Vol. 22, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, May 1977, p. 540-547 Copyright ©D 1977 American Society for Microbiology
Intracellular Organization of Bacteriophage T7 DNA: Analysis of Parental Bacteriophage T7 DNA-Membrane and DNA-Protein Complexes RON HIEBSCH AND MELVIN S. CENTER* Division of Biology, Kansas State University, Manhattan, Kansas 66506 Received for publication 3 December 1976
After infection of Escherichia coli with bacteriophage T7, the parental DNA
forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Brij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000, and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.
associated with certain proteins to form a structure which exhibits a fast sedimentation rate in sucrose gradients.
After infection ofEscherichia coli with bacteriophage T7, both the parental and newly synthesized DNA exhibit a fast sedimentation rate in neutral sucrose gradients (5). Subsequent studies concerned with analyzing newly synthesized T7 DNA strongly suggested an association of the DNA with host membranes (17). Similar findings for many other phage systems have also been described (24). Recently, it has been shown that newly synthesized T7 DNA can be isolated which is essentially free of membrane material but the DNA still has a 600S value (23). An electron microscope study indicates that the sedimentation rate of this DNA may be due to the presence of a highly condensed, compact structure (23). The data obtained thus far suggest that the intracellular DNA of T7 may have features similar to the E. coli chromosome which exists as a compact, folded structure attached to the cell envelope (18, 19, 26, 29). In the case of E. coli it appears that RNA and protein may have a major role in maintaining the compactness of the DNA (7, 18, 19). In continuing our studies on the intracellular organization of the T7 chromosome, we have observed that the infecting parental DNA associates with host membranes to form a stable complex which can be purified by centrifugation in cesium chloride gradients. In the present report we describe some properties of the complex and a detailed analysis of the protein composition of this material. We also show that most of the membrane lipid can be removed from the complex, but that the DNA remains
MATERIALS AND METHODS Bacterial strains. E. coli B was used as the host for T7 phage infection. E. coli 011' (ATCC 27214) was the permissive host for amber mutants. Bacteriophage. Bacteriophage T7 was obtained from W. F. Studier. All experiments were carried out with T7 am28, a mutant in gene 5. This phage mutant will be designated by gene number. Preparation of phage. T7 phage was prepared essentially as described by Studier (27). Phage containing 32p_ or 3H-labeled DNA were purified in cesium chloride density gradients (4) before use. Other materials. [3H]thymidine (14.3 Ci/mmol), [3H]uridine (28 Ci/mmol), and 32p were obtained from Schwarz Bio Research. [35S]methionine (470 Ci/mmol) and [2-3H]glycerol (7 Ci/mmol) were purchased from New England Nuclear Corp. Brij-58 was obtained from Atlas Chemical Industries and Sarkosyl-NL97 was from Geigy. Other reagents used were as described previously (5, 17). Infection with T7 and preparation of cell lysates. In the present experiments E. coli B was infected with radioactively labeled T7 phage bearing an amber mutation in gene 5, the structural gene for a subunit of the T7 DNA polymerase holoenzyme (16). The gene 5 protein is essential for viral DNA synthesis (16, 27). Thus, in these experiments the fate of intracellular parental T7 DNA could be followed under conditions which avoided the presence of replicative intermediates such as concatemers (11). E. coli B was grown in M9 medium (1) supplemented with 0.2% Casamino Acids. Unless indicated otherwise, cell infection was carried out at 30°C. When the density reached 2 x 108 cells/ml, the 540
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culture was infected with 32P- or 3H-labeled phage at a multiplicity of infection of 5. At 10 min after infection, cell cultures were mixed with an equal volume of 0.15 M NaCl-0.015 M EDTA and then centrifuged. The cells were lysed by the method of Knippers and Sinsheimer (12). The pellet from 2 x 109 infected cells was suspended in 0.3 ml of a 20% sucrose solution prepared in 0.05 M Tris-hydrochloride (pH 8.0). To this solution 0.1 ml of lysozyme (2 mg/ml in 0.25 M Tris-hydrochloride, pH 8.0) and 0.1 ml of 0.01 M EDTA were added, and incubation was carried out for 30 min at 00. At the end of the incubation period, 0.15 ml of a 5% solution of Brij-58 was added. After the solution was held for 20 min at 00C, lysis was complete. Labeling host membranes. E. coli B was grown as described above, and when the cell density reached 2.0 x 108 cells/ml [2-3H]glycerol, a radioactive label specific for E. coli membranes (8), was added to a final concentration of 1.6 ,uCi/ml. Twenty minutes later the cells were infected with 32P-labeled T75. At 10 min after infection cell lysates were prepared. Labeling host and viral proteins. E. coli B was grown in Tris-glucose medium (28) containing 0.05% Casamino Acids. When the cell density reached 2.5 x 108 cells/ml, [35S]methionine was added to a final concentration of 4 ttCi/ml. After 20 min the cells were infected with 3H-labeled T75 phage, and at 10 min after infection cell lysates were prepared. Sedimentation analysis of intracellular parental T7 DNA. (i) Cesium chloride density gradients. Portions of the cell lysates were centrifuged in a stepwise cesium chloride density gradient in the Spinco SW50.1 rotor for 2 h at 40,000 rpm and 5°C. In our initial studies (data described in Fig. 1, 2, and 3) step gradients were prepared by layering 1.5 ml each of cesium chloride solutions having densities of 1.3, 1.46, and 1.6 g/ml. The cesium chloride solutions were prepared in 0.01 M Tris-hydrochloride (pH 8.0). In all other experiments lysates were centrifuged under identical conditions except that the density of the top layer was reduced to 1.2 g/ml. Under these conditions of centrifugation the T7 DNA-membrane complex sediments to a position in the gradient which allows its separation from free DNA and lowmolecular-weight material. After the first cesium chloride centrifugation, the membrane complex can be detected in the gradient tube as a white band when viewed against a black background. When the complex was to be isolated for subsequent use, the band was taken up in a plastic pipette and then dialyzed against 0.02 M Tris-hydrochloride (pH 7.6) for 5 h at 4°C. The dialyzed membrane complex was further purified by centrifugation in a second cesium chloride gradient under identical conditions described above. At the end of the centrifugation, four-drop fractions were collected from the bottom of the tube and a 0.02-ml portion was used for radioactivity determination. The DNA-membrane complex was isolated and then dialyzed as described above. The purified membrane complex was used for most of the experiments described below. Polyacrylamide gel electrophoresis. Proteins labeled with [35S]methionine were examined by electrophoresis on 10% polyacrylamide slab gels with a
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FIG. 1. Sedimentation analysis of intracellular parental T7 DNA. E. coli B was infected with 32Jp labeled T75, and at 10 min after infection cell lysates were prepared. A portion of the lysate (0.1 ml) was mixd with 3H-labeled reference T7 DNA, and the solution was centrifuged in a cesium chloride step gradient as described in the text (A). In a separate but identical experiment lysates were centrifuged in a cesium chloride step gradient and the 32P-labeled T7 DNA present in the low-density portion of the gradient was isolated and dialyzed for 5 h against 0.02 M Tris-hydrochloride (pH 7.6). The dialyzed material was mixed with 3H-labeled reference T7 DNA and then centrifuged in a 5 to 20% sucrose gradient containing a 1.0-ml cesium chloride shelf (1.1 g of cesium chloride per ml of 20% sucrose). Sucrose solutions were prepared in 20 mM Tris-hydrochloride (pH 7.6)-0.2 mM EDTA. Centrifugation was for 30 min at 30,000 rpm and 5°C in the Spinco SW50.1 rotor (B). Three-drop fractions were collected directly into scintillation vials containing glass-fiber filter paper. After the filters were dried the radioactivity was determined. Symbols: x, 32P-labeled T7 DNA; *, 3H-labeled reference T7 DNA.
