EARLY ENZYME SYNTHESIS AND ITS CONTROL IN E. COLI

VOL. 48, 1962 BIOCHEMISTRY: WIBERG ET AL. 293 3Wood, H. G., and R. Stjernholm, these PROCEEDINGS, 47, 289 (1961). 4Stjernholm, R., and H. G. Wood, Fed. Proc., 20, 235 (1961). 5 Beck, W. S., and S. Ochoa, J. Biol. Chem., 232, 931 (1958). 6 Smith, R. M., and K. J. Monty, Biochem. Biophys. Research Commun., 1, 105 (1959); Stadt- man, E. R., P. Overath, H. Eggerer, and F. Lynen, Biochem. Biophys. Research Commun., 2, 1 (1960); Lengyel, P., R. Mazumder, and S. Ochoa, these PROCEEDINGS, 46, 1312 (1960); Gurnani, S., S. P. Mistry, and B. C. Johnson, Biochim. et Biophys. Acta, 38, 187 (1960); Stern, J. R., and D. L. Friedman, Biochem. Biophys. Research Commun., 2, 82 (1960). 7 Munch-Petersen, A., and H. A. Barker, J. Biol. Chem., 230, 649 (1958). 8 Eggerer, H., P. Overath, F. Lynen, and E. R. Stadtman, J. Am. Chem. Soc., 82, 2643 (1960). 9 Phares, E. F., M. V. Long, and S. F. Carson, Bact. Proc., 61, 184 (1961). 10 Kosower, E. M., personal communication. 11 Beck, W. S., M. Flavin, and S. Ochoa, J. Biol. Chem., 229, 997 (1957). 12 Swim, H. E., and L. 0. Krampitz, J. Bact., 67, 426 (1954). 13 Lagerkvist, U., Acta Chem. Scand., 7, 114 (1953). 14 Bueding, E., and H. W. Yale, J. Biol. Chem., 193, 411 (1951). 15 Stadtman, E. R., in Methods in Enzymology, eds. S. P. Colowick and N. 0. Kaplan (New York: Academic Press, 1957), vol. 3, pp. 931-941. 16 Simon, E. J., and D. Shemin, J. Am. Chem. Soc., 75, 2520 (1953). 17 Phares, E. F., Arch. Biochem., 33, 173 (1951). 18 Stjernholm, R., and H. G. Wood, these PROCEEDINGS, 47, 303 (1961). 19 Barker, H. A., Federation Proc., 20, 956 (1961). 20 Stadtman, E. R., and P R. Vagelos, International Symposium on Enzyme Chemistry (Tokyo and Kyoto: Pan-Pacific Press, 1957), p. 86. 21 Rendina, G., and M. J. Coon, J. Biol. Chem., 225, 523 (1957). 22 Giovanelli, J., and P. K. Stumpf, J. Biol. Chem., 231, 411 (1958). 23 Lipmann, F., and L. C. Tuttle, J. Biol. Chem., 159, 21 (1945). 24 Stadtman, E. R., J. Am. Chem. Soc., 77, 5765 (1955). 25 Vagelos, P. R., J. M. Earl, and E. R. Stadtman, J. Biol. Chem., 234, 765 (1959). 26 Vagelos, P. R., and J. M. Earl, J. Biol. Chem., 234, 2272 (1959). EARLY ENZYME SYNTHESIS AND ITS CONTROL IN E. COLI INFECTED WITH SOME AMBER MUTANTS OF BACTERIOPHAGE T4* BY JOHN S. WIBERG, MARIE-LUISE DIRKSEN, RICHARD H. EPSTEINJt S. E. LURIA, AND JOHN M. BUCHANAN DIVISION OF BIOCHEMISTRY, DEPARTMENT OF BIOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY Communicated January 2, 1962 The "amber" (am) mutants of bacteriophage T4 are a recently discovered class of mutants that can replicate in certain derivatives of Escherichia coli strain K-12 but not in E. coli B. ' The bacterial strains that support growth of the am mutants, thus making their isolation and propagation possible, are called the "permissive" hosts. The biochemical basis of this permissiveness has not yet been clarified. With some of the am mutants, infection of E. coli B leads to a subnormal production of phage DNA; with others, DNA is formed but infection is abortive because of failure of late stages of phage development.2 It is known that infection of E. coli with a T-even bacteriophage leads to the ap- pearance of several new enzyme activities and to an increase in several other enzyme activities, all related to synthesis of phage DNA ("early enzymes").312 These 294 BIOCHEMIST'RY: WIBERG ET AL. PROc. N. A. S. changes are detectable 3 to 5 minutes after infection, and the enzyme activities in- crease until 10 to 15 minutes; then the increase stops. If, however, the infecting phage has been treated with doses of UV light that prevent phage production and synthesis of phage DNA,3 13, 14 then the early enzyme synthesis continues for a longer period. 