such a high rate that a culture grown from a slant on one day might be strongly restrictive, whereas a culture grown from the same slant on the next day might be almost wild type. There- fore, it is not possible to show a "typical" experiment. However, two features are consistent among all experimental results.

1. A minority (0-25%) of the cells infected with a mixture of go-9H and go+ phage yields any progeny phages at all at 40".

2. Those cells that do yield progeny at 40" produce small bursts (20-30%

312 J. L. JENSEN A N D M. SUSMAN

of the 30" burst sizes). Among the phages produced by these cells, the go-9H phages outnumber the go+ about three to one.

The results are consistent with the conclusion that go+ is dominant in mixed infection with go-SH, and that it performs a dominant "suicidal" function. How- ever, in cells that happen by chance to be infected with a majority of go-9H parental phages, the level of this suicidal function is sufficiently reduced to allow the production of some progeny phage. Of course, other interpretations are also compatible with the data; hence, the question of dominance must be considered unresolved.

DNA synthesis in E. coli phd-9H infected with T4Dgo+ or go-9H phages: Synthesis of DNA in phd+ and phd-9H cells infected with go+ or go-9H phage was measured by following the incorporation of 3H-thymidine into TCA-precipitable material. The results of these experiments are shown in Figures 3 and 4. It can be seen that at 30" (Figure 3) the amounts of DNA made in phd+ cells infected with go+ and phd-9H cells infected with go+ or go-9H are virtually identical. The results for go-9H in phd+ were superimposable (data not shown).

At 40", however, (Figure 4) there is a three- to five-fold reduction in the amount of DNA made in go+-infected phd-9H as compared with the amount of DNA made in go-9H-infected phd-9H. The results for go4 in phd+ were the same as those for go-9H in phd+ (data not shown). A small but reproducible

701 i

' 10 20 30 40 50 60 MINUTES DEVELOPMENT, 30"

FIGURE 3.-DNA synthesis in phdf and phd-9H bacteria infected with go+ and go-9H phages at 30". The procedure is described in MATERIALS AND METHODS. A T4 DNA-negative control (phd-9H infected with go-9H am43 mutant) was included to give a measure of the maximum amount 3f background due to any surviving bacteria.

T4-RESTRICTIVE MUTANTS O F E. Coli 313

h-4 40 9 H phage t e* bocterla

e - o @om43 phage+pM-9Hbacleria

MINUTES DEVELOPMENT, 40'

FIGURE 4.-DNA synthesis in phd+ and phd-9H bacteria infected with go+ and go-9H phages at 40". The procedure is described in MATERIALS AND METHODS. A T4 DNA-negative control (phd-SH infected with a go-9H am43 mutant) was included to give a measure of the maximum amount of background due to any surviving bacteria.

reduction in the amount of DNA synthesized at 40' (Figures 3 and 4) is also seen in go-9H-infected phd-9H when compared with go-9H-infected phd + cells. This agrees with the previous conclusion, based on the relative burst sizes at 40" of go-9H-infected phd+ and phd-SH, that the go-9H mutation only partially compensates for the phage growth restriction imposed by phd-9H.

The quantity of DNA made by go+ phage in phd-9H bacteria at 40" is -20% of that made by go-9H. This is significantly more than the amount of DNA synthesized by the DNA-negative control. This reduction in DNA synthesis is not in itself sufficient to account for the difference in burst size between go+ and go-9H in phd-9H cells at high temperature. In light of this consideration, the DNA made in go+-infected phd-9H cells was studied in order to determine its stability and size relative to normal replicative T4 DNA.

Figures 5 and 6 show the results of pulse and pulse-chase experiments with phage-infected phd-9H bacteria. It can be seen (Figure 5 ) that the reduction in the net accumulation of DNA in go+-infected phd-9H cells at 40" is reflected in a lower instantaneous rate of DNA synthesis throughout the latent period. The pulse-chase data in Figure 6 show that there is no detectable breakdown into acid-soluble nucleotides of the DNA made by go+ phage in phd-9H bac-

314

5000

4500

4000 W I- 2 3500- 2 LL w 3000- a U7 5 2500- 3

-

2000 s

I500

1000

J. L. JENSEN AND M. SUSMAN

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-

-

-

-

-

1 gofphagetphd-9H / bacteria-

500; I I 10 20 30 40 50 60 70

MINUTES DEVELOPMENT,40"