5% stacking gel as described by Laemmli (13). Proteins were denatured in sodium dodecyl sulfate (SDS) before electrophoresis as described by McMillen and Consigli (14). The fixed gels were dried under vacuum and autoradiograms were prepared using RP Royal "X-Omat" film. In some cases the gels were subjected to fluorography as described by Bonner and Laskey (3). Marker proteins labeled with 14C by reductive alkylation (14) were generously provided by R. A. Consigli. These proteins consisted of bovine serum albumin with a molecular weight of 68,000 (68K); ovalbumin, 43K; chymotrypsinogen, 25K; and cytochrome c, 12.4K. Tracings of the autoradiograms were made with a Joyce-Loebl microdensitometer.
RESULTS Evidence that intracellular parental T7 DNA is associated with host membranes. (i) Sedimentation of T7 DNA in cesium chloride density gradients. Previously we have pro-
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FIG. 2. Separation of host and intracellular T7 DNA in cesium chloride gradients. E. coli B was grown in M9 medium containing 0.2% Casamino Acids to a density of 2 x 108 cells/ml. [3H]thymidine was added to the culture (2 uCilml), and the cells were grown for an additional 60 min. The cells were centrifuged and suspended in fresh M9 medium at 250C and then infected with 32P-labeled T75 phage (multiplicity of infection = 5). At 15 min after infection, cell lysates were prepared and a portion of the lysate (0.1 ml) was centrifuged in a cesium chloride step gradient as described in the text. Acid-insoluble radioactivity was determined in three-drop fractions collected from the bottom of the tube. Symbols: x, 32P-labeled intracellular T7 DNA; *, 3H-labeled host DNA.
vided evidence that replicating bacteriophage T7 DNA is associated with host membranes (17). In the present experiments we provide similar findings for parental T7 DNA and show that this membrane complex is stable to cesium chloride gradient centrifugation. This latter finding has facilitated the purification of the DNA-membrane complex and its separation from extraneous proteins and nucleic acids. The sedimentation properties of intracellular parental T7 DNA in a cesium chloride gradient are shown in Fig. 1A. About 60% of the DNA sediments to a position in the gradient having a density of 1.3 g/ml. This DNA, which represents 60 to 70% of the recovered radioactivity in various experiments, is completely separated from the exogenous DNA marker (Fig. 1A). If the parental T7 DNA which exhibits a low density in cesium chloride is isolated, dialyzed, and recentrifuged in sucrose, most of this material has an extremely fast sedimentation rate (Fig. 1B).
Additional studies have been carried out to determine if intracellular parental T7 DNA is separated from host DNA after cesium chloride gradient centrifugation. This should be the case since previous studies have shown that after T7 infection the host DNA is released from its association with cell membranes (21, 25). E. coli B was labeled with [3H]thymidine and then infected with 32P-labeled T75 phage. Cell lysates were prepared and centrifuged in cesium chloride density gradients. In this experiment infected cells were collected before a time when
extensive degradation of host DNA had occurred. There is an essentially complete separation of labeled host DNA from the major portion of the intracellular parental T7 DNA (Fig. 2). We have also carried out studies to analyze the sedimentation pattern of intracellular parental T7 DNA and RNA pulse-labeled during T7 infection. E. coli B was infected with 32P_ labeled T75 phage, and at 15 min after infection (250C) the cells were pulse-labeled for 1 min with [3H]uridine. Preliminary experiments have demonstrated that T7 RNA synthesis is maximal at 15 to 17 min after infection. Cell lysates were prepared and then centrifuged in neutral sucrose containing a cesium chloride shelf. About 70% of the DNA but only 10% of the pulse-labeled RNA exhibits a fast sedimentation rate in the sucrose gradient (Fig. 3A). When the fast-sedimenting DNA and RNA are isolated and recentrifuged in a cesium chloride
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FIG. 3. Sedimentation analysis of intracellular T7 DNA and RNA. A 5-ml culture of E. coli B was grown in M9 medium supplemented with 0.2% Casamino Acids to a density of 3.5 x 108 cells/ml. Then the cells were infected at 25°C with 32P-labeled T75 phage. The infected cells were labeled with [3H]uridine (2 ,uCilml) for 1 min beginning at 15 min after infection. At the end of the labeling period, cell lysates were prepared and then mixed with 2 volumes of 0.05 M Tris-hydrochloride (pH 8.0). A portion of this solution was centrifuged in a 5 to 20% sucrose gradient containing a cesium chloride shelf. Centrifugation was carried out in the Spinco SW27 rotor for 30 min at 20,000 rpm and 5°C. Sucrose solutions were prepared in 0.01 M Tris-hydrochloride (pH 7.6)-0.001 M EDTA. Fractions (1 ml) were collected and the acid-insoluble radioactivity in a 0.1-ml sample was determined (A). Fractions 6 and 7 from the sucrose gradient were pooled and dialyzed. A portion of the dialyzed material (0.3 ml) was centrifuged for 17 h in a cesium chloride gradient as described in the text, and the radioactivity in the collected fractions was determined as described in the legend to Fig. lB. Symbols: x, 32P-labeled intracellular T7 DNA; 0, 3H-labeled RNA.
INTRACELLULAR STRUCTURE OF T7 DNA
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gradient, all of the DNA and 50% of the labeled RNA cosediment as low-density material (Fig. 3B). The nature of the RNA that cosediments with the DNA is unknown at the present time. (ii) Cosedimentation of host membrane and intracellular parental T7 DNA. The studies described above show that a major portion of the intracellular parental T7 DNA sediments in cesium chloride gradients as low-density material. Studies were therefore carried out to determine if the sedimentation rate of this DNA was due to its association with host membranes. Evidence that this is the case is provided by the finding that 3H-labeled host membranes and 32P-labeled parental T7 DNA cosediment in cesium chloride density gradients (Fig. 4). The association between T7 DNA and host membranes appears to be quite stable because, if the complex from the first gradient is isolated, dialyzed, and recentrifuged under identical conditions, DNA and membranes still exhibit the same sedimentation rate. The DNA associated with membrane material is susceptible to degradation by pancreatic DNase, and the complex is completely disrupted by treatment with 1% Sarkosyl for 30 min at 30°C. (iii) Protein composition of the T7 DNAmembrane complex. E. coli B was grown in TCG (Tris-Casamino Acids-glucose) medium, and host and viral proteins were labeled with P5S]methionine as described in Materials and Methods. At 10 min after infection, cell lysates were prepared and then centrifuged in a cesium chloride gradient. The DNA-membrane complex was isolated and dialyzed, and a portion of this material was centrifuged in a second
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cesium chloride gradient. The- second centrifugation results in the removal of about 70% of the labeled protein present in the original complex (Fig. 5). However, a significant fraction of the radioactively labeled protein still cosediments with the 3H-labeled T7 DNA. The DNA-membrane complex isolated from the first cesium chloride gradient and fractions 17 and 22-23 from the second gradient (Fig. 5) were analyzed for protein after polyacrylamide gel electrophoresis (Fig. 6). Proteins in the gel were identified by autoradiographic techniques. The membrane complex isolated from the first cesium chloride gradient consists of a complex mixture of some 25 polypeptides (Fig. 6D). Further purification of the complex results in the selective removal of several proteins (Fig. 6B). An examination of the protein pattern of the purified complex (Fig. 6C) indicates that it has become enriched for a 54K protein doublet and a major 32K protein. The 32K protein in particular appears to retain an essentially complete association with the complex during its purification. Several minor proteins are also present in the purified complex, and the most notable of these are two polypeptides with molecular weights of 29,000 and 24,000 (Fig. 6C). Additional studies have demonstrated that the DNA-membrane complex is stable to a third cesium chloride gradient centrifugation and that the protein composition of this material is identical to that of the complex shown in Fig. 6C. Essentially identical results as those described above have been obtained when [14C]leucine is used in place of P15S]methionine for labeling proteins. An exper100
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FIG. 4. Sedimentation analysis of host cell membranes and T7 DNA. E. coli B (10-ml culture) was labeled with [2-3H]glycerol and infected with 32p_ labeled T75 phage. Lysates were prepared and centrifuged in a cesium chloride step gradient as described in the text. Fractions from the gradient were collected into tubes and a 0.025-ml sample was used for radioactivity determination. Symbols: x, 32P-labeled intracellular T7 DNA; 0, 3H-labeled membranes. The arrow indicates the position to which marker T7 DNA sediments.
