JOURNAL OF VIRLoGY, Aug. 1975, p. 315-321 Copyright 0 1975 American Society for Microbiology
Vol. 16, No. 2
Printed in U.S.A.
N-Glycosidase Activity in Extracts of Bacillus subtilis and Its Inhibition After Infection with Bacteriophage PBS2 KENNETH MINTON Laboratory of Experimental Oncology, Department of Pathology and Department of Biology,* Stanford ERROL C. FRIEDBERG, ANN K. GANESAN,*
AND
University, Stanford, California 94305 Received for publication 5 March 1975
We have detected in crude extracts of Bacillus subtilis an N-glycosidase activity which catalyzes the release of free uracil from DNA of the subtilis phage PBS2 labeled with [3IH ]uridine. This DNA contains deoxyuridine instead of thymidine. The enzyme is active in the presence of 1.0 mM EDTA and under these conditions Escherichia coli or T7 DNA labeled with [3H]thymidine is not degraded to labeled acid-soluble products. The activity resembles an N-glycosi-
dase from E. coli which releases free uracil from DNA containing deaminated cytosine residues. Both enzymes in crude extracts are active in the presence of EDTA, do not require dialyzable co-factors, and have the same pH optimum. They differ in that the enzyme from E. coli is more sensitive to heat, sulfhydryl reagents, and salt. The enzyme from B. subtilis is inactive on DNA containing 5-bromouracil or hydroxymethyluracil. Extracts of PBS2-infected B. subtilis lose the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity in extracts of uninfected cells in vitro. ura-1) was obtained from A. T. Ganesan, Department of Genetics, Stanford University. This strain, as well as strains E. coli K-12 (JG 139) and B-3, were maintained on agar plates containing brain heart infusion broth (Difco). Phage stocks. Phage PBS2 (a gift of Alan Price, Department of Biological Chemistry, University of Michigan) was prepared by infecting motile log-phase B. subtilis cells grown in Penassay broth (Difco antibiotic medium 3), at a multiplicity of 3. Motile cells were selected as described by Price and Cook (7). After infection the culture was shaken vigorously at 37 C for 3 h and then allowed to stand overnight at this temperature. The lysate was treated with chloroform and the phage was harvested by differential centrifugation. Phage SPOl (provided by Cliff 0. Yehle, Department of Genetics, Stanford University) was prepared in the same manner except that motile cells were not specifically selected and lysis was complete after shaking at 37 C for 3 h. Both PBS2 and SPOl phage were titered as described by Price and Cook (7). Isotopically labeled DNA. PBS2 DNA was labeled with [6-8H Juridine. Motile SB5 cells, selected as described above, were inoculated into 20.0 ml of Davis minimal broth (Difco), 21.2 g/liter, supplemented with 0.5% glucose, 0.1% Casamino Acids, 1.0 mM histidine, 0.5 mM tryptophan, and 20 usg of uracil. This culture was incubated overnight at 37 C and then added to 1 liter of the same medium. At a density of approximately 5 x 107/ml, cells were MATERIALS AND METHODS infected with phage PBS2 at a multiplicity of 3 and, Bacterial stocks. B. subtilis SB5 (try C2, his Al, after 5 min of incubation, 10 mCi of [6-3H juridine 315
An N-glycosidase has been partially purified from extracts of Escherichia coli (4). This enzyme releases uracil from DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers. The DNA of the bacteriophage PBS2 contains deoxyuridine instead of thymidine (8). Since Bacillus subtilis is the natural host for this phage we wished to determine whether extracts of B. subtilis, like those of E. coli, contain an activity against uracil-containing DNA. Additionally, we wished to determine whether the enzyme activity described in extracts of E. coli discriminates between DNA containing uracil paired with guanine (in cytosine deaminated DNA) and that containing uracil paired with adenine as exists in PBS2 DNA. In the experiments to be described, we provide evidence for an Nglycosidase activity and compare its properties to those of the E. coli enzyme. In addition, we show that after infection of B. subtilis with phage PBS2 an inhibitor is produced which can inactivate the N-glycosidase present in extracts of uninfected cells. Inhibition in vivo of the activity that attacks uracil-containing DNA requires protein synthesis after phage infection.
