Cellular Site in Bacillus Subtilis of a Nuclease Which Preferentially Degrades Single-Stranded Nucleic Acids H

Cellular Site in Bacillus subtilis of a Nuclease Which Preferentially Degrades Single-Stranded Nucleic Acids H. C. BIRNBOIM Department ofBiochemistry, Albert Einstein College ofMedicine, New York, New York Received for publication 8 November 1965

ABSTRACT BIRNBOIM, H. C. (Albert Einstein College of Medicine, New York, N.Y.). Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids. J. Bacteriol. 91:1004-1011. 1966.-A nuclease, identified by a marked preference for single-stranded nucleic acids, has been demonstrated in extracts of Bacillus subtilis. The enzyme was associated with the cell wall-membrane fraction of mechanically disrupted cells and was released from cells which had been converted to protoplasts by lysozyme. The nuclease activity prepared by the latter procedure was found to be activated and solubilized by treatment with trypsin. The enzyme had about 2% activity on native deoxyribonucleic acid (DNA) as compared with denatured DNA. By use of CsCl analytical density gradient ultracentrifugation, this preparation was shown to degrade denatured DNA selectively in mixtures of native and denatured DNA.

The cellular location of certain enzymes in studied, and the partially purified enzyme was bacteria has been the subject of several recent characterized with respect to substrate and re- investigations. In Escherichia coli, several phos- requirements for optimal activity. phatases and nucleases (6, 10, 11, 12) have been shown to be released from bacterial cells when MATERIALS AND METHODS converted to spheroplasts. The localization of Bacterial strain. B. subtilis 168-2, a transformable these enzymes at or near the cell surface has been strain requiring tryptophan and leucine, was used for proposed. In Bacillus licheniformis (previously studies on enzyme distribution and purification. B. classified as B. subtilis), a penicillinase has been subtilis 1681>, which requires thymine and trypto- found attached to the protoplast membrane phan, was used for the preparation of radioactive (3, 4). In B. subtilis, Momose et al. (8) demon- DNA. strated a 5'-nucleotidase, the major portion of Media and growth conditions. V-Y broth [Difco which could be released from cells upon conver- Veal Infusion broth, 2.5% (w/v); Difco yeast extract, sion to protoplasts with lysozyme. In strains of 0.5% (w/v)] was the routine medium used for growing B. subtilis for enzyme isolation. Volumes of 1 Mycoplasma, several enzymes, including a ribo- liter were inoculated from a single colony and incu- nuclease and a deoxyribonuclease have been bated in 2-liter flasks at 37 C in a New Brunswick found associated with cell membranes (13). gyrotory floor shaker for 16 hr. Under these condi- In this paper, we present evidence for the exist- tions, the culture reached the early stationary phase ence in B. subtilis of a nuclease characterized by of growth, about 3 g (wet weight) of bacteria per liter. a marked preference for single-stranded deoxy- Reagents. Lysozyme (egg white), three-times ribonucleic acid (DNA), as compared with native, crystallized, B grade, was a product of Calbiochem. double-stranded DNA, as a substrate. In mechan- Tris(hydroxymethyl)aminomethane (Tris) was Trizma ically disrupted cells, about one-half of the of the Sigma Chemical Co., St. Louis, Mo. Analytical "cell reagent-grade sucrose was obtained from Mallinckrodt enzyme was found associated with the wall- Chemical Works, New York, N.Y. Trypsin (Worth- membrane" pellet. When cells were converted to ington Biochemical Corp., Freehold, N.J.) was used protoplasts by treatment with lysozyme, the en- without further purification. Diethylaminoethyl zyme was released and recovered in the super- (DEAE) cellulose (Carl Schleicher and Schuell Co., natant fraction. The association of the nuclease Keene, N.H.) was prepared for use by washing with 1 with