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FIG. 5. Sedimentation analysis of intracellular parental T7 DNA and labeled proteins. E. coli B (10ml culture) was grown in TCG medium, and host and viral proteins were labeled with [35S]methionine as described in the text. Cell lysates were prepared and then centrifuged in a cesium chloride step gradient (density, 1.2 to 1.6 g/ml). The DNA-membrane complex was isolated from the gradient and dialyzed for 5 h against 0.02 M Tris-hydrochloride (pH 7.6). The dialyzed material was centrifuged in a second cesium chloride step gradient, and fractions were collected into tubes. A 0.02-ml sample was used for radioactivity determination. Symbols: x, 3H-labeled intracellular T7 DNA; 0, 35S-labeled protein.
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The protein composition of this material was also found to be identical to that of DNA-membrane complexes isolated from T7-infected cells (Fig. 6C). Thus, this seems to indicate that all proteins present in the purified T7 DNA membrane complex are derived from the host. Alternatively, certain T7 and host proteins could have identical electrophoretic mobilities. (iv) Effect of detergents on DNA-membrane association. DNA-membrane complexes containing 32P-labeled T7 DNA and 3H-labeled membranes were incubated in the presence of 1% Brij-58 or Sarkosyl and then centrifuged in neutral sucrose gradients. As shown in Fig. 7, Brij and Sarkosyl have quite different effects on the sedimentation rate of the membraneassociated DNA. Sarkosyl causes the complete disruption of the complex, solubilizing the
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FIG. 6. Polyacrylamide gel electrophoresis of proteins associated with the T7 DNA-membrane complex. 35S-labeled proteins isolated from the cesium chloride density gradient described in the legend to Fig. 5 were subjected to SDS-polyacrylamide gel electrophoresis as described in the text. (A) Protein markers; (B) fractions 22-23 from the second cesium chloride gradient; (C) fraction 17 from the second cesium chloride gradient; (D) DNA-membrane complex isolated from the first cesium chloride gradient. iment has also been carried out in which cells were infected with 3H-labeled T75 phage, and at 2 min after infection proteins were labeled with [35Slmethionine for 10 min. Membrane com-
plexes purified through two cesium chloride gradients were analyzed after electrophoresis in 10% polyacrylamide gels. The total protein composition was essentially identical to that for isolated complexes labeled with [35S]methionine before and during T7 infection (Fig. 6C). In a parallel experiment, uninfected cells were labeled with [35S]methionine and host membranes in sheared lysates were purified through two cesium chloride gradients.
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FIG. 7. Effect of detergents on the sedimentation rate of membrane-associated T7 DNA. DNA-membrane complexes prepared as described in the legend to Fig. 4 were purified through two cesium chloride gradient centrifugations. Portions of the isolated complex (0.05 ml) were mixed with 0.1 ml of 0.01 M Tris-hydrochloride (pH 7.6) and either 0.05 ml of 5 % Brij-58 or 0.05 ml of 5% Sarkosyl. The solutions were incubated at 32°C for 30 min and then centrifuged in a 5 to 20% neutral sucrose gradient containing a 1.0-ml cesium chloride shelf. Centrifugation was carried out as described in the legend to Fig. 1. (A) Incubation in the absence of detergent; (B and C) incubation with Brij and Sarkosyl, respectively. Symbols: x, 32P-labeled intracellular T7 DNA; *, 3H-labeled host membranes.
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membrane, and releasing the DNA which now sediments near the top of the gradient (Fig. 7C). In contrast, after incubation with Brij-58, 95% of the 3H-labeled membrane is removed C) ?o from the complex, but the DNA still exhibits a fast sedimentation rate in the sucrose gradient 2 - 4 .45O a. 12(Fig. 7B). If the fast-sedimenting Brij-treated U,. 3 with Pronase material is isolated and incubated or RNase, the DNA still exhibits a fast sedimen-2 tation rate. The DNA is, however, converted to acid-soluble material after treatment with pan10 5 15 creatic DNase. After treatment of the complex FRACTION NUMBER with Brij, the resulting DNA structure has an S value of about 1,000 compared to internal T7 FIG. 8. Sedimentation of Brij treated DNA-membrane complex in sucrose gradients. Analysis ofDNA phage (453S) or T4 phage (1,OOOS) markers. (v) RNA composition of Brij-treated DNA- and protein. E. coli B (10-ml culture) was grown in membrane complex. Additional studies have TCG medium, and host and viral proteins were lawith [35S]methionine as described in the text. been carried out in which [3H]uridine was beled The DNA-membrane complex was purified through added to a cell culture 15 min before infection. two chloride gradient centrifugations. A porThe cells were infected with 32P-labeled T75 tion cesium of the isolated complex (0.2 ml) was treated with phage, and 10 min after infection lysates were Brij-58 as described in the legend to Fig. 8. The Brijprepared. The membrane complex purified treated material was centrifuged in a neutral sucrose through two cesium chloride gradients was gradient as described in the legend to Fig. 1. Symtreated with Brij and centrifuged in neutral bols: x, 3H-labeled T7 DNA; 0, 35S-labeled proteins. sucrose. It was found that only 3% of the labeled RNA associated with the membrane comDISCUSSION plex after the second cesium chloride gradient Our studies on the intracellular organization cosedimented with the DNA complex. (vi) Protein composition of the Brij-treated of bacteriophage T7 DNA continue to demonDNA-membrane complex. DNA-membrane strate an association of the chromosome with complexes containing 35S-labeled protein and host membranes. In the present work we have 3H-labeled T7 DNA were prepared as described shown that intracellular parental T7 DNA sediin Materials and Methods. The purified com- ments in cesium chloride gradients as low-denplex was treated with Brij and then centrifuged sity material coincident with host membranes. in a neutral sucrose gradient. After Brij treat- Of particular importance in these experiments ment about 55% of the labeled protein still asso- is the evidence suggesting that the intracelciates with the DNA as fast-sedimenting mate- lular DNA does not become trapped in memrial (Fig. 8). Additional studies have been car- brane vesicles during preparation of the cell ried out in which the membrane complex was lysate. Evidence against the idea of nonspecific treated with Brij and the solution was sepa- trapping is provided by the finding that the rated by centrifugation into a pellet (DNA-pro- membrane complex can be isolated under contein complex) and supernatant fractions. The ditions which allow it to be separated from proteins present in each fraction were analyzed endogenous host DNA and the bulk of RNA after electrophoresis in an SDS-polyacrylamide labeled during T7 infection. The parental T7 DNA-membrane complex is gel (Fig. 9). Before Brij treatment the membrane complex had an electrophoretic profile quite stable and can be purified by centrifugaessentially identical to that shown in Fig. 6C. tion in successive cesium chloride gradients. In However, as shown in Fig. 9B, Brij treatment of previous studies we have observed that centrifthe complex results in the selective removal of ugation of membrane complexes containing several protein components which are now newly synthesized T7 DNA in cesium chloride present in the supernatant fraction. The 34K for time periods similar to those described in protein present in the supernatant fraction is a the present work results in the dissociation of major component of the complex. We have the DNA from membranes (17). These results found, however, that complete resolution of the suggest that the nature of the linkage involved 34K and 32K proteins is only observed after in maintaining parental and newly synthesized electrophoresis of the supernatant and pellet DNA-membrane attachment may be quite diffractions obtained after Brij treatment of the ferent. The T7 DNA-membrane complex purified through two cesium chloride gradient cencomplex. b
x
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FIG. 9. SDS-gel electrophoresis of isolated DNAprotein complex. DNA-membrane complexes were isolated and treated with Brij-58 as described in the legend to Fig. 8. After Brij treatment the solution was centrifuged at 15,000 rpm for 30 min in the Sorval SE-12 rotor. The pellet was taken up in 0.05 ml of 10-3 phosphate buffer (pH 7.5). The proteins in the supernatant were precipitated with 5% trichloroacetic acid, and the resulting pellet was washed with cold acetone and then taken up in 0.05 ml of 10-3 M phosphate buffer (pH 7.5). The proteins in the pellet (A) and those from the supernatant (B) were electrophoresed in an SDS-polyacrylamide gel as described in the text. Autoradiogram was traced with a JoyceLoebl microdensitometer.