316
FRIEDBERG, GANESAN, AND MINTON
(18.5 Ci/mmol) was added. The culture was incubated for 2 to 3 h at 37 C with vigorous shaking and then kept at the same temperature overnight without shaking to allow complete lysis. An alternative labeling procedure was modified from that of Hunter et al. (3). Motile B. subtilis (strain SB5) were grown in 200 ml of nutrient broth (Difco) supplemented with 20 gg of uracil per ml. When the culture was in early log phase the cells were harvested by centrifugation and resuspended in fresh nutrient broth with 5 gg of uracil per ml. When the culture reached a density of 5 x 101 cells/ml PBS2 phage was added at a multiplicity of 3, followed after 5 min by 5.0 mCi of [6- 3H]uridine. Phage were harvested by differential centrifugation. The final phage pellet was resuspended in 5.0 ml of a solution of NaCl, 0.1 mM. The phage suspension was placed onto preformed gradients made by layering equal volumes of CsCl at densities of 17, 1.5, and 1.3 in centrifuge tubes (9). Sedimentation was at 37,000 rpm for 90 min in a Beckman SW50.1 rotor at 5 C. Phage sedimented as a band visible to the naked eye and were removed from the gradients and dialyzed against 0.01 M potassium phosphate buffer, pH 7.0. DNA was extracted with cold phosphate-buffered phenol and dialyzed against 0.01 M potassium phosphate buffer, pH 7.0. 3H-labeled SPOl phage were prepared by infecting strain SB5 with phage SPOl at a multiplicity of about 2 in Davis minimal broth supplemented as described above. Ten minutes after phage infection, [6- 3H ]uridine was added and the cultures were incubated until lysis was complete as judged by clarification of the culture. Phage were harvested by differential centrifugation and DNA was extracted as described above. The DNA was dialyzed against standard saline citrate buffer. The specific activity of the DNA preparations varied between 49,400 and 60,400 counts/min per Aig. [3H ]thymine- and [3H ]adeninelabeled phage T7 DNA and [3H ]thymine-labeled E. coli DNA were prepared as described previously (1). [3H ]adenine-labeled DNA was prepared with [83H ]adenosine. The specific activity of the [3H ]thymine and [3H ]adenine T7 DNA were 84,000 and 320,000 counts/min per ug, respectively. [3H ]bromouracil-containing DNA from WI-38 cells transformed by SV40 was a gift of C. A. Smith, Biology Department, Stanford University. Cell-free extracts. Extracts of uninfected cells were prepared from cells grown to exponential phase in Penassay broth, brain heart infusion broth, or yeast-tryptone extract. For extracts of phage-infected cells infection was carried out at a multiplicity of about 6. In the case of phage PBS2, motile cells were selected as described above. Chloramphenicol (150 ug/ml) was added to the cultures 10 min after infection with phage. Controls consisted of cells infected in the presence of chloramphenicol. Cells were harvested by centrifugation at 5,000 x g and washed in 10 mM potassium phosphate buffer, pH 7.0, or 10 mM Tris-hydrochloride buffer pH 8.0 at 4 C. Cells were resuspended in either of these buffers and sonically treated with a Bronwill Biosonik III sonifier. The sonic extract was centrifuged at 15,000 x g for 15 min at 4 C and the supernatant was stored as crude
J. VIROL. extract. When extracts were stored
thawing resulted in
a
frozen, repeated progressive loss of activity.
Extracts maintained at 4 C were stable for at least a month. Enzyme assays. Activity was assayed by two methods. (i) Acid-soluble labeled product. Incubation mixtures (0.1 to 0.3 ml) contained labeled PBS2 DNA (3 to 10 nmol as nucleotide); EDTA, 1 to 10 mM; potassium phosphate buffer, pH 7.0, 10 mM or Tris-hydrochloride buffer, pH 8.0, 10 mM; and varying amounts of crude extract. Reactions were terminated by the successive addition of carrier bovine serum albumin (50 gg) and cold trichloroacetic acid (final concentration, 5%). After standing for 15 min at 4 C, the reaction tubes were centrifuged at 5,000 x g for 5 min and the supernatant was saved. Radioactivity in aliquots (0.05 to 0.2 ml) of the acid-soluble fraction was determined by adding either 10 ml of a scintillation mixture consisting of two parts Omnifluor (New England Nuclear Corp.) in toluene base (5.04 mg/ml) plus one part of Triton X-100, or 5 ml of Aquasol (New England Nuclear Corp.), and counting samples in a Beckman LS-250 liquid scintillation spectrometer. (ii) This-layer chromatography. Reactions were terminated by placing samples onto ice and immediately spotting 0.005- to 0.010-ml aliquots of the incubation mixture on polyethyleneimine thin-layer plates. The plates were developed for a distance of 10.0 cm with water, using cold uracil as a marker. After drying the plates with a warm air blower, 0.5-cm fractions were processed for radioactivity determination using the Scotch tape overlay method previously described (2). Radioactivity was eluted by placing segments of the plates