the cell wall-membrane of B. subtilis was N NaOH, water, 1 N HCI, 1 N NaOH, and water, and 1004 VOL. 91, 1966 NUCLEASE OF BACILLUS SUBTILIS 1005 was finally suspended in 0.05 M Tris-HCl (pH 8). nucleic acid preparation of E. coli was used. It was Columns were packed under 10 psi of pressure and prepared by the method of Marmur (7) up to the washed with the starting buffer. Sephadex G-100 was first ethyl alcohol precipitation, after which the purchased from Pharmacia, Uppsala, Sweden. Carbo- spooled nucleic acid was dissolved in 0.1 X SSC, and wax 4000 (polyethylene glycol) was a product of the absorbance at 260 m, was adjusted to approxi- Union Carbide Chemical Co., New York, N.Y. All mately 100. The preparation was heated at 80 C for 10 other chemicals were reagent grade. SSC refers to a min to decrease any nuclease activity that might be solution of 0.15 M NaCl and 0.015 M sodium citrate associated with the carrier. at pH 7; 0.1 X SSC refers to a 1 in 10 dilution of it. Definition of unit. One enzyme unit was defined as Competent cells. B. subtilis 168-2 was made compe- the amount of enzyme which, under the conditions tent by a method similar to that of Anagnostopoulos described above, converted 1 m,umole of denatured and Spizizen (1). DNA to a form soluble in ethyl alcohol-HCl. Enzyme Preparation of radioactive DNA. B. subtilis 168T activity was proportional to enzyme concentration in was grown in 400 ml of the minimal salt medium of the range of 0.2 to 2 units. For studies on the distri- Spizizen (15) containing the following: DL-tryptophan, bution of enzyme in fractionated bacterial cells, assays 100 jug/ml; casein hydrolysate (Difco), 0.05% (w/v); were carried out at pH 8.4. For studies on the purifi- yeast extract (Difco), 0.1% (w/v); glucose, 1.25% cation and properties of the enzyme, the assay was (w/v); nonradioactive thymine, 0.5 jg/ml; thymine-2- performed at pH 9.2. Specific activity was defined as C14, 35 Aug, specific activity 14.8 mc/mmole (Calbio- enzyme units per milligram of protein. chem). After 15 hr of growth at 37 C, the cells were Protein was estimated by the method of Lowry et harvested by centrifugation, and DNA was prepared al. (5) with bovine serum albumin as standard. by the method of Marmur (7), except that the final isopropanol precipitation step was omitted. The yield RESULTS of DNA was approximately 3 mg. It had a specific radioactivity of 2,500 counts per min per Ag, counted Distribution ofnuclease activity in bacterial cells on a gas-flow counter. None of the radioactivity was (with denatured DNA as substrate). Bacterial cul- in ribonucleic acid (RNA), as judged by the absence tures, harvested at the beginning of the stationary of acid-soluble counts after treatment with pancreatic phase of growth, were used for studies on the ribonuclease or with 0.3 N KOH at 37 C for 15 hr. The DNA, dissolved in 0.1 X SSC, was stored at 4 C cellular distribution of nuclease activity. In the over chloroform. three separate experiments reported in Table 1, Assay for nuclease activity with denatured DNA as the nuclease activity of the culture supematant substrate. B. subtilis DNA-thymine-2-Cl" (114,g/ml in fluid and the bacterial cells was determined. The 0.1 X SSC) was denatured by heating at 100 C for 10 bacterial cells were fractionated either by con- min, then cooled quickly in an ice-water bath. An version to protoplasts with lysozyme or by dis- assay mixture was prepared by combining 0.1 ml ofde- ruption in a French pressure cell. natured DNA, 0.1 ml of CaCi2 (0.1 M), and 0.8 ml of Protoplasts. One-half of a washed bacterial buffer [either 0.1 M sodium glycylglycinate (pH 8.4) pellet was suspended at a concentration of 2 g or 0.1 M sodium glycinate (pH 9.2)]. A 0.1-ml amount M of this was combined with 0.01 ml of enzyme (0.2 to (wet weight) per 10 ml of 0.5 sucrose contain- 2 units) and incubated at 37 C for 30 min. The re- ing 0.05 M Tris-HCl (pH 8.2), 0.01 M divalent action