15-18 The am mutants of T4 provide a tool for the study of the genetic control and regu- lation of the synthesis of the early enzymes. Specifically, it may be possible, on the one hand, to refer specific abnormalities in phage DNA synthesis to specific blocks in the synthesis of early enzymes and, on the other hand, to correlate certain stages in phage development with changes in the kinetics of accumulation of the early enzymes. The present paper reports results with seven of the am mutants. For one of these, the failure to induce phage DNA synthesis is explained by specific biochemical blocks; remarkably, two of the early enzyme activities, out of seven tested, are lost. In addition, a comparison of phage mutants reveals a consistent pattern of quantitative relationship between DNA synthesis and the cessation of the increase in the level of the early enzyme activities. Materials and Methods. t-The am mutants used in this work were derived from phage T4D by treatment with HNO2, 0.5 M, pH 5.0, at 210C. Stocks of the am mutants were grown in the permissive host strain E. coli CR63 by standard techniques.19 T4D, used as the normal control, is a spontaneous revertant from am 82 and will be referred to as am+; stocks of it were also grown in E. coli CR63. The phage were purified as follows: bacterial debris was removed by centrifuga- tion for 30 min at 5,000 X g, and phage was collected from the supernatant suspension by cen- trifugation at 00 for one hr at 20,000 X g. The pellets were resuspended by covering them with several ml of a buffer solution (containing, per liter, 100 ,umoles of sodium phosphate, pH 7.5, 100 ,umoles MgCl2, and 10 mg of gelatin); after storage in the cold for several days, homogeneous suspensions were obtained by brief agitation. The medium for phage assay contained, per liter: Bacto agar, 10 gm; Bacto tryptone, 13 gm.; NaCl, 8 gm; sodium citrate, 2 gm; and glucose (autoclaved separately), 1.3 gm. Top agar for phage assay contained, per liter: Bacto agar, 6 gm; Bacto tryptone, 10 gm; NaCl, 8 gm; sodium citrate, 2 gm; and glucose (autoclaved sepa- rately), 3 gm. dCMP, dCTP, and dTMP were purchased from the California Corporation for Biochemical Research. dUMP, ATP, and UDP-glucose were purchased from the Sigma Chemical Company. C14-labeled UDP-glucose was prepared and generously provided by P. W. Robbins. C14-labeled formaldehyde was purchased from New England Nuclear Corporation. The T2-DNA used in the HMC-,3 glucosyl transferase assay was the gift of P. F. Davison. Tetrahydrofolic acid was prepared by the method of Blakley.20 The molarity of all Tris buffers refers to the total Tris concentration; that of all ammonium formate buffers refers to the total formic acid-formate concentration. Preparation of C'4-labeled dHMP and unlabeled dHMP: C14-labeled dHMP was prepared enzymatically by a modification of the method of Flaks and Cohen.2' A 1.2 ml aliquot of an extract of E. coli B (harvested 20 min after infection with T2-bacteriophage and prepared as were the concentrated extracts of sets A and B described in the following section) was added to 20 ml of a reaction mixture similar to that previously described for the assay of dCMP hydroxy- methylase'5 but modified by doubling the concentrations of formaldehyde and tetrahydrofolate. The mixture was incubated for 3 hr at 370 and then chilled. Then 8 ml of 1 M formaldehyde and 1.8 ml of 60% trichloroacetic acid were added. The resulting precipitate was removed by cen- trifugation and the trichloroacetic acid in the supernatant fluid was removed by extraction three times with two volumes of ether. After removal of residual ether by aeration, the solution was neutralized with 1 M NH40H and applied to a column of Dowex-1-formate ion exchange resin, 1 X 22 cm. The column was washed with 2 liters of water and elution was begun with 0.015 M ammonium formate, pH 3. By this procedure, the dHMP was completely separated from un- reacted dCMP and was eluted in the region between 400 and 570 ml of effluent. The effluents containing dHMP were concentrated on a rotary evaporator and the residual water and am- VOL. 48, 1962 BIOCHEMISTRY: WIBERG ET AL. 295 monium formate were removed in vacuo at room