trifugations contains a reproducible set of proteins which are derived from host membranes. The protein pattern is still complex and several distinct polypeptides can be identified. Certain major proteins are present and these have molecular weights of 54K, 34K, and 32K. Recent studies indicate that two proteins having molecular weights of 80K and 56K may be present at the site of attachment of E. coli DNA to membranes (20). Possibly the proteins of similar molecular weights observed in the purified T7 membrane complex are identical to those described by Portalier and Worcel (20) and may be involved in both E. coli and T7 DNA membrane attachment.
Treatment ofthe purified membrane complex with Brij-58 releases 95% of the lipid-containing material and selectively removes several distinct protein components. The major protein present in the complex after Brij treatment has a molecular weight of 32K and may be identical to the 31K polypeptide which makes up close to 50% of the total outer membrane protein (20). This would be consistent with the finding that nonionic detergents solubilize the inner membrane of the cell envelope, in contrast to the outer membrane which is resistant to similar treatment (22). Thus, it seems that treatment of the membrane complex with Brij results in the formation of a DNA-protein complex in which most proteins are probably derived from the outer E. coli cell membrane. It has been found, however, that the sedimentation rate of the Brij-treated complex is resistant to treatment with Pronase, suggesting that factors in addition to protein contribute to the high S value of this material. The sedimentation rate of the complex may be explained if the DNA exists as a highly condensed compact structure whose conformation is maintained by protease-resistant substances. Recent studies have shown that in T7-infected cells replicating DNA may exist as a highly compact globular structure containing up to 30 phage equivalents of DNA (2, 23). Similar types of condensed structures have also been described for the E. coli chromosome (18, 19, 26, 29) and parental and newly synthesized T4 DNA (6, 9, 10). The condensed intracellular DNA in T4- (6, 9, 10) and T7-infected (23) cells has been isolated in a form which is essentially free of membrane material, and these structures are resistant to treatment with proteolytic enzymes. It seems indicated, however, that the absence of DNA-membrane association may be related to the use of strong ionic detergents in preparing lysates or to the prolonged incubation of detergent-treated lysates at room temperature (6, 9, 10, 23). In the latter case (23) this may be similar to the finding that the presence of the membrane-bound or membrane-free E. coli chromosome is dependent on the temperature at which lysates are prepared (19, 30). Lysis of cells at 250C results in the formation of the membrane-free chromosome. The evidence obtained thus far suggests that in T7-infected cells both the parental and newly synthesized DNA are membrane bound and that the DNA associated with membranes may have a compact structure. ACKNOWLEDGMENT
This investigation was supported by Public Health
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Service research grant AI-10238 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virus resistant mutants of Escherichia coli strain "B." Proc. Natl. Acad. Sci. U.S.A. 32:120-128. 2. Bernstein, C., and H. Bernstein. 1974. Coiled rings of DNA released from cells infected with bacteriophages T7 or T4 or from uninfected Escherichia coli. J. Virol. 13:1346-1355. 3. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:8388. 4. Center, M. S., and C. C. Richardson. 1970. An endonuclease induced after infection ofEscherichia coli with bacteriophage T7. I. Purification and properties of the enzyme. J. Biol. Chem. 245:6285-6291. 5. Center, M. S. 1972. Replicative intermediates of bacteriophage T7 DNA. J. Virol. 10:115-123. 6. Curtis, M. J., and B. Alberts. 1976. Studies on the structure of intracellular bacteriophage T4 DNA. J. Mol. Biol. 102:793-816. 7. Drlica, K., and A. Worcel. 1975. Conformational transitions in the Escherichia coli chromosome: analysis by viscometry and sedimentation. J. Mol. Biol. 98:393411. 8. Green, E. W., and M. Schaechter. 1972. The mode of segregation of the bacterial cell membrane. Proc. Natl. Acad. Sci. U.S.A. 69:2312-2316. 9. Hamilton, S., and D. E. Pettijohn. 1976. Properties of condensed bacteriophage T4 DNA isolated from Escherichia coli infected with bacteriophage T4. J. Virol. 19:1012-1027. 10. Huberman, J. A. 1968. Visualization of replicating mammalian and T4 bacteriophage DNA. Cold Spring Harbor Symp. Quant. Biol. 33:509-524. 11. Kelly, T. J., Jr., and C. A. Thomas, Jr. 1969. An intermediate in the replication of bacteriophage T7 DNA molecules. J. Mol. Biol. 44:459-475. 12. Knippers, R., and R. S. Sinsheimer. 1968. Process of infection with bacteriophage 46X174. XX. Attachment of the parental DNA of bacteriophage 4X174 to a fast sedimenting cell component. J. Mol. Biol. 34:17-29. 13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:660-685. 14. McMillen, J., and R. A. Consigli. 1974. Characterization of polyoma DNA-protein complexes. I. Electrophoretic identification of the proteins in a nucleoprotein complex isolated from polyoma-infected cells. J. Virol. 14:1326-1336.