was stopped by chilling on ice, and 0.1 ml of cation (shown in Table 1), and 0.5 mg/ml of cold carrier DNA (described below) was added lysozyme. After 30 to 45 min of incubation at followed by 0.5 ml of cold ethyl alcohol-HCl (1 vol- 37 C (at which time microscopic examination ume of 95% ethyl alcohol-i volume of 2 N HCl). A showed all bacteria to be spherical), the suspen- flocculent precipitate formed which was removed by sion was centrifuged at 39,000 X g for 30 min. centrifugation at 10,000 X g for 5 min at 2 C. Sam- The supernatant fluid was designated "protoplast ples of 0.5 ml of the clear supernatant fluid were supernatant fraction." The pellet was suspended removed and dried in stainless-steel planchets under a 0.05 M Tris-HCl heat lamp. The dried samples were counted in a in an equal volume of (pH 8.2) Nuclear Chicago gas-flow low-background counter. containing 0.01 M divalent cation. This fraction Assay for nuclease activity with native DNA as was designated "protoplast lysate." substrate. The method used was similar to the above The distribution of nuclease activity in these except that unheated (native) DNA was added in- fractions is shown in Table 1. In experiments 1 stead of denatured DNA. and 2 of Table 1, the majority of the nuclease Blanks incubated as above but containing no en- was released into the 39,000 X g supematant zyme and either denatured or native DNA contained fluid during the preparation of protoplasts. The about 2 and 0.75%, respectively, of the total counts attendant 10-fold increase in specific activity added. release of the was The use of ethyl alcohol-hydrochloric acid as pre- suggests that the enzyme cipitant permitted the counting of acid-soluble C1"- selective and not simply the result of lysis of a nucleotides with minimal self-absorption losses on a part of the cells. gas-flow counter. However, it required a larger amount Pressure cell disruption. The second half of a of carrier than usual and, for the purpose, a crude bacterial pellet was suspended at a concentration 1006 BIRNBOIM J. BACTERIOL. of 2 g (wet weight) per 10 ml of cold 0.05 M marked increase in nuclease activity (CW-M, as Tris-HCI (pH 8.2), 0.01 M divalent cation (shown compared with lysed CW-M, Table 1) as well as in Table 1). The suspension was chilled on ice, a release of about one-half of the enzyme into a and then was passed through a cold French pres- 100,000 X g, 30 min, supernatant fraction (data sure cell (American Instrument Co., Inc., Silver not shown). Spring, Md.) at 20,000 psi. The crushed cells The CW-M and lysed CW-M fractions of ex- were centrifuged at low speed (8,000 x g, 5 min) periment 3, Table 1, were also tested for nuclease to remove unbroken bacteria, and the turbid activity with native DNA as substrate at the same supernatant fluid was removed and centrifuged at pH. The rate of activity was about 10% that seen 39,000 x g for 30 min. The clear supernatant when denatured DNA was used as substrate, and fluid was designated "soluble extract." The pellet it also increased after lysozyme treatment. appeared to contain three layers analogous to Nuclease activity of intact bacterial cells. The those described by Salton (14). The top, brown experiment reported in Table 2 was designed to layer was designated "membrane fragments." show that part of the nuclease associated with The intermediate, whitish layer (cell wall-mem- washed cells was accessible to denatured DNA, brane fraction) was washed once by suspension even though the cells remained "intact." At the in 0.05 M Tris-HCl (pH 8.2), and centrifugation times indicated, a 10-ml sample was removed at 23,000 x g for 15 min (CW-M wash). The from a culture growing in V-Y broth, and the washed pellet (CW-M) was treated with lysozyme cells were washed once by centrifugation and (0.5 mg/ml) for 30 min at 37 C (lysed CW-M of suspended in 2.0 ml of 0.05 M imidazole buffer Table 1). The bottom layer of the 39,000 X g (pH 