temperature. The residue was dissolved in 10 ml of water and was stored at - 200. The yield of Ct-labeled dHMP based on the original dCMP was 41%. Yields as high as 57% have been obtained when additional formaldehyde and tetra- hydrofolate were added and the incubation prolonged. Unlabeled dHMP was prepared similarly from unlabeled formaldehyde instead of CI4-labeled formaldehyde. Preparation of extracts from infected bacteria: Two of the three sets of extracts (A and B) were made in identical fashion. Extracts of Set A were prepared from E. coli B infected with T4 am +, am 82, or am 122; those of Set B from E. coli B infected with am+, am 81, am 116, or am 130; those of Set C from E. coli B infected with am+, am 17, or am 90. Cells of E. coli B were grown with vigorous aeration at 370 in the glycerol casamino acid medium of Fraser and Jerrel22 to a concentration of 1.3 X 109 cells per ml. Aeration was then stopped and the culture was stored at room temperature for 30 to 45 min; under these conditions, no further increase in turbidity occurred. Aliquots of 1,300 ml were placed in bottles and warmed to 370, and L-tryptophan was added to a concentration of 10 mg per liter. Vigorous aeration was begun through sintered glass tubes. After 5 min, 4 phage per bacterium were added, and 4 min later, the same amount of phage was added again. The actual multiplicity of infection 3 min after the first infection was calcu- lated to be 2.4 from measurements of surviving cells.23 At various times after infection, aliquots of 130 ml were removed and chilled rapidly by admixture with 60 gm of frozen medium. All subsequent steps were carried out at 4°. The cells were collected by centrifugation at 6,000 X g for 10 min, resuspended with a Potter-Elvehjem homogenizer in 0.02 M Tris acetate, pH 7.5, and the final volume was adjusted to 4 ml. This suspension was frozen in liquid nitrogen and forced through a Hughes press24 to break the cells. The supernatant fluid obtained by centrifuga- tion of the extracts for 30 min at 11,000 X g was used for the DNA polymerase assays. For dCTPase assay on extracts in Set B, these extracts were diluted 1 to 8 with 0.02 M Tris acetate buffer, pH 7.5. For all other assays, the extracts were diluted 1 to 40 with 0.02 M Tris acetate buffer, pH 7.5. Both diluted extracts were centrifuged for 10 min at 11,000 X g; the supernatant fluids were used in the enzyme assays. For the third set (Set C), the infection was performed similarly except that 100 ml of bacterial suspension were used per vessel; the aliquots were removed at various times after infection, pipetted into liquid nitrogen, and forced through a Hughes press. The supernatant fluids ob- tained by centrifugation of the extracts at 700 X g for 3 min were used for all assays. Phage production was determined at about 65 min after the initial infection. Aliquots of 0.1 ml of the cultures were diluted into 100 ml of dilution broth (0.8% Difco nutrient broth powder in 0.5% NaCl) previously shaken with 0.3 ml of chloroform. These diluted samples were left at room temperature for at least 30 min, after which aliquots were plated on E. coli CR63. DNA assay: Trichloroacetic acid was added in the cold to aliquots of the diluted extracts to a concentration of 5%. After 10 min, the precipitate was collected by centrifugation at 1,200 X g for 10 min and washed once with cold 5% trichloroacetic acid and twice with cold 95% ethanol. Residual ethanol was removed in vacuo. The DNA content of the residue was determined by the Ceriotti procedures with the modification that the indole-HCl reagent was added directly to the residue. The standard was the T2-DNA preparation used as primer in the HMC-,3 glucosyl transferase assay below. Enzyme assays: The assay for dCMP hydroxymethylase has been described previously.'5 The DNA polymerase assay is a modification of the procedure of Bollum26 and will be presented in detail elsewhere. It involves the measurement of the rate of incorporation