into scintillation vials containing 0.2 ml of 0.5 N NaCl. After 15 min 0.8 ml of water was added followed by 10.0 ml of scintillation mixture. In this chromatographic system nucleotides or polynucleotides are retained at the origin whereas nucleosides and bases migrate away. Activity was measured by determining the percentage of radioactivity at the R, of a uracil standard relative to the total radioactivity on the plate. All radioactivity was concentrated in two areas: at the origin or at the R, of uracil. No radioactivity was detected at the R, of deoxyuridine which was readily resolved from uracil. Other thin-layer chromatographic systems. Various bases, nucleosides, and nucleotides were separated by one-dimensional thin-layer chromatography on silica gel (Polygram Sil G/UV 254, Brinkmann Instruments, Inc.) or on cellulose (Polygram Cell 300/UV 254, Brinkmann Instruments, Inc.). The solvents used were the top phase of a mixture of ethylacetate:n-propanol:water (4:1:2), or isopropanol: concentrated hydrochloride:water (170:41:39). In all instances in which labeled material was chromatographed, radioactivity was eluted using the Scotch tape overlay method described above. Except with polyethyleneimine plates which required elution with salt, water (0.5 to 1.0 ml) was used to elute radioactivity from the thin-layer matrix. Materials. [6- 3H ]uridine, [5- 3H ]deoxyuridine, [methyl- 3H ]thymidine, and [5- 3H ]-5'-deoxyuridylate
N-GLYCOSIDASE IN B. SUBTILIS
VOL. 16, 1975
317
were purchased from Schwarz/Mann. Thin-layer plates on plastic backing (20 by 20 cm) were purchased from the Brinkmann Instrument Co. Unlabeled bases, nucleosides, and nucleotides were obtained from Calbiochem and Schwarz/Mann.
RESULTS Initial experiments demonstrated degradation of PBS2 DNA by measuring acid-soluble label released from DNA labeled with [3H ]uridine. Figure 1 shows the kinetics of this reaction with extracts of B. subtilis. In this experiment, after 20 min of incubation, 32.7% of the label in DNA was rendered acid soluble. With longer incubation times or the use of higher concentrations of extract, it was possible to release close to 100% of the radioactivity into the acid-soluble fraction. By contrast, using phage T7 DNA labeled with [3H ]thymidine,
Vol. 16, No. 2
Printed in U.S.A.
N-Glycosidase Activity in Extracts of Bacillus subtilis and Its Inhibition After Infection with Bacteriophage PBS2 KENNETH MINTON Laboratory of Experimental Oncology, Department of Pathology and Department of Biology,* Stanford ERROL C. FRIEDBERG, ANN K. GANESAN,*
AND
University, Stanford, California 94305 Received for publication 5 March 1975
We have detected in crude extracts of Bacillus subtilis an N-glycosidase activity which catalyzes the release of free uracil from DNA of the subtilis phage PBS2 labeled with [3IH ]uridine. This DNA contains deoxyuridine instead of thymidine. The enzyme is active in the presence of 1.0 mM EDTA and under these conditions Escherichia coli or T7 DNA labeled with [3H]thymidine is not degraded to labeled acid-soluble products. The activity resembles an N-glycosi-
dase from E. coli which releases free uracil from DNA containing deaminated cytosine residues. Both enzymes in crude extracts are active in the presence of EDTA, do not require dialyzable co-factors, and have the same pH optimum. They differ in that the enzyme from E. coli is more sensitive to heat, sulfhydryl reagents, and salt. The enzyme from B. subtilis is inactive on DNA containing 5-bromouracil or hydroxymethyluracil. Extracts of PBS2-infected B. subtilis lose the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity in extracts of uninfected cells in vitro. ura-1) was obtained from A. T. Ganesan, Department of Genetics, Stanford University. This strain, as well as strains E. coli K-12 (JG 139) and B-3, were maintained on agar plates containing brain heart infusion broth (Difco). Phage stocks. Phage PBS2 (a gift of Alan Price, Department of Biological Chemistry, University of Michigan) was prepared by infecting motile log-phase B. subtilis cells grown in Penassay broth (Difco antibiotic medium 3), at a multiplicity of 3. Motile cells were selected as described by Price and Cook (7). After infection the culture was shaken vigorously at 37 C for 3 h and then allowed to stand overnight at this temperature. The lysate was treated with chloroform and the phage was harvested by differential centrifugation. Phage SPOl (provided by Cliff 0. Yehle, Department of Genetics, Stanford University) was prepared in the same manner except that motile cells were not specifically selected and lysis was complete after shaking at 37 C for 3 h. Both PBS2 and SPOl phage were titered as described by Price and Cook (7). Isotopically labeled DNA. PBS2 DNA was labeled with [6-8H Juridine. Motile SB5 cells, selected as described above, were inoculated into 20.0 ml of Davis minimal broth (Difco), 21.2 g/liter, supplemented with 0.5% glucose, 0.1% Casamino Acids, 1.0 mM histidine, 0.5 mM tryptophan, and 20 usg of uracil. This culture was incubated overnight at 37 C and then added to 1 liter of the same medium. At a density of approximately 5 x 107/ml, cells were MATERIALS AND METHODS infected with phage PBS2 at a multiplicity of 3 and, Bacterial stocks. B. subtilis SB5 (try C2, his Al, after 5 min of incubation, 10 mCi of [6-3H juridine 315