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15. McMillen, J., and R. A. Consigli. 1974. In vitro radioisotopic labeling or proteins associated with purified polyoma virions. J. Virol. 14:1627-1629. 16. Modrich, P., and C. C. Richardson. 1975. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Bacteriophage T7 DNA polymerase: an enzyme composed of phage- and host-specified subunits. J. Biol. Chem. 250:5515-5522. 17. Pacumbaba, R. P., and M. S. Center. 1973. Association of replicating bacteriophage T7 DNA with bacterial membranes. J. Virol. 12:855-861. 18. Pettijohn, D. E., and R. Hecht. 1973. RNA molecules bound to the folded bacterial genome stabilize DNA folds and segregate domains of supercoiling. Cold Spring Harbor Symp. Quant. Biol. 38:31-41. 19. Pettijohn, D., R. Hecht, 0. G. Stonington, and T. Stamato. 1973. Factors stabilizing DNA folding in bacterial chromosomes, p. 145-162. In R. Wells and R. Inmau (ed.), DNA synthesis in vitro. University Park Press, Baltimore, Md. 20. Portalier, R., and A. Worcel. 1976. Association of the folded chromosome with the cell envelope of E. coli: characterization of the proteins at the DNA-membrane attachment site. Cell 8:245-255. 21. Sadowski, P. D., and C. Kerr. 1970. Degradation of Escherichia coli B deoxyribonucleic acid after infection with deoxyribonucleic acid-defective amber mutants of bacteriophage T7. J. Virol. 6:149-155. 22. Schnaitman, C. A. 1971. Effect of ethylenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolated cell walls ofEscherichia coli. J. Bacteriol. 108:553-563. 23. Serwer, P. 1974. Fast sedimenting bacteriophage T7 DNA from T7-infected Escherichia coli. Virology 59:70-88. 24. Siegel, P. J., and M. Schaechter. 1973. The role of the host cell membrane in the replication and morphogenesis of bacteriophages. Annu. Rev. Microbiol. 27:261-282. 25. Silberstein, S., M. Inouye, and F. W. Studier. 1975. Studies on the role of bacteriophage T7 lysozyme during phage infection. J. Mol. Biol. 96:1-11. 26. Stonington, O., and D. Pettijohn. 1971. The folded genome of Escherichia coli isolated in a protein-DNARNA complex. Proc. Natl. Acad. Sci. U.S.A. 68:6-9. 27. Studier, F. W. 1969. The genetics and physiology of bacteriophage T7. Virology 39:562-574. 28. Thomas, C. A., Jr., and J. Abelson. 1966. The isolation and characterization of DNA from bacteriophage. In G. L. Cantoni and D. R. Davies (ed.), Procedures in nucleic acid research. Harper & Row, New York. 29. Worcel, A., and E. Burgi. 1974. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J. Mol. Biol. 82:91-105.
JOURNAL OF VIROLOGY, May 1977, p. 540-547 Copyright ©D 1977 American Society for Microbiology
Intracellular Organization of Bacteriophage T7 DNA: Analysis of Parental Bacteriophage T7 DNA-Membrane and DNA-Protein Complexes RON HIEBSCH AND MELVIN S. CENTER* Division of Biology, Kansas State University, Manhattan, Kansas 66506 Received for publication 3 December 1976
After infection of Escherichia coli with bacteriophage T7, the parental DNA
forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Brij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000, and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.
associated with certain proteins to form a structure which exhibits a fast sedimentation rate in sucrose gradients.
After infection ofEscherichia coli with bacteriophage T7, both the parental and newly synthesized DNA exhibit a fast sedimentation rate in neutral sucrose gradients (5). Subsequent studies concerned with analyzing newly synthesized T7 DNA strongly suggested an association of the DNA with host membranes (17). Similar findings for many other phage systems have also been described (24). Recently, it has been shown that newly synthesized T7 DNA can be isolated which is essentially free of membrane material but the DNA still has a 600S value (23). An electron microscope study indicates that the sedimentation rate of this DNA may be due to the presence of a highly condensed, compact structure (23). The data obtained thus far suggest that the intracellular DNA of T7 may have features similar to the E. coli chromosome which exists as a compact, folded structure attached to the cell envelope (18, 19, 26, 29). In the case of E. coli it appears that RNA and protein may have a major role in maintaining the compactness of the DNA (7, 18, 19). In continuing our studies on the intracellular organization of the T7 chromosome, we have observed that the infecting parental DNA associates with host membranes to form a stable complex which can be purified by centrifugation in cesium chloride gradients. In the present report we describe some properties of the complex and a detailed analysis of the protein composition of this material. We also show that most of the membrane lipid can be removed from the complex, but that the DNA remains
MATERIALS AND METHODS Bacterial strains. E. coli B was used as the host for T7 phage infection. E. coli 011' (ATCC 27214) was the permissive host for amber mutants. Bacteriophage. Bacteriophage T7 was obtained from W. F. Studier. All experiments were carried out with T7 am28, a mutant in gene 5. This phage mutant will be designated by gene number. Preparation of phage. T7 phage was prepared essentially as described by Studier (27). Phage containing 32p_ or 3H-labeled DNA were purified in cesium chloride density gradients (4) before use. Other materials. [3H]thymidine (14.3 Ci/mmol), [3H]uridine (28 Ci/mmol), and 32p were obtained from Schwarz Bio Research. [35S]methionine (470 Ci/mmol) and [2-3H]glycerol (7 Ci/mmol) were purchased from New England Nuclear Corp. Brij-58 was obtained from Atlas Chemical Industries and Sarkosyl-NL97 was from Geigy. Other reagents used were as described previously (5, 17). Infection with T7 and preparation of cell lysates. In the present experiments E. coli B was infected with radioactively labeled T7 phage bearing an amber mutation in gene 5, the structural gene for a subunit of the T7 DNA polymerase holoenzyme (16). The gene 5 protein is essential for viral DNA synthesis (16, 27). Thus, in these experiments the fate of intracellular parental T7 DNA could be followed under conditions which avoided the presence of replicative intermediates such as concatemers (11). E. coli B was grown in M9 medium (1) supplemented with 0.2% Casamino Acids. Unless indicated otherwise, cell infection was carried out at 30°C. When the density reached 2 x 108 cells/ml, the 540
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culture was infected with 32P- or 3H-labeled phage at a multiplicity of infection of 5. At 10 min after infection, cell cultures were mixed with an equal volume of 0.15 M NaCl-0.015 M EDTA and then centrifuged. The cells were lysed by the method of Knippers and Sinsheimer (12). The pellet from 2 x 109 infected cells was suspended in 0.3 ml of a 20% sucrose solution prepared in 0.05 M Tris-hydrochloride (pH 8.0). To this solution 0.1 ml of lysozyme (2 mg/ml in 0.25 M Tris-hydrochloride, pH 8.0) and 0.1 ml of 0.01 M EDTA were added, and incubation was carried out for 30 min at 00. At the end of the incubation period, 0.15 ml of a 5% solution of Brij-58 was added. After the solution was held for 20 min at 00C, lysis was complete. Labeling host membranes. E. coli B was grown as described above, and when the cell density reached 2.0 x 108 cells/ml [2-3H]glycerol, a radioactive label specific for E. coli membranes (8), was added to a final concentration of 1.6 ,uCi/ml. Twenty minutes later the cells were infected with 32P-labeled T75. At 10 min after infection cell lysates were prepared. Labeling host and viral proteins. E. coli B was grown in Tris-glucose medium (28) containing 0.05% Casamino Acids. When the cell density reached 2.5 x 108 cells/ml, [35S]methionine was added to a final concentration of 4 ttCi/ml. After 20 min the cells were infected with 3H-labeled T75 phage, and at 10 min after infection cell lysates were prepared. Sedimentation analysis of intracellular parental T7 DNA. (i) Cesium chloride density gradients. Portions of the cell lysates were centrifuged in a stepwise cesium chloride density gradient in the Spinco SW50.1 rotor for 2 h at 40,000 rpm and 5°C. In our initial