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Defective Amber Mutants of Bacteriophage T71 JOURNAL OF VIROLOGY, Aug. 1970, p. 149-155 Vol. 6, No. 2 Copyright © 1970 American Society for Microbiology Printed in U.S.A. Degradation of Escherichia coli B Deoxyribonucleic Acid After Infection with Deoxyribonucleic Acid- Defective Amber Mutants of Bacteriophage T71 PAUL D. SADOWSKI2 AND CAROL KERR Division of General and Experimental Pathology, Department of Pathology, University of Toronto, Toronto, Ontario, Canada Received for publication 27 April 1970 The degradation of bacterial deoxyribonucleic acid (DNA) was studied after in- fection ofEscherichia coli B with DNA-negative amber mutants of bacteriophage T7. Degradation occurred in three stages. (i) Release of the DNA from a rapidly sedi- menting cellular structure occurred between 5 and 6 min after infection. (ii) The DNA was cleaved endonucleolytically to fragments having a molecular weight of about 2 x 106 between 6 and 10 min after infection. (iii) These fragments of DNA were reduced to acid-soluble products between 7.5 and 15 min after infection. Stage 1 did not occur in the absence of the gene 1 product (ribonucleic acid polymerase sigma factor), stage 2 did not occur in the absence of the gene 3 product (phage T7- induced endonuclease), and stage 3 did not occur in the absence of the gene 6 product. It has long been known that Escherichia coli duced ribonucleic acid (RNA) polymerase sigma deoxyribonucleic acid (DNA) is degraded after factor which is necessary for subsequent synthesis infection with T-even (8-10, 12, 14, 15) and T- of most phage proteins (22, 23). Gene 3 is the odd bacteriophages (5, 12, 13, 16, 19). Recently, structural gene for a T7-induced endonuclease the breakdown of cellular DNA after infection involved with breakdown of host-cell DNA (4). It with bacteriophage T4 has been extensively has been suggested that gene 6 codes for an exo- studied. Mutants defective in genes 46 and 47 fail nuclease which is involved in breakdown of cellu- to degrade host DNA to acid-soluble material lar DNA (4). Mutants in gene 1, 3, or 6 do not (26). Sedimentation studies have suggested that, degrade cellular DNA to acid-soluble material after T4 infection, E. coli DNA first undergoes (4,7). endonucleolytic breakdown to fragments of DNA We have studied the breakdown of E. coli B with a molecular weight of greater than 106 (2, DNA after infection with T7 phages bearing 11, 25). These fragments are then reduced directly amber mutations in genes 1 to 6. The results re- to acid-soluble material, a process which requires ported in this paper show that the breakdown of the action of genes 46 and 47 (11). Two endo- cellular DNA to acid-soluble material occurs in nucleases which may be involved in the initial three discrete stages which require the action of stage of degradation of cellular DNA have been gene 1, gene 3, and gene 6, respectively. purified from T4 phage-infected bacteria (17, 18). MATERIALS AND METHODS The genetics and physiology of bacteriophage Bacteria. E. coli B, the nonpermissive host for T7 T7 have received considerable attention recently amber mutants, was obtained from J. Wiberg. E. coli (7, 20, 21). Amber mutants in 19 distinct com- BBW/1 (7) and E. coli 011' (20), the permissive hosts plementation groups have been isolated and the for T7 amber mutants, were obtained from R. Haus- genes have been ordered on a linear genetic map. mann and F. W. Studier, respectively. Bacteriophage. T7 am+, T7 am H280 (gene 1), and The genes are numbered sequentially from left to T7 am H131 (gene 4) were obtained from R. Haus- right, and mutants in genes 1 to 6 are defective in mann (7), whereas T7 am 23 (gene 1), T7 am 64 phage DNA synthesis. Gene 1 specifies a T7-in- (gene 2), T7 am 29 (gene 3), T7 am 20 (gene 4), T7 were the I Supported by the Medical Research Council of Canada and am 28 (gene 5), and T7 am 147 (gene 6) the S. W. Stedman Foundation. gift of F. W. Studier (20). Double amber mutants were 2 Medical Research Council of Canada Scholar. constructed by using the procedure for genetic crosses 149 150 SADOWSKI AND KERR J. VIROL. described by Studier (20). Wild-type T4D was from equal volume of the phosphate buffer portion of GCA the collection of J. Hurwitz. medium containing 0.05 M EDTA before sucrose Chemicals. Unlabeled deoxyadenosine (dA) and gradient centrifugation. deoxythymidine (dT) were purchased from Schwarz Sedimentation analysis. In agreement with Kutter BioResearch. Thymine-2-_4C (59 mCi/mmole) and and Wiberg (11), we found that, at early times after thymidine-methzyl-3H (21 Ci/mmole) were obtained