FIGURE 5.-Rate of DNA synthesis in phd-9H bacteria infected with go+ and go-9H phages at 40". The procedure is described in MATERIALS AND METHODS. Phd-9H host cells were infected with go+ and go-9H phages at 40". At the indicated times, samples were withdrawn from the nonradioactive adsorption tube and diluted into labelling medium (growth tubes). Incorporation was allowed to proceed for two min, then aliquots from the radioactive growth tubes were withdrawn for determination of TCA-precipitable counts incorporated during the 2-min pulse. A go+ am43 control was included to measure background due to bacterial survivors. Points on the graph are plotted at the times the 2-min pulses were initiated.

teria at high temperature. The data in Figures 5 and 6 suggest that the lower net accumulation of DNA in go+-infected phd-9H cells is not due to instability of DNA made, as reflected by breakdown into acid-soluble nucleotides.

In order to determine whether normal T4 replicative DNA was made under restrictive conditions, the sizes of T4 molecules synthesized in go+ and go-9H- infected phd-9H and phd + bacteria were examined, using neutral sucrose gradient velocity sedimentation. The results of these experiments are shown in Figure 7. Significant differences are found. In extracts of go+-infected phd+ cells, the four species of T4 DNA found by ALTMAN and LERMAN (1970) and others (FRANKEL 1966a,b) are found: the bottom component (fraction 1-10) , which consists of membrane-bound T4 DNA (ALTMAN and LERMAN 1970; FRANKEL 1966a,b) ; the phage component (haction 11-20) consisting of mature, packaged T4 DNA that co-sediments with infective phage (Figure 8) ; the fast sedimenting

T4-RESTRICTIVE MUTANTS O F a??. C o l i

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315

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3 '15 20 25 30 35

MINUTES DEVELOPMENT, 40" FIGURE 6.-Stability of DNA made in phd-SH bacteria infected with go-9H or go+ phages

at 40". The procedure is described in MATERIALS AND METHODS. Host cells of phd-9H genotype were infected with go-9H and go+ phages at 40". At two min after infection, cells were diluted into the growth tube medium (labelling medium), and phage development was allowed to proceed for 13 min. At 15 min after infection, two aliquots were removed from the radioactive growth tube. One aliquot was diluted five-fold into the growth tube medium and labelling allowed to proceed as before (control tube). The second aliquot was diluted into medium containing 3 mg/ml cold thymidine (chase tube). At the indicated times, samples were removed from both the control and chase tubes, and the amount of TCA-precipitable material was determined. The chase data are given by the solid lines. The control data are given by the broken lines. A go+ am43 control was included to measure background due to bacterial survivors.

fraction (fractions 21-38), which consists of T4 concatemeric DNA of hetero- geneous molecular weight (ALTMAN and LERMAN 1970; FRANKEL 1966a) and the slow component (fractions 39-48) consisting of unpackaged mature T4DNA that co-sediments with the T4 marker DNA. In extracts from go-9H-infected phd-9H cells incubated at 40°, the bottom component, phage component and slow sedimenting fractions are present in nearly normal quantities, but the fast- sedimenting component is greatly reduced. In extracts from go+-infected phd-9H


1500Fp- k

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FRACTION NUMBER

FIGURE 7.-Sedimentation profiie of T4 DNA extracted from phd+ and phd-9H bacteria infected with go-9H and go+ phage. The procedure is from ALTMAN and LERMAN (1970). The lysates for this set of gradients were taken at 18 min after infection at 40". The gradients run with lysates of go-9H and go+-infected phd-9H represent results from the same experiment. The gradient run with the lysate of go+-infected phd+ cells was done separately. The distribution of the 1%-labelled marker DNA is the distribution of the marker run with the go-9H- infected phd-9H lysate. The distribution was similar in the other lysates, and the marker peaked in fraction 46 out of 48 (shown by the arrow) in the three gradients. The infective phage peak in the gradients is indicated by the star. 85% of the plaque-forming phage falls between fractions 11 and 20 (FIGURE 8 ) ; these data are from gradients run with go-9H-infected phd-9H cells, which were similar for gradients run with go-infected phd+ cells.

cells incubated at 40°, the bottom component is greatly reduced and the phage component and fast-sedimenting fractions are absent. Virtually all of the newly synthesized T4 DNA is found at the top of the gradient. These results suggest that the phd-9H mutation may interfere with the production or stability of high molecular weight species of T4 DNA seen in normal phage infections (FRANKEL 1966a,b).