7.0) containing 0.001 M MnCl2. A 0.01-ml pellet (intact bacteria) was combined with the amount of the suspension was added directly to 8,000 X g pellet and suspended in the same Tris an assay mixture (pH 8.4; see Materials and buffer. This suspension was treated with lysozyme Methods) containing denatured C`4-DNA ("In- (0.5 mg/ml) for 30 min at 37 C, and the nuclease tact cell suspension," Table 2). As an estimate of activity of the lysate was determined (Bacterial the maximal amount of lysis of the cells that pellet of Table 1). Intact bacteria and the cell might have occurred during incubation with the wall-membrane fraction were identified on the radioactive DNA, another sample of cells was basis of phase-contrast microscopic examination. concomitantly incubated under similar conditions The distribution of nuclease activity (assayed (no DNA) for an equal length of time. These at pH 8.4) in these fractions is shown in Table 1. cells were then chilled and centrifuged at 10,000 x In these experiments, the cells were harvested at g for 5 min, and the nuclease activity of the the early stationary phase of growth (16 hr). As supernatant fluid was assayed ("Incubated cell much enzyme was found to be associated with supernatant fraction," Table 2). To determine the cells as was found in the broth supernatant the total cellular nuclease activity, a third sample fluid. It may be seen that roughly one-half of the of cells, similarly washed, was treated with lyso- total enzyme of mechanically disrupted cells was zyme (0.5 mg/ml) for 30 min at 37 C before assay recovered in the 39,000 x g pellet. Treatment of for nuclease activity ("Lysed cells"). The data of the CW-M fraction with lysozyme led to a Table 2 show that the "Intact cells" manifested

TABLE 1. Distribution of nuclease activity in Bacillus subtilis* Protoplast Cells disrupted by French press Broth Cell -Mmrn Expt no.t supernatant wash Protoplast Membrane fluid supernatant Protoplast Soluble fragments CW M Lysed Bacterial ffractionfractionlysate extract and~~~~~washCW-M CW-M pellet

1 40,200 676 52,400 24,700 22,200 10,800 18,200 28,500 16,900 (887) (74) (79) (397) (597) 2 31,700 210 80,300 9,650 19,750 4,600 25,500 16,700 3 28,000 495 - 13,230 5,400 5,830 11,800 6,390 (52.4) (276) (561) * Total units of nuclease activity (assayed at pH 8.4 with denatured DNA as substrate, as described in the text) recovered from 1 liter of broth supernatant fluid or cells, fractionated either as protoplasts or disrupted in the French press. Numbers in parentheses refer to the corresponding specific activity. t The media for fractionating cells contained, in addition to the buffer described in the text, the following divalent cations at a concentration of 0.01 M: experiments 1 and 2, Mn++; experiment 3, Mg++ (in each case as the chloride salt). VOL. 912 1966 NUCLEASE OF BACILLUS SUBTILIS 1007

TABLE 2. Nuclease activity of intact and lysed cells during growth of culture*

Nuclease activity Absorbance Time of growthmgbat 600 a Broth supernatant Wash Intact cell Incubated Wah cells cell fluid suspension Lysed fsupernatantraction hr 2.4 0.924 3.4 1 5.4 28.5 2.3 (33.3) (177) 4 1.764 11.2 1 12.0 64.2 0.6 (41.4) (221) 6 2.184 36.2 3.6 35.3 85.7 1.7 (98) (238) 12 3.549 40.4 6 48.6 130 1.5 (91.7) (245) 50 3.339 925 27.6 67.0 158 9.7 (93) (209) * Expressed as total units of nuclease activity (pH 8.4, denatured DNA as substrate) per milliliter of culture. The numbers in parentheses refer to specific activity (units per milligram of protein). t Absorbance at 600 miA determined on dilutions of the culture into the original medium to read in the the range 0.050 to 0.200. 