infection, the recovery of labeled cellular DNA was from Amersham/Searle. The 3H-thymidine was diluted poor when the lysate was sedimented in a neutral, 5 to a specific activity of 3.0 Ci/mmole with unlabeled to 20% sucrose gradient even if the gradient was thymidine before use. Lysozyme was the product of formed over a "shelf" of 60% sucrose. This appears Worthington Biochemical Corp., and sodium dodecyl to be due to the fact that before infection and early sulfate (SDS) was purchased from Sigma Chemical after infection the DNA is bound to a very rapidly Co. sedimenting structure which sediments through the Growth of bacteriophage. Stocks of T7 amber 60% sucrose to the bottom of the tube. When such mutants were grown on the permissive host as samples were sedimented through 20 to 60% sucrose described by Hausmann and Gomez (7) or Studier gradients at 5 C, about 60% of the radioactivity was (20). T7+ or T4D+ DNA was labeled with 14C by recovered in the gradient fractions. An additional growing the phage on E. coli BBW/1 whose DNA had 20% could be eluted from the tube with 0.3 N NaOH been labeled with thymine-2-14C. An overnight culture and appeared to have sedimented to the bottom. grown in glycerol-Casamino Acids (GCA) medium (6) Neutral sucrose gradients contained 0.1 M NaCl and was diluted 1:50 into GCA medium containing 250 0.05 M EDTA (pH 8.5), whereas the alkaline gradients Ag of dA per ml and 2 ,uCi of thymine-2-_4C per ml. contained 0.7 M NaCI, 0.3 N NaOH, and 10-3 M The cells were grown at 37 C to a cell density of about EDTA. The 20 to 60% gradients were 3.2 ml in 109/ml and infected with 0.1 phage per cell. Aeration volume, wheras the 5 to 20% gradients were 3.0 ml in was continued at 37 C. Cells infected with T7 phage volume and were formed over a 0.2-ml "shelf" of 60% lysed in 40 min, whereas cells infected with T4 phage sucrose. For alkaline gradients, 0.1 ml of 0.5 M NaOH were lysed with chloroform 4 hr after infection. The was layered on top of the gradient before application phages were purified by differential centrifugation. of the sample. Labeling of bacterial DNA. Experiments were done The entire lysate (0.2 ml) was applied to the in a reciprocating water bath shaker at 37 C. A 10-ml gradient. With 20 to 60% gradients, the lysate was culture of E. coli B was labeled with 3H-dT exactly as gently poured onto the top of the gradient to minimize described by Kutter and Wiberg (11). Under these shear degradation. The sample was applied to the 5 conditions, the 3H-dT was incorporated into cold tri- to 20% gradients with a sawed-off Pasteur pipette. chloroacetic acid-precipitable material at a linear rate Neutral gradients were formed at 5 C and centrifuged until about 20%0 of the label had been incorporated. immediately; alkaline gradients were formed at room When the cell titer was 3 X 108 to 5 X 108/ml, the temperature, and the sample was allowed to stand on cells were washed once in GCA medium and were top of the gradient for 20 min at room temperature suspended in 10 ml of GCA medium containing 62.5 before centrifugation. Centrifugation was done in ,g of dT per ml and 250 ug of dA per ml. After shaking polyallomer tubes of the IEC SB405 rotor at 35,000 for 2 min at 37 C, the cells were infected with the rev/min (140,000 X g) or 54,000 rev/min (300,000 X appropriate T7 phage at a multiplicity of infection of g) at 5 C. The bottom of the tube was punctured, and about 10. the gradient was fractionated into 26 to 29 fractions Incorpoation of 3H-dT into bacterial DNA and the with a peristaltic pump. To each fraction were added amount of acid-insoluble radioactivity remaining after 50 jg of bovine serum albumin and cold 5% trichloro- infection were followed by precipitating 0.1 -ml samples acetic acid. The sample was deposited on a filter disc with 5% trichloroacetic acid. Each sample was (Millipore, type HA, or Whatman 3MM paper), filtered onto a membrane filter (type HA; Millipore which was washed with I% trichloroacetic acid and Corp., Bedford, Mass.) or a Whatman 3MM paper dried. Radioactivity was determined with a liquid disc; the filter was washed with 1% trichloroacetic scintillation spectrometer set for counting 3H in the acid and dried, and the radioactivity was determined presence of 4C. The approximate efficiency for count- in a liquid scintillation counter. ing of the Millipore filters was 35% for tritium and Preparation of cell lysates. The method used was 70% for '4C, whereas for the paper filters the approxi- identical to the procedure of Kutter and Wiberg (11), mate efficiency was 20%o for tritium and 40% for 1"C.
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