T4-RESTRICTIVE M U T A N T S O F E . Coli 31 7

&-A gotinfected phd'cells M - 90-9H infectedphd-9H - cells W W oa aa

no 30

FRACTION NUMBER FIGURE 8.-Distribution of infective phage in the gradients run with go-9H-infected pM-SH

cells and go+-infected phd+ cells. Each fraction from the gradients shown in FIGURE 7 was assayed for infective phage. The data are plotted as the percent of the total phage recovered from the gradient found in each sample.

Protein synthesis in phd+ and phd-9H bacteria infected with go+ and go-9H phage: Phage proteins made in phd+ and phd-9H bacteria infected with go+ and go-9H phage at high temperature where labelled with 14C-amino acids as described in MATERIALS AND METHODS. The labelled proteins were examined on polyacrylamide gels. The results are shown in Figures 9, 10 and 11.

Figure 9 shows the patterns of early phage protein synthesis in phd+ and phd-9H cells infected with go+ or go-9H phage at 40". The infection in lane A is with a phage that carries an amber mutation in gene 43, the gene for DNA polymerase. Thus, we can be certain that the band pattern seen in lane A is a prereplicative (early) labelling pattern. The band pattern seen in all of the infections is identical to that seen in the infection of phd+ bacteria by go+ phage. It appears that there is no abnormality of early protein synthesis in phd-9H bacteria at elevated temperature.

Figures 10 and 11 show the pattern of post-replicative T4 protein synthesis at 40" in go+ and go-9H-infected phd-9H host cells and in go+-infected phd+ host cells. P18, the T4 sheath protein, appears to be present in all lysates. No differences are observed between the banding patterns in go-9H-infected phd-9H and go+-infected phd+. Other late proteins (see bands in Figure 11) are absent in lysates made under restrictive conditions: P34 (data not shown), P7, P37 and P23* are consistently absent from lysates of phd-9H infected at high temperature by go+ phage. P34 and P37 are major components of T4 tail fibers;


FIGURE g.-Pre-replicative protein synthesis in phd+ and pM-9H bacteria infected with go-9H and go+ phage. The general labelling procedure and electrophoresis are described in MATERIALS AND METHODS. Host cells were infected with phage at 40". Mixed 14C-amino acids were added at three min after infection. Labelling was terminated at seven min after infection by the addition of ''cold" casamino acids. All samples were labelled on the same day, and the results are from a single slab gel. The position of T4 P43 is indicated by the arrow in lane A. The origin of these gels is at the top. (A) go-9H am43 phage + pM+ bacteria; (B) g d phage 4- phd+ bacteria; (C) go+ phage + pM-9H bacteria; (D) go-9H phage 4- pM-9H bacteria; (E) uninfected phd-9H bacteria.

T4-RESTRICTIVE MUTANTS OF E. Coli -

319

FIGURE 10.-Postreplicative protein synthesis in pM+ and pM-9H bacteria infected with go-9H and go+ phage. The general labelling procedure and electrophoresis are described in MATERIALS AND METHODS. Host cells were infected with phage at 40". Mixed W-amino acids were added at 12 min after infection. Labelling was terminated at 16 min after infection by the addition of cold casamino acids. The origin of these gels is at the top.

Identification of P7, P37 and P18. Lanes A through D are from one gel; lanes E through H are from a second gel. The positions of P37, P7 and PI8 are indicated by the arrows in lanes A, B, and E, respectively. (A) go+ am37 phage + phd+ bacteria; (B) go+ am7 phage 4- phd+ bacteria; (C) go+ phage + pM-9H bacteria; (D) go+ phage + phd+ bacteria; (E) go+ am28 phage + pM+ bacteria; (F) go-9H phage + pM+ bacteria; (G) go-9H phage + phd-9H bacteria; (H) uninfected phd-9H bacteria.

P7 is a component of the base plate; and P23*, a cleavage product of P23 (the major head protein), is formed during maturation of the phage head.