An N-glycosidase has been partially purified from extracts of Escherichia coli (4). This enzyme releases uracil from DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers. The DNA of the bacteriophage PBS2 contains deoxyuridine instead of thymidine (8). Since Bacillus subtilis is the natural host for this phage we wished to determine whether extracts of B. subtilis, like those of E. coli, contain an activity against uracil-containing DNA. Additionally, we wished to determine whether the enzyme activity described in extracts of E. coli discriminates between DNA containing uracil paired with guanine (in cytosine deaminated DNA) and that containing uracil paired with adenine as exists in PBS2 DNA. In the experiments to be described, we provide evidence for an Nglycosidase activity and compare its properties to those of the E. coli enzyme. In addition, we show that after infection of B. subtilis with phage PBS2 an inhibitor is produced which can inactivate the N-glycosidase present in extracts of uninfected cells. Inhibition in vivo of the activity that attacks uracil-containing DNA requires protein synthesis after phage infection.
316
FRIEDBERG, GANESAN, AND MINTON
(18.5 Ci/mmol) was added. The culture was incubated for 2 to 3 h at 37 C with vigorous shaking and then kept at the same temperature overnight without shaking to allow complete lysis. An alternative labeling procedure was modified from that of Hunter et al. (3). Motile B. subtilis (strain SB5) were grown in 200 ml of nutrient broth (Difco) supplemented with 20 gg of uracil per ml. When the culture was in early log phase the cells were harvested by centrifugation and resuspended in fresh nutrient broth with 5 gg of uracil per ml. When the culture reached a density of 5 x 101 cells/ml PBS2 phage was added at a multiplicity of 3, followed after 5 min by 5.0 mCi of [6- 3H]uridine. Phage were harvested by differential centrifugation. The final phage pellet was resuspended in 5.0 ml of a solution of NaCl, 0.1 mM. The phage suspension was placed onto preformed gradients made by layering equal volumes of CsCl at densities of 17, 1.5, and 1.3 in centrifuge tubes (9). Sedimentation was at 37,000 rpm for 90 min in a Beckman SW50.1 rotor at 5 C. Phage sedimented as a band visible to the naked eye and were removed from the gradients and dialyzed against 0.01 M potassium phosphate buffer, pH 7.0. DNA was extracted with cold phosphate-buffered phenol and dialyzed against 0.01 M potassium phosphate buffer, pH 7.0. 3H-labeled SPOl phage were prepared by infecting strain SB5 with phage SPOl at a multiplicity of about 2 in Davis minimal broth supplemented as described above. Ten minutes after phage infection, [6- 3H ]uridine was added and the cultures were incubated until lysis was complete as judged by clarification of the culture. Phage were harvested by differential centrifugation and DNA was extracted as described above. The DNA was dialyzed against standard saline citrate buffer. The specific activity of the DNA preparations varied between 49,400 and 60,400 counts/min per Aig. [3H ]thymine- and [3H ]adeninelabeled phage T7 DNA and [3H ]thymine-labeled E. coli DNA were prepared as described previously (1). [3H ]adenine-labeled DNA was prepared with [83H ]adenosine. The specific activity of the [3H ]thymine and [3H ]adenine T7 DNA were 84,000 and 320,000 counts/min per ug, respectively. [3H ]bromouracil-containing DNA from WI-38 cells transformed by SV40 was a gift of C. A. Smith, Biology Department, Stanford University. Cell-free extracts. Extracts of uninfected cells were prepared from cells grown to exponential phase in Penassay broth, brain heart infusion broth, or yeast-tryptone extract. For extracts of phage-infected cells infection was carried out at a multiplicity of about 6. In the case of phage PBS2, motile cells were selected as described above. Chloramphenicol (150 ug/ml) was added to the cultures 10 min after infection with phage. Controls consisted of cells infected in the presence of chloramphenicol. Cells were harvested by centrifugation at 5,000 x g and washed in 10 mM potassium phosphate buffer, pH 7.0, or 10 mM Tris-hydrochloride buffer pH 8.0 at 4 C. Cells were resuspended in either of these buffers and sonically treated with a Bronwill Biosonik III sonifier. The sonic extract was centrifuged at 15,000 x g for 15 min at 4 C and the supernatant was stored as crude
J. VIROL. extract. When extracts were stored
thawing resulted in
a
frozen, repeated progressive loss of activity.