studies (data described in Fig. 1, 2, and 3) step gradients were prepared by layering 1.5 ml each of cesium chloride solutions having densities of 1.3, 1.46, and 1.6 g/ml. The cesium chloride solutions were prepared in 0.01 M Tris-hydrochloride (pH 8.0). In all other experiments lysates were centrifuged under identical conditions except that the density of the top layer was reduced to 1.2 g/ml. Under these conditions of centrifugation the T7 DNA-membrane complex sediments to a position in the gradient which allows its separation from free DNA and lowmolecular-weight material. After the first cesium chloride centrifugation, the membrane complex can be detected in the gradient tube as a white band when viewed against a black background. When the complex was to be isolated for subsequent use, the band was taken up in a plastic pipette and then dialyzed against 0.02 M Tris-hydrochloride (pH 7.6) for 5 h at 4°C. The dialyzed membrane complex was further purified by centrifugation in a second cesium chloride gradient under identical conditions described above. At the end of the centrifugation, four-drop fractions were collected from the bottom of the tube and a 0.02-ml portion was used for radioactivity determination. The DNA-membrane complex was isolated and then dialyzed as described above. The purified membrane complex was used for most of the experiments described below. Polyacrylamide gel electrophoresis. Proteins labeled with [35S]methionine were examined by electrophoresis on 10% polyacrylamide slab gels with a
400300
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FIG. 1. Sedimentation analysis of intracellular parental T7 DNA. E. coli B was infected with 32Jp labeled T75, and at 10 min after infection cell lysates were prepared. A portion of the lysate (0.1 ml) was mixd with 3H-labeled reference T7 DNA, and the solution was centrifuged in a cesium chloride step gradient as described in the text (A). In a separate but identical experiment lysates were centrifuged in a cesium chloride step gradient and the 32P-labeled T7 DNA present in the low-density portion of the gradient was isolated and dialyzed for 5 h against 0.02 M Tris-hydrochloride (pH 7.6). The dialyzed material was mixed with 3H-labeled reference T7 DNA and then centrifuged in a 5 to 20% sucrose gradient containing a 1.0-ml cesium chloride shelf (1.1 g of cesium chloride per ml of 20% sucrose). Sucrose solutions were prepared in 20 mM Tris-hydrochloride (pH 7.6)-0.2 mM EDTA. Centrifugation was for 30 min at 30,000 rpm and 5°C in the Spinco SW50.1 rotor (B). Three-drop fractions were collected directly into scintillation vials containing glass-fiber filter paper. After the filters were dried the radioactivity was determined. Symbols: x, 32P-labeled T7 DNA; *, 3H-labeled reference T7 DNA.
5% stacking gel as described by Laemmli (13). Proteins were denatured in sodium dodecyl sulfate (SDS) before electrophoresis as described by McMillen and Consigli (14). The fixed gels were dried under vacuum and autoradiograms were prepared using RP Royal "X-Omat" film. In some cases the gels were subjected to fluorography as described by Bonner and Laskey (3). Marker proteins labeled with 14C by reductive alkylation (14) were generously provided by R. A. Consigli. These proteins consisted of bovine serum albumin with a molecular weight of 68,000 (68K); ovalbumin, 43K; chymotrypsinogen, 25K; and cytochrome c, 12.4K. Tracings of the autoradiograms were made with a Joyce-Loebl microdensitometer.
RESULTS Evidence that intracellular parental T7 DNA is associated with host membranes. (i) Sedimentation of T7 DNA in cesium chloride density gradients. Previously we have pro-
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FIG. 2. Separation of host and intracellular T7 DNA in cesium chloride gradients. E. coli B was grown in M9 medium containing 0.2% Casamino Acids to a density of 2 x 108 cells/ml. [3H]thymidine was added to the culture (2 uCilml), and the cells were grown for an additional 60 min. The cells were centrifuged and suspended in fresh M9 medium at 250C and then infected with 32P-labeled T75 phage (multiplicity of infection = 5). At 15 min after infection, cell lysates were prepared and a portion of the lysate (0.1 ml) was centrifuged in a cesium chloride step gradient as described in the text. Acid-insoluble radioactivity was determined in three-drop fractions collected from the bottom of the tube. Symbols: x, 32P-labeled intracellular T7 DNA; *, 3H-labeled host DNA.
vided evidence that replicating bacteriophage T7 DNA is associated with host membranes (17). In the present experiments we provide similar findings for parental T7 DNA and show that this membrane complex is stable to cesium chloride gradient centrifugation. This latter finding has facilitated the purification of the DNA-membrane complex and its separation from extraneous proteins and nucleic acids. The sedimentation properties of intracellular parental T7 DNA in a cesium chloride gradient are shown in Fig. 1A. About 60% of the DNA sediments to a position in the gradient having a density of 1.3 g/ml. This DNA, which represents 60 to 70% of the recovered radioactivity in various experiments, is completely separated from the exogenous DNA marker (Fig. 1A). If the parental T7 DNA which exhibits a low density in cesium chloride is isolated, dialyzed, and recentrifuged in sucrose, most of this material has an extremely fast sedimentation rate (Fig. 1B).
Additional studies have been carried out to determine if intracellular parental T7 DNA is separated from host DNA after cesium chloride gradient centrifugation. This should be the case since previous studies have shown that after T7 infection the host DNA is released from its association with cell membranes (21, 25). E. coli B was labeled with [3H]thymidine and then infected with 32P-labeled T75 phage. Cell lysates were prepared and centrifuged in cesium chloride density gradients. In this experiment infected cells were collected before a time when
extensive degradation of host DNA had occurred. There is an essentially complete separation of labeled host DNA from the major portion of the intracellular parental T7 DNA (Fig. 2). We have also carried out studies to analyze the sedimentation pattern of intracellular parental T7 DNA and RNA pulse-labeled during T7 infection. E. coli B was infected with 32P_ labeled T75 phage, and at 15 min after infection (250C) the cells were pulse-labeled for 1 min with [3H]uridine. Preliminary experiments have demonstrated that T7 RNA synthesis is maximal at 15 to 17 min after infection. Cell lysates were prepared and then centrifuged in neutral sucrose containing a cesium chloride shelf. About 70% of the DNA but only 10% of the pulse-labeled RNA exhibits a fast sedimentation rate in the sucrose gradient (Fig. 3A). When the fast-sedimenting DNA and RNA are isolated and recentrifuged in a cesium chloride
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FIG. 3. Sedimentation analysis of intracellular T7 DNA and RNA. A 5-ml culture of E. coli B was grown in M9 medium supplemented with 0.2% Casamino Acids to a density of 3.5 x 108 cells/ml. Then the cells were infected at 25°C with 32P-labeled T75 phage. The infected cells were labeled with [3H]uridine (2 ,uCilml) for 1 min beginning at 15 min after infection. At the end of the labeling period, cell lysates were prepared and then mixed with 2 volumes of 0.05 M Tris-hydrochloride (pH 8.0). A portion of this solution was centrifuged in a 5 to 20% sucrose gradient containing a cesium chloride shelf. Centrifugation was carried out in the Spinco SW27 rotor for 30 min at 20,000 rpm and 5°C. Sucrose solutions were prepared in 0.01 M Tris-hydrochloride (pH 7.6)-0.001 M EDTA. Fractions (1 ml) were collected and the acid-insoluble radioactivity in a 0.1-ml sample was determined (A). Fractions 6 and 7 from the sucrose gradient were pooled and dialyzed. A portion of the dialyzed material (0.3 ml) was centrifuged for 17 h in a cesium chloride gradient as described in the text, and the radioactivity in the collected fractions was determined as described in the legend to Fig. lB. Symbols: x, 32P-labeled intracellular T7 DNA; 0, 3H-labeled RNA.