It is possible, but by no means certain, that P23 itself is also missing from the restricted lysates. The doubt arises from some variability in the band patterns observed among lysates of phd-9H cells infected with go+ phage at high temperature. Lanes E and F in Figure 11 illustrate the range of variation. Both are go+ infections, but they were grown on different days. The go+ lysate in lane E appears identical to the lysate (in lane D) of a gene 23 amber mutant labelled at the same time. The similarity oE the two patterns would strongly support the conclusion that P23 is lacking in the restrictive infections. However, lane F,


A B C. D E F

FIGURE 11.-Identification of P23 and P23*. The results are from a single gel. The positions of P23 and PU* are indicated by the curved and straight arrows, respectively. (A) go-9H phage 4- phd-9H bacteria; (B) go+ am23 phage 4- phd+ bacteria; (C) go+ phage + pM+ bacteria; (D) go+ am23 phage + phd-9H bacteria; (E) go+ phage + phd-9H bacteria; (F) go+ phage 4- pM-9H bacteria.

The band remaining near the position of P23 is the co-migrating P12.


although unlike the control lysates from phd+ bacteria (lanes B and C), does have a heavy band in the neighborhood of P23. We must suspend judgment on the presence of the genes 23 product in these lysates until we can account for these variations in band patterns.

DISCUSSION

This paper describes the isolation and characterization of a bacterial mutant that restricts the growth of bacteriophage T4 and of a phage mutant that over- comes this restriction. The evidence presented suggests that a host product is necessary for both normal T4 DNA synthesis and for the synthesis of certain late T4 proteins.

Nitrosoguanidine mutagenesis often results in the production of clusters of mutations on replicating DNA, presumably at the replication fork (CERDA- OLMEDO, HANAWALT and GUEROLA 1968). Although the phd-9H host mutation has not yet been mapped, the observation that a number of T4 go mutants, ones that overcome both the restrictions of DNA synthesis and the restriction of late protein synthesis, map as closely linked mutations is consistent with the idea that a single host mutation may be responsible for both effects. This conclusion is also supported by the observation that stocks of phd-9H are unstable and revert to phd+ at a high rate.

The screening procedure for the phd-9H mutant was designed to select for mutations that were nonlethal to the host. The phd-9H mutant bacterium studied in this paper, in fact, appears by most criteria to be little different from its phd+ parent. The host function defined by the phd-9H mutation may be one that is relatively nonessential for host growth or that is overproduced by the wild-type host cell. Alternatively, the specific mutation introduced into the phd-9H cells may be one that does not interfere with the normal bacterial function, but that affects the “modifiability” of some host function by specific action of infecting T4 phage.

The go-9H mutation shows 5 to 6 percent recombination with the a m N116 mutation in gene 39. Therefore, it may be in a relatively “dispensable” region of the T4 map, or, possibly, at the promoter end of gene 39. In any event, the go-9H mutation does not show delayed bursts or reduced DNA synthesis in phd+ at 30” (relative to go+ in phd+) that might be expected if the go mutation were in gene 39. Second, YEGIAN et al. (1971) have shown that amber mutants in T4 gene 39 show a low initial rate of DNA synthesis. The rate eventually rises until it approaches the rate seen in wild-type controls. Although the rate of DNA synthesis observed in wild-type infection of phd-9H is lower than that seen in infections with the compensating go mutant, the rate remains fairly constant, even at late times after infection, and no phage production is observed even after two hours at the nonpermissive temperature. Thus, the phenotype of wild-type phage restricted by phd-9H bacteria is not the typical “DNA-delayed” phenotype of gene 39 mutants.

During normal T4 DNA synthesis (for review see KORNBERG 1974), the par-

322 J. L. JENSEN A N D M. SUSMAN

ental DNA becomes attached to the host cell membrane, and newly synthesized daughter molecules remain covalently attached to the parental DNA, resulting in the formation of long concatemers. Initiation of new rounds of DNA synthesis can occur before the completion of the previous round, allowing for a rapid increase in the rate of synthesis. The host cell-membrane-attached T4 DNA and the concatemeric molecules are identifiable as high molecular weight intermediates in sucrose gradient velocity sedimentation. DNA synthesis in go+-infected phd-9H at the nonpermissive temperature is characterized by an abnormally low rate of synthesis (see Figures 3,4 and 5) and by the reduction or absence of the high molecular weight replicative intermediates (defined in these experiments as the bottom component and the fast-sedimenting component) seen in all infections of phd+ and, albeit in reduced quantities, with go-9H-infected phd-9H (see figure 7). The apparent reduced rate of DNA synthesis does not appear to be due to a gross instability of the DNA (Fig. 6). The pulse-chase experiment equipment shown in Figure 6 shows that under restrictive conditions there is no extensive degradation of replicating T4 DNA into acid-soluble fragments. How- ever, the possibility that DNA breaks down into fragments somewhat smaller than mature T4 marker DNA cannot be excluded by the sucrose gradient sedimentation profile seen in Figure 7.