Extracts maintained at 4 C were stable for at least a month. Enzyme assays. Activity was assayed by two methods. (i) Acid-soluble labeled product. Incubation mixtures (0.1 to 0.3 ml) contained labeled PBS2 DNA (3 to 10 nmol as nucleotide); EDTA, 1 to 10 mM; potassium phosphate buffer, pH 7.0, 10 mM or Tris-hydrochloride buffer, pH 8.0, 10 mM; and varying amounts of crude extract. Reactions were terminated by the successive addition of carrier bovine serum albumin (50 gg) and cold trichloroacetic acid (final concentration, 5%). After standing for 15 min at 4 C, the reaction tubes were centrifuged at 5,000 x g for 5 min and the supernatant was saved. Radioactivity in aliquots (0.05 to 0.2 ml) of the acid-soluble fraction was determined by adding either 10 ml of a scintillation mixture consisting of two parts Omnifluor (New England Nuclear Corp.) in toluene base (5.04 mg/ml) plus one part of Triton X-100, or 5 ml of Aquasol (New England Nuclear Corp.), and counting samples in a Beckman LS-250 liquid scintillation spectrometer. (ii) This-layer chromatography. Reactions were terminated by placing samples onto ice and immediately spotting 0.005- to 0.010-ml aliquots of the incubation mixture on polyethyleneimine thin-layer plates. The plates were developed for a distance of 10.0 cm with water, using cold uracil as a marker. After drying the plates with a warm air blower, 0.5-cm fractions were processed for radioactivity determination using the Scotch tape overlay method previously described (2). Radioactivity was eluted by placing segments of the plates into scintillation vials containing 0.2 ml of 0.5 N NaCl. After 15 min 0.8 ml of water was added followed by 10.0 ml of scintillation mixture. In this chromatographic system nucleotides or polynucleotides are retained at the origin whereas nucleosides and bases migrate away. Activity was measured by determining the percentage of radioactivity at the R, of a uracil standard relative to the total radioactivity on the plate. All radioactivity was concentrated in two areas: at the origin or at the R, of uracil. No radioactivity was detected at the R, of deoxyuridine which was readily resolved from uracil. Other thin-layer chromatographic systems. Various bases, nucleosides, and nucleotides were separated by one-dimensional thin-layer chromatography on silica gel (Polygram Sil G/UV 254, Brinkmann Instruments, Inc.) or on cellulose (Polygram Cell 300/UV 254, Brinkmann Instruments, Inc.). The solvents used were the top phase of a mixture of ethylacetate:n-propanol:water (4:1:2), or isopropanol: concentrated hydrochloride:water (170:41:39). In all instances in which labeled material was chromatographed, radioactivity was eluted using the Scotch tape overlay method described above. Except with polyethyleneimine plates which required elution with salt, water (0.5 to 1.0 ml) was used to elute radioactivity from the thin-layer matrix. Materials. [6- 3H ]uridine, [5- 3H ]deoxyuridine, [methyl- 3H ]thymidine, and [5- 3H ]-5'-deoxyuridylate
N-GLYCOSIDASE IN B. SUBTILIS
VOL. 16, 1975
317
were purchased from Schwarz/Mann. Thin-layer plates on plastic backing (20 by 20 cm) were purchased from the Brinkmann Instrument Co. Unlabeled bases, nucleosides, and nucleotides were obtained from Calbiochem and Schwarz/Mann.
RESULTS Initial experiments demonstrated degradation of PBS2 DNA by measuring acid-soluble label released from DNA labeled with [3H ]uridine. Figure 1 shows the kinetics of this reaction with extracts of B. subtilis. In this experiment, after 20 min of incubation, 32.7% of the label in DNA was rendered acid soluble. With longer incubation times or the use of higher concentrations of extract, it was possible to release close to 100% of the radioactivity into the acid-soluble fraction. By contrast, using phage T7 DNA labeled with [3H ]thymidine,