of label from C'4- labeled dHTP into acid-insoluble material by an enzymatic system to which denatured calf thymus DNA6 is added as primer. dCTPase assay: This assay is a modification of that of Koerner, Smith, and Buchanan.27 The reaction mixture contained in 1 ml: enzyme; Tris acetate buffer, pH 9.1, 160 Mmoles; mag- nesium acetate, 5 jmoles; and dCTP, 0.45 emole. After incubation for 60 min at 370, the reac- tion was stopped by addition of 0.2 ml of 0.05 M EDTA, pH 8. The reaction mixture was ap- plied to a 3.3 X 30 mm column of Dowex-1-8X-formate ion exchange resin (200-400 mesh). After adsorption, the column was washed with 1 ml of water followed by 3 ml of 0.01 M ammonium formate buffer, pH 4.3. Elution of dCMP was then accomplished with 3 ml of 0.06 M ammonium formate buffer, pH 2.8. The amount of dCMP was determined by measurement of the absorb- ancy at 280 ma. 296 BIOCHEMISTRY: WIBERG ET AL. PROC. N. A. S. dTMP synthetase assay: The reaction mixture was identical with that used for the dCMP hydroxymethylase assay, with the exception that dUMP was substituted for dCMP. The mix- ture contained, in 0.5 ml: enzyme; Tris acetate buffer, pH 8.0, 25 /Amoles; EI)TA, pH 8.0, 10jumoles; dUMP, 0.7 jmole; 2-mercaptoethanol, 10 /Amoles; tetrahydrofolate, 0.45 JAmole; and CL4-labeled formaldehyde, 0.5 smole (total radioactivity, 200,000 cpm). After incubation for 45 min at 370, the reaction was stopped as follows: the sample was chilled, 1 ml of a cooled solution containing 200 Mmoles of formaldehyde and 20 m/Lmoles of carrier dTMP was immediately added, and without delay the mixture was applied to a 3.3 X 50 mm column of I)owex-1-8X-formate ion- exchange resin (200-400 mesh) in the cold. After adsorption, the column was washed with 5 ml of water at room temperature followed by 4 ml of 0.1 M ammonium formate buffer, pH 4.35. Elution of dTMP was accomplished with an additional 8 ml of the same buffer. The entire eluate containing the dTMP was dried by collecting it directly on an aluminum planchet placed on a hot plate, and the radioactivity was measured. dTMP kinase assay: Enzyme and reagents were incubated under the conditions described by Lehman, Bessman, Simms, and Kornberg.28 The reaction mixture contained, in 1 ml: enzyme; ATP, 1.4 jpmoles; MgCl2, 18 ,umoles; Tris acetate buffer, pH 7.5, 40 /Amoles; and C14-labeled dTMP, 72 mjumoles (total radioactivity, 10,000 cpm). After incubation for 30 min at 370, the reaction was stopped by addition of 0.5 ml of 0.072 M EDTA, pH 8. The reaction mixture was applied to a 3.3 X 30 mm column of Dowex-1-8X-formate ion-exchange resin (200-400 mesh). After adsorption, the column was washed with 1 ml of water followed by 10 ml of 0.25 M am- monium formate, pH 4.35. Elution of dTDP and dTTP was accomplished with 4 ml of 4.0 M ammonium formate, pH 4.35. An aliquot was evaporated on a stainless steel planchet and the radioactivity was measured. dHMP kinase assay: Enzyme and reagents were incubated under essentially the conditions described by Somerville, Ebisuzaki, and Greenberg.4 The reaction mixture contained, in 0.6 ml: enzyme; ATP, 2 hmoles; MgCl2, 5 /Amoles; Tris chloride buffer, pH 8.0, 10 jsmoles; and C14- labeled dHMP, 37 m~umoles (total radioactivity, 3,300 cpm). After incubation for 15 min at 370, the reaction was stopped by the addition of 0.4 ml of 0.03 M formic acid. The reaction mixture was applied to a 3.3 X 100 mm column of Dowex-50W-4X ion-exchange resin (hydrogen form, 200-400 mesh), and the effluent was collected directly on an aluminum planchet placed on a hot plate. Then 1.5 ml of 0.01 M formic acid was applied to the column, the eluate was collected on the same planchet, and the radioactivity was measured. HMC-f3-glucosyl transferase assay: Enzyme and reagents were incubated as described by Kornberg, Zimmerman, and Kornberg.8 The reaction mixture contained, in 0.2 ml: enzyme; Tris acetate buffer, pH 7.5, 20 Mmoles; MgCl2, 5 umoles; DNA from bacteriophage T2, 11 myA moles