The absence of a rapid rise in the rate of DNA synthesis in go+-infected phd-9H at 40" may reflect a disturbance in the normally rapid reinitiation of DNA synthesis. Alternatively, the replicative intermediate formed in go+ - infected phd-9H at 40" may be unstable at high temperature, and this in turn may affect the rate of DNA synthesis. The reduction in the quantity of fast- sedimenting component in go-9H-infected phd-9H suggests that the defect in DNA synthesis is only partially compensated for by the go-9H mutation.

In these experiments, the synthesis of pre-replicative and post-replicative (HOSODA and LEVINTHAL 1968; O'FARRELL and GOLD 1973a,b) T4 proteins was examined. In go+-infected phd-SH, early protein synthesis appears to be normal at 40°, but specific late proteins are absent. These include the products of T4 genes 34, 37, 7 , 23* and possibly 23. P34 and P37 are T4 tail-fiber components (EDGAR and WOOD 1966). P7 is a component of the T4 baseplate (KING 1968). The cleavage product of P23-called P23*-is the major T4 head structural protein (LAEMMLI 1970); it, too, is missing in go+-infected phd-9H at 40". On the other hand, P18, the major sheath protein (KING 1968), appears to be present.

DNA synthesis is abnormal in go+-infected phd-9H at high temperature. This defect in replication might cause the transcription of late genes to be abnormal (BOLLE et al. 1968), with the result that certain specific late proteins would not be produced. The normal production of early proteins and the production of certain late proteins argue against a generally enhanced level of protease or ribonuclease activity in phd-9H at high temperature.

Other investigators have isolated host mutants of E. coli that restrict bacteriophage growth. Most relevant to this paper are the hd 590 mutants isolated by SIMON, SNOVER and DOERMANN (1974) and the HTC-803 mutant isolated


by TAKAHASHI (1978), both of which have been described previously. Wild-type infection of hd 590 mutants results in a low rate of T4 DNA synthesis and the absence of certain late proteins. The compensating go-590 phage mutants map near T4 genes 39 and 31. In contrast, we find that the 11 independent go mutants examined in this work map near gene 39. In addition, SIMON, SNOVER and DOERMANN (1974) did not observe any abnormalities in the molecular weight of the replicating DNA found in wild-type T4-infected hd 590 mutants at high temperature. T4 proteins P7 and P34 are missing in T4-infected hd 590 mutants, but P23 (and P23*) are present. The phenotypic differences between go+- infected phd-9H hosts and go+-infected hd 590 hosts suggest that phd-9H and hd 590 may represent different host mutations.

Wild-type infection of HTC-803 mutants results in a delayed DNA synthesis phenotype, qualitatively normal (although possibly quantitatively abnormal) production of early and late T4 RNA's, and the absence of viable T4 heads and tails. Specific tail-fiber and head proteins are absent in T4D-infected phd-SH, but we do not conclude that there is a delay in T4 DNA synthesis under restrictive conditions. Thus, we conclude that the mutant described here is different from those previously described.

We are especially grateful to WILLIAM MCCLAIN for suggestions and for his reading of the manuscript and to BARBARA SUSMAN for technical assistance.

LITERATURE CITED

ADELBERG, E., M. MANDEL and G. CHEIN-CHING-CHEN, 1965 Optimal conditions for mutagenesis by N-methyl-N-nitro-N nitrosoguanidine in E. coli Kl2. Biochem. and Biophys. Res. Commun. 18: 788-795.

Kinetics and intermediates in the intracellular synthesis of bacteriophage T4 deoxyribonucleic acid. J. Mol. Biol. 50 : 235-261.

Transcription during bacteriophage T4 development: Requirements for late messenger synthesis. J. Mol. Biol. 33 :

Effect of host cell wall material on the adsorbability of cofactor-requiring T4. J. Virol. 4: 94-108.

Mutagenesis of the replication point by nitrosoguanidine: map and pattern of replication of the E. coli chromosome. J. Mol. Biol. 33: 705-719.

Abortive bacteriophage T4 head assembly in mutants in E. coli. J. Mol. Biol. 76: 61-87. -. , 1975 Host mutant (tab-D) -induced inhibition of bacteriophage T4 late transcription. I. Isolation and phenotypic characterization of the mutants. J. Mol. Biol. 96: 579-600,

Genetic structure of bacteriophage T4 as described by recombination studies of {actors influencing plaque morphology. Genetics 38: 79-90.