INTRACELLULAR STRUCTURE OF T7 DNA
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gradient, all of the DNA and 50% of the labeled RNA cosediment as low-density material (Fig. 3B). The nature of the RNA that cosediments with the DNA is unknown at the present time. (ii) Cosedimentation of host membrane and intracellular parental T7 DNA. The studies described above show that a major portion of the intracellular parental T7 DNA sediments in cesium chloride gradients as low-density material. Studies were therefore carried out to determine if the sedimentation rate of this DNA was due to its association with host membranes. Evidence that this is the case is provided by the finding that 3H-labeled host membranes and 32P-labeled parental T7 DNA cosediment in cesium chloride density gradients (Fig. 4). The association between T7 DNA and host membranes appears to be quite stable because, if the complex from the first gradient is isolated, dialyzed, and recentrifuged under identical conditions, DNA and membranes still exhibit the same sedimentation rate. The DNA associated with membrane material is susceptible to degradation by pancreatic DNase, and the complex is completely disrupted by treatment with 1% Sarkosyl for 30 min at 30°C. (iii) Protein composition of the T7 DNAmembrane complex. E. coli B was grown in TCG (Tris-Casamino Acids-glucose) medium, and host and viral proteins were labeled with P5S]methionine as described in Materials and Methods. At 10 min after infection, cell lysates were prepared and then centrifuged in a cesium chloride gradient. The DNA-membrane complex was isolated and dialyzed, and a portion of this material was centrifuged in a second
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cesium chloride gradient. The- second centrifugation results in the removal of about 70% of the labeled protein present in the original complex (Fig. 5). However, a significant fraction of the radioactively labeled protein still cosediments with the 3H-labeled T7 DNA. The DNA-membrane complex isolated from the first cesium chloride gradient and fractions 17 and 22-23 from the second gradient (Fig. 5) were analyzed for protein after polyacrylamide gel electrophoresis (Fig. 6). Proteins in the gel were identified by autoradiographic techniques. The membrane complex isolated from the first cesium chloride gradient consists of a complex mixture of some 25 polypeptides (Fig. 6D). Further purification of the complex results in the selective removal of several proteins (Fig. 6B). An examination of the protein pattern of the purified complex (Fig. 6C) indicates that it has become enriched for a 54K protein doublet and a major 32K protein. The 32K protein in particular appears to retain an essentially complete association with the complex during its purification. Several minor proteins are also present in the purified complex, and the most notable of these are two polypeptides with molecular weights of 29,000 and 24,000 (Fig. 6C). Additional studies have demonstrated that the DNA-membrane complex is stable to a third cesium chloride gradient centrifugation and that the protein composition of this material is identical to that of the complex shown in Fig. 6C. Essentially identical results as those described above have been obtained when [14C]leucine is used in place of P15S]methionine for labeling proteins. An exper100
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FIG. 4. Sedimentation analysis of host cell membranes and T7 DNA. E. coli B (10-ml culture) was labeled with [2-3H]glycerol and infected with 32p_ labeled T75 phage. Lysates were prepared and centrifuged in a cesium chloride step gradient as described in the text. Fractions from the gradient were collected into tubes and a 0.025-ml sample was used for radioactivity determination. Symbols: x, 32P-labeled intracellular T7 DNA; 0, 3H-labeled membranes. The arrow indicates the position to which marker T7 DNA sediments.
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FIG. 5. Sedimentation analysis of intracellular parental T7 DNA and labeled proteins. E. coli B (10ml culture) was grown in TCG medium, and host and viral proteins were labeled with [35S]methionine as described in the text. Cell lysates were prepared and then centrifuged in a cesium chloride step gradient (density, 1.2 to 1.6 g/ml). The DNA-membrane complex was isolated from the gradient and dialyzed for 5 h against 0.02 M Tris-hydrochloride (pH 7.6). The dialyzed material was centrifuged in a second cesium chloride step gradient, and fractions were collected into tubes. A 0.02-ml sample was used for radioactivity determination. Symbols: x, 3H-labeled intracellular T7 DNA; 0, 35S-labeled protein.
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The protein composition of this material was also found to be identical to that of DNA-membrane complexes isolated from T7-infected cells (Fig. 6C). Thus, this seems to indicate that all proteins present in the purified T7 DNA membrane complex are derived from the host. Alternatively, certain T7 and host proteins could have identical electrophoretic mobilities. (iv) Effect of detergents on DNA-membrane association. DNA-membrane complexes containing 32P-labeled T7 DNA and 3H-labeled membranes were incubated in the presence of 1% Brij-58 or Sarkosyl and then centrifuged in neutral sucrose gradients. As shown in Fig. 7, Brij and Sarkosyl have quite different effects on the sedimentation rate of the membraneassociated DNA. Sarkosyl causes the complete disruption of the complex, solubilizing the
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FIG. 6. Polyacrylamide gel electrophoresis of proteins associated with the T7 DNA-membrane complex. 35S-labeled proteins isolated from the cesium chloride density gradient described in the legend to Fig. 5 were subjected to SDS-polyacrylamide gel electrophoresis as described in the text. (A) Protein markers; (B) fractions 22-23 from the second cesium chloride gradient; (C) fraction 17 from the second cesium chloride gradient; (D) DNA-membrane complex isolated from the first cesium chloride gradient. iment has also been carried out in which cells were infected with 3H-labeled T75 phage, and at 2 min after infection proteins were labeled with [35Slmethionine for 10 min. Membrane com-
plexes purified through two cesium chloride gradients were analyzed after electrophoresis in 10% polyacrylamide gels. The total protein composition was essentially identical to that for isolated complexes labeled with [35S]methionine before and during T7 infection (Fig. 6C). In a parallel experiment, uninfected cells were labeled with [35S]methionine and host membranes in sheared lysates were purified through two cesium chloride gradients.
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FIG. 7. Effect of detergents on the sedimentation rate of membrane-associated T7 DNA. DNA-membrane complexes prepared as described in the legend to Fig. 4 were purified through two cesium chloride gradient centrifugations. Portions of the isolated complex (0.05 ml) were mixed with 0.1 ml of 0.01 M Tris-hydrochloride (pH 7.6) and either 0.05 ml of 5 % Brij-58 or 0.05 ml of 5% Sarkosyl. The solutions were incubated at 32°C for 30 min and then centrifuged in a 5 to 20% neutral sucrose gradient containing a 1.0-ml cesium chloride shelf. Centrifugation was carried out as described in the legend to Fig. 1. (A) Incubation in the absence of detergent; (B and C) incubation with Brij and Sarkosyl, respectively. Symbols: x, 32P-labeled intracellular T7 DNA; *, 3H-labeled host membranes.