of DNA-phosphorus, assayed by the method of Chen et al.29; and C14-labeled UDP-glu- cose, 10 mumoles. The rate of incorporation of C'L4-labeled glucose into DNA was determined by adapting the filter paper technique of Bollum.68 During the course of the incubation at 37°, aliquots of 25 ,IA were removed and placed on treated filter paper disks (Schleicher and Schuell Co., No. 595). The treatment involved placing 25 ,.d of 0.45 M EDTA, pH 8, on each disk, which was then dried. EDTA was added to the papers in order to ensure the immediate cessa- tion of the reaction. The dried disks were then treated with trichloroacetic acid and alcohol according to Bollum's procedure and the radioactivity was determined. Infection of E. coli with T4 and Its Am Mutants.-Phage production and synthesis of DNA: Measurements of phage production by E. coli B infected with T4 am+ TABLE 1 PHAGE PRODUCTION 65 MINUTES AFTER INFECTION OF E. coli B BY T4 am + AND BY Amber MUTANTS _-Phage Production per Infected Cell (E. coli B)-- Infecting phage Set A Set B Set C T4 am+ 302 240 270 am 82 0.2 am 122 0.1 am 81 9 am 116 87 am 130 9 am 17 13 am 90 4 VOL. 48, 1962 BIOCHEMISTRY: WIBERG ET AL. 297 and seven am mutants at 65 minutes after infection are given in Table 1. Five of the mutants were selected because they were known to be deficient in DNA synthesis; the other two mutants (am 90 and am 17) can cause synthesis of phage DNA in E. coli B but are defective in some other steps needed for production of infective virus. Table 1 shows that two mutants, am 82 and am 122, are completely unable to reproduce in E. coli B. The other three mutants that are defective in DNA synthesis (am 81, am 116, and am 130) multiply at least to a limited extent. By reference to Figure 1, it may be seen that phage production by these five mu- tants is roughly related to the level of DNA production in infected cells. 500- 10A 0~~~~~~~~~~~~~ 400 8- O O~~~~~~~~~~~0Xox03 0 5 0FG .-omto fezmsatrifc <100 - ___ 0J D0. 2~~~~~~~~00 0 2 0 4 0 6 MINUTES AFTER INFECTION tion with am 82. 0 = dCMP hydroxymeth-ylase. * = dTMP synthetase. o = dHMP FIG. 1. Formation of DNA by E. coli B kinase. A = dTMPkinase. X = dCTPase. after infection by T4 am+ and am mutants. In this and subsequent figures (3, 4, and 5), A: o = T4 am+; X = am 82; * = am the horizontal dotted line represents the level 122. B: o = am 17; V = am 90; /\ = of enzyme activities 15 minutes after infection am 130; v = am 116; O = am 81. with T4 am+. Infection with mutants am 17 and am 90 leads to production of very little phage, although DNA production is normal (see Fig. iB). The course of DNA synthesis after infection with T4 am+ and seven am mutants is illustrated in Figure 1. The values for DNA formation with am 17 and am 90 and with T4 am+ are identical. Two mutants, am 82 and am 122, are essentially incapable of inducing synthesis of DNA. With am 81, DNA synthesis is negligible during the first 20 min, but later there is a slow but significant rate of synthesis. The two remaining mutants, am 130 and am 116, induce DNA synthesis with kinetics strikingly different from that observed with T4 am+~. In cells infected with am 130, DNA synthesis starts at the normal rate but stops abruptly at about 15 minutes after infection. With am 116, DNA synthesis is delayed until approxi- mately 20 minutes and then commences at a rate comparable to that observed with cells infected with normal T4 phage. 298 BIOCHEMISTRY: WJBERG ET AL. PROC. N. A. S. Formation of early enzymes: Extracts of cells infected either by T4 am+ or by one of the am mutants were made from samples taken at suitable intervals and were examined for the levels of several enzyme activities. Figures 2 through 6 present plots of enzyme activity versus time after infection. Data for dCTPase, dCMP 500k 500- 0 400k I ~~~~~~~~~> 400 1300 < N~~~~~~~~~~~~~~~~~~~~Ix TSAT RINFIO W200-~~ ~ ~ ~~~~~~0 w w 1200 -z >100 - 0 0 10 20 30 40 50 60 0 _____ MINUTES AFTER INFECTION 0 10 20 30 40 50 6