Mapping experiments with r mutants of bacteriophage T4D. Genetics 47: 179-186.

Morphogenesis of bacteriophage T4 in extracts of mutant- infected cells. Proc. Natl. Acad. Sci. US. 55: 498-505.

ALTMAN, S. and L. LERMAN, 1970

BOLLE, A., R. H. EPSTEIN, W. SALZER and E. P. GEIDUSCHEK, 1968

339-362.

BROWN, D. and T. ANDERSON, 1969

CERDA-OLMEDO, E., P. C. HANNAWALT and N. GUEROLA, 1968

COPPO, A., A. MANZI, J. PULITZER and H. TAKAHASHI, 1973

DOERMANN, A. H. and M. B. HILL, 1953

EDGAR, R. S., R. P. FEYNMAN, S. KLEIN, I. LIELAUSIS and C. M. STEINBERG, 1962.

EDGAR, R. S. and W. B. WOOD, 1966

324 J . L. J E N S E N AND M. SUSMAN

FRANKEL, F., 1966a The absence of mature phage DNA molecules from the replicating pool of T-even infected E. coli. J. Mol. Biol. 18: 109-126. 1966b Studies on the nature of replicating DNA in T4-infected E. coli. J. Mal. Biol. 18: 127-143.

A bacterial mutant affecting lambda development. pp. 733-738. In: The Bacteriophage Lambda. Edited by A. D. HERSHEY, Cold Spring Harbor Laboratory, New York.

FRIEDMAN, D. I., C. JOLLY and R. MURAL, 1973 Interference with the expression of N gene function of phage A in a mutant of E. coli. Virology 51 : 216-926.

GEORGOPOULOS, C. P., 1971a A bacterial mutation affecting N function. pp. 639-645. In: The Bacteriophage Lambda. Edited by A. D. HERSHEY, Cold Spring Harbor Laboratory, New York. --, 1971b Bacterial miitants in which the gene N function of bacteriophage lambda is blocked have an altered RNA polymerase. Proc. Natl. Acad. Sci. U.S. 68:

GEORGOPOULOS, C. P., R. HENDRIX, A. D. KAISER and W. WOOD, 1972 Role of the host cell in bacteriophage morphogenesis: Effects of a bacterial mutation on T4 head assembly. Nature New Biology 239: 3841.

HASELKORN, R. 2nd F. SMITH, 1969 Proteins associated with ribosomes in T4-infected E. coli. Cold Spring Harbor Symp. on Quant. Biology 34: 91-94.

HASELKORN, R., M. VOGEL and R. BROWN, 1969 Conservation of the rifampicin sensitivity of transcription during T4 development. Nature 221 : 836-838.

HOMYK, T. and J. WEIL, 1974 Deletion analysis of two nonessential regions of the T4 genome Virology 61: 505-523.

HORVITZ, H. R., 1974a Contrcrl by bacteriophage T4 of two sequential phosphorylations of the alpha subunit of E . coli RN-4 polymerase. J. Mol. Biol. 90: 727-738. - , 1974b Bacteriophage T4 mutants deficient in alteration and modification of the E. coli RNA polymerase. J. Mol. Bid. 90 : 739-750.

HOSODA, J. and C. LEVINTHAL, 1968 Protein synthesis by E. coli infected with bacteriophage T4D. Virology 34: 709-727.

KING, J., 1968 KORNBERG, A., 1974

LAEMMLI, U. K., 1970 Cleavage Gf structural proteins during the assembly of the head of

LAEMMLI, U. K., F. BEGUIN and G. GUJER-KELLENBERGER, 1970 A factor preventing the major

LURIA, S. E. and M. L. HUMAN, 1952 A non-hereditary, host-induced variation of bacterial

MARCHIN, G. L., M. M. COMER and F. C. NEIDHARDT, 1972 Viral modification of the valyl transfer ribonucleic acid synthetase of E. coli. J. Biol. Chem. 247: 5132-5145.

MONTGOMERY, D. L. and L. R. SNYDER, 1973 A negative effect of /3-glucosylation on T4 growth in certain RNA polymerase mutants of E . coli: Genetic evidence implicating pyrimidine- rich sequences of DNA in transcription. Virology 53: 349L358.