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membrane, and releasing the DNA which now sediments near the top of the gradient (Fig. 7C). In contrast, after incubation with Brij-58, 95% of the 3H-labeled membrane is removed C) ?o from the complex, but the DNA still exhibits a fast sedimentation rate in the sucrose gradient 2 - 4 .45O a. 12(Fig. 7B). If the fast-sedimenting Brij-treated U,. 3 with Pronase material is isolated and incubated or RNase, the DNA still exhibits a fast sedimen-2 tation rate. The DNA is, however, converted to acid-soluble material after treatment with pan10 5 15 creatic DNase. After treatment of the complex FRACTION NUMBER with Brij, the resulting DNA structure has an S value of about 1,000 compared to internal T7 FIG. 8. Sedimentation of Brij treated DNA-membrane complex in sucrose gradients. Analysis ofDNA phage (453S) or T4 phage (1,OOOS) markers. (v) RNA composition of Brij-treated DNA- and protein. E. coli B (10-ml culture) was grown in membrane complex. Additional studies have TCG medium, and host and viral proteins were lawith [35S]methionine as described in the text. been carried out in which [3H]uridine was beled The DNA-membrane complex was purified through added to a cell culture 15 min before infection. two chloride gradient centrifugations. A porThe cells were infected with 32P-labeled T75 tion cesium of the isolated complex (0.2 ml) was treated with phage, and 10 min after infection lysates were Brij-58 as described in the legend to Fig. 8. The Brijprepared. The membrane complex purified treated material was centrifuged in a neutral sucrose through two cesium chloride gradients was gradient as described in the legend to Fig. 1. Symtreated with Brij and centrifuged in neutral bols: x, 3H-labeled T7 DNA; 0, 35S-labeled proteins. sucrose. It was found that only 3% of the labeled RNA associated with the membrane comDISCUSSION plex after the second cesium chloride gradient Our studies on the intracellular organization cosedimented with the DNA complex. (vi) Protein composition of the Brij-treated of bacteriophage T7 DNA continue to demonDNA-membrane complex. DNA-membrane strate an association of the chromosome with complexes containing 35S-labeled protein and host membranes. In the present work we have 3H-labeled T7 DNA were prepared as described shown that intracellular parental T7 DNA sediin Materials and Methods. The purified com- ments in cesium chloride gradients as low-denplex was treated with Brij and then centrifuged sity material coincident with host membranes. in a neutral sucrose gradient. After Brij treat- Of particular importance in these experiments ment about 55% of the labeled protein still asso- is the evidence suggesting that the intracelciates with the DNA as fast-sedimenting mate- lular DNA does not become trapped in memrial (Fig. 8). Additional studies have been car- brane vesicles during preparation of the cell ried out in which the membrane complex was lysate. Evidence against the idea of nonspecific treated with Brij and the solution was sepa- trapping is provided by the finding that the rated by centrifugation into a pellet (DNA-pro- membrane complex can be isolated under contein complex) and supernatant fractions. The ditions which allow it to be separated from proteins present in each fraction were analyzed endogenous host DNA and the bulk of RNA after electrophoresis in an SDS-polyacrylamide labeled during T7 infection. The parental T7 DNA-membrane complex is gel (Fig. 9). Before Brij treatment the membrane complex had an electrophoretic profile quite stable and can be purified by centrifugaessentially identical to that shown in Fig. 6C. tion in successive cesium chloride gradients. In However, as shown in Fig. 9B, Brij treatment of previous studies we have observed that centrifthe complex results in the selective removal of ugation of membrane complexes containing several protein components which are now newly synthesized T7 DNA in cesium chloride present in the supernatant fraction. The 34K for time periods similar to those described in protein present in the supernatant fraction is a the present work results in the dissociation of major component of the complex. We have the DNA from membranes (17). These results found, however, that complete resolution of the suggest that the nature of the linkage involved 34K and 32K proteins is only observed after in maintaining parental and newly synthesized electrophoresis of the supernatant and pellet DNA-membrane attachment may be quite diffractions obtained after Brij treatment of the ferent. The T7 DNA-membrane complex purified through two cesium chloride gradient cencomplex. b
x
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FIG. 9. SDS-gel electrophoresis of isolated DNAprotein complex. DNA-membrane complexes were isolated and treated with Brij-58 as described in the legend to Fig. 8. After Brij treatment the solution was centrifuged at 15,000 rpm for 30 min in the Sorval SE-12 rotor. The pellet was taken up in 0.05 ml of 10-3 phosphate buffer (pH 7.5). The proteins in the supernatant were precipitated with 5% trichloroacetic acid, and the resulting pellet was washed with cold acetone and then taken up in 0.05 ml of 10-3 M phosphate buffer (pH 7.5). The proteins in the pellet (A) and those from the supernatant (B) were electrophoresed in an SDS-polyacrylamide gel as described in the text. Autoradiogram was traced with a JoyceLoebl microdensitometer.
trifugations contains a reproducible set of proteins which are derived from host membranes. The protein pattern is still complex and several distinct polypeptides can be identified. Certain major proteins are present and these have molecular weights of 54K, 34K, and 32K. Recent studies indicate that two proteins having molecular weights of 80K and 56K may be present at the site of attachment of E. coli DNA to membranes (20). Possibly the proteins of similar molecular weights observed in the purified T7 membrane complex are identical to those described by Portalier and Worcel (20) and may be involved in both E. coli and T7 DNA membrane attachment.
Treatment ofthe purified membrane complex with Brij-58 releases 95% of the lipid-containing material and selectively removes several distinct protein components. The major protein present in the complex after Brij treatment has a molecular weight of 32K and may be identical to the 31K polypeptide which makes up close to 50% of the total outer membrane protein (20). This would be consistent with the finding that nonionic detergents solubilize the inner membrane of the cell envelope, in contrast to the outer membrane which is resistant to similar treatment (22). Thus, it seems that treatment of the membrane complex with Brij results in the formation of a DNA-protein complex in which most proteins are probably derived from the outer E. coli cell membrane. It has been found, however, that the sedimentation rate of the Brij-treated complex is resistant to treatment with Pronase, suggesting that factors in addition to protein contribute to the high S value of this material. The sedimentation rate of the complex may be explained if the DNA exists as a highly condensed compact structure whose conformation is maintained by protease-resistant substances. Recent studies have shown that in T7-infected cells replicating DNA may exist as a highly compact globular structure containing up to 30 phage equivalents of DNA (2, 23). Similar types of condensed structures have also been described for the E. coli chromosome (18, 19, 26, 29) and parental and newly synthesized T4 DNA (6, 9, 10). The condensed intracellular DNA in T4- (6, 9, 10) and T7-infected (23) cells has been isolated in a form which is essentially free of membrane material, and these structures are resistant to treatment with proteolytic enzymes. It seems indicated, however, that the absence of DNA-membrane association may be related to the use of strong ionic detergents in preparing lysates or to the prolonged incubation of detergent-treated lysates at room temperature (6, 9, 10, 23). In the latter case (23) this may be similar to the finding that the presence of the membrane-bound or membrane-free E. coli chromosome is dependent on the temperature at which lysates are prepared (19, 30). Lysis of cells at 250C results in the formation of the membrane-free chromosome. The evidence obtained thus far suggests that in T7-infected cells both the parental and newly synthesized DNA are membrane bound and that the DNA associated with membranes may have a compact structure. ACKNOWLEDGMENT
This investigation was supported by Public Health
VOL. 22, 1977
INTRACELLULAR STRUCTURE OF T7 DNA
Service research grant AI-10238 from the National Institute of Allergy and Infectious Diseases.
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