Bacteriophage T4 gene expression: Evidence for two classes of prereplicstive cistrons. J. Biol. Chem. 248: 5502-5511. - , 1973b Tran- scription and translation of prereplicative bacteriophage T4 genes in uitro. J. Biol. Chem. 248: 5512-5519.

Inactive T4 progeny virus formation in a temperature

--,

FRIEDMAN, D. I., 1971

2977-2981.

Assembly of the tail 3f bacteriophage T4. 5. Mol. Biol. 32: 231-262. DNA Synthesis. Freeman, San Francisco. pp. 274-281.

bacteriophage T4,. Ncture 227: 680-685.

head protein of bacteriophage T4 from random aggregation. J. Mol. Biol. 47: 69-85.

viruses. J. Bacteriol. 64: 557-569.

O'FARRELL, P. Z. and L. GOLD, 1973a

PULITZER, J. and M. YANAGIDA, 1971 sensitive mutant of E. coli K12. Virology 45: 539-554.

T4-RESTRICTIVE MUTANTS O F E. Coli 325 RAPPAPORT, H., M. RUSSEL and M. SUSMAN, 1974 Some acridine-resistant mutations of bacterio-

phage T4D. Genetics 78: 579-592. RATNER, D., 1974 Bacteriophage T4 transcriptional control gene 55 codes for a protein bound

to Escherichia coli RNA polymerase. J. Mol. Biol. 89: 803-807. RODRIGUEZ, A. 1976 Isolation and characterization of mutants in a pair of Escherichia coli and

T4 genes whose interaction is essential for phage development. Ph.D. Thesis, Vanderbilt University, Nashville, Tennessee.

SIMON, L., 1969 The infection of E. coli by T2 and T4 bacteriophages as seen in the electron microscope. 111. Membrane-associated intracellular bacteriophages. Virology 38: 285-296.

SIMON, L., D. SNOVER and A. H. DOERMANN, 1974 A bacterial mutation affecting T4 DNA synthesis and tail production. Nature 252 : 451-455.

SNYDER, L., 1972 An RNA polymerase mutant of E coli defective in the T4 viral transcription program. Virology 50: 39-03. - , 1973 Change in RNA polymerase associated with the shutoff of host transcription by T4. Nature New Biol. 243: 131-134.

SNYDER, L. and D. MONTGOMERY, 1974 Inhibition of T4 growth by an RNA polymerase mutation o€ E. coli: Physiological and genetic analysis of the effects during phage development. Virology 62: 184-196.

STEINBERG, C. and R. EDGAR, 1962 A critical test of a current theory of genetic recombination in bacteriophage T4. Genetics 47: 187-208.

STEVENS, A., 1972 New small polypeptides associated with DNA-dependent RNA polymerase of E. coli after infection with bacteriophage T4. Proc. Natl. Acad. Sci. US. 69: 603-608.

SUEOKA, N., T. KANO-SUEOKA and W. J. GARTLAND, 1966 Modification of tRNA and regulation of protein synthesis. Cold Spring Harbor Symp. on Quant. Biol. 31: 571-582.

SYMONDS, N., K. A. STACEY, S. W. GLOVER, J. SCHELL and S. SILVER, 1963 The chemical basis for a case of host induced modification in phage T2. Biochem. and Biophys. Res. Commun. 12 : 220-222.

TAKAHASHI, H., 1978 Genetic and physiological characterization of E. coli K12 mutants (tab C) which induce abortive infection of bacteriophage T4. Virology 87: 256-265.

TARAHASHI, H., A. COPPO, A. MANZI, G. MATIRE and J. F. PULITZER, 1975 Design of a system of conditional lethal mutations (trrb/k/com) affecting protein-protein interactions in bacteriophage T4-infected Escherichia coli. J. Mol. Biol. 96: 563-569.

TAKANO, T. and T. KAKEFUDA, 1972 Involvement of a bacterial factor in morphogenesis of bacteriophage capsid. Nature New Biology 239: 34-37.

TRAVERS, A., 1970 RNA polymerase and T4 development. Cold Spring Harbor Symp. on Quant. Biol. 35: 241-251.

YEGIAN, C., M. MUELLER, G. SELZER, V. Russo and F. STAHL, 1971 Properties of the DNA- delay mutants of bacteriophage T4. Virology 46: 900-919.

Corresponding editor: G. MOSIG

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of - Genetics · T4 mutants that overcome this block were isolated by the three groups. All of these mutations map in T4 gene 31 [involved in the ordered assembly of T4 head proteins