10 to 40% of the nuclease activity of correspond- CaCl2, 0.001 M MnCl2], and less than 40% of the ing cells which had first been lysed with lysozyme. nuclease activity was recovered (Table 3 and 4). The results suggest that part of the cell-bound When this suspension was centrifuged at 100,000 nuclease was located at or near the cell surface. X g for 30 min, about one-half of the nuclease Nuclease activity in the "incubated cell super- activity remained in the supematant fluid. How- natant fraction" was presumably a reflection of ever, as is shown in Table 3 and 4, treatment of lysis of some of the cells during the incubation, the ammonium sulfate precipitate with 1 mg/ml although possibly it could have represented con- of trypsin for 50 min at 37 C resulted in a three- tamination by enzyme from the broth supernatant fold increase in nuclease activity, the majority of fluid or excretion of enzyme by the cell, particu- which was soluble after centrifugation at 100,000 larly in the case of the 50-hr sample. X g for 30 min. Trypsin alone had almost no Table 2 also shows that up to 12 hr of growth deoxyribonuclease activity, as measured under the enzyme associated with the cells was greater the same conditions of salt and pH as the B. than that found in the broth supematant fluid; subtilis nuclease. Incubation of the ammonium between 12 and 50 hr, the broth supernatant en- sulfate precipitate without trypsin for an equal zyme increased considerably, and the cell-bound length of time resulted in a definite increase in enzyme changed very little. activity but less than that seen with trypsin Further purification ofthe protoplast supernatant treatment. nuclease. The nuclease recovered in the 39,000 x Preincubation of the protoplast supernatant g supernatant fraction after conversion of early fluid for 30 min at 37 C (without first precipitat- stationary phase cells to protoplasts was ex- ing with ammonium sulfate) resulted in a vari- amined further. Additional centrifugation at able, slight increase in activity. As is shown in 100,000 x g for 30 min did not sediment the Table 3, preincubation of protoplast supernatant enzyme, but attempts at purification by am- fluid with trypsin (1 mg/ml) for 50 min at 37 C monium sulfate fractionation showed that, after resulted in a threefold increase in activity, as did its precipitation, the enzyme was associated with trypsin treatment of the ammonium sulfate pre- a turbid suspension and could not be brought into cipitate. solution. Because trypsin was reported to have A purification procedure for nuclease from solubilized penicillinase from B. licheniformis protoplast supernatant fluid is shown in Table 4. membranes (3, 4), its action on the insoluble B. Fractions were assayed for nuclease activity with subtilis nuclease preparation was studied (Table denatured DNA as substrate at pH 9.2, as de- 3). scribed in Materials and Methods. Protoplast supernatant fluid was brought to An amount of 10 g (wet weight) of cells was 65% saturation with solid ammonium sulfate at suspended in 50 ml of a medium containing 0.5 M 0 C. The precipitate which formed did not redis- sucrose, 0.05 M Tris-HCl (pH 7.4), 0.05 M NaCl, solve in buffer [0.05 M Tris-HCl (pH 7.5), 0.01 M 0.01 M MgC92, and 0.5 mg/ml of lysozyme, and 1008 BIRNBOIM J. BACTRIOL.

TABLE 3. Effect of trypsin on protoplast for 30 min. The supematant fluid was brought to supernatant fluid nuclease 65% saturation with solid ammonium sulfate, and after 15 min at 0 C the precipitate Relative activity* which Treatment formed was collected by centrifugation. The precipitate was suspended in 3 ml of 0.05 M Expt 1 Expt 2 Tris-HCl, (pH 7.4), 0.01 M CaC12, and 0.001 M (a) Protoplast supernatant MnCl2, and was incubated with trypsin (1 mg/ml) fluid nuclease...... 100 100 at 37 C for 50 min; the mixture was then cen- (b) (a) 50 min, 37 C, tryp- trifuged at 100,000 X g for 20 min at 2 C. sin ..315 This supematant fluid was diluted to 20 ml with (c) Ammonium sulfate pre- the Tris buffer and run onto a DEAE cellulose cipitate of (a) ...... 12 40 column (1.2 by 13 cm) equilibrated with the same (d) (c) 50 min, 37 C, no buffer. The column was washed with 80 ml of trypsin ...... 21 the buffer followed by an equal volume of 0.05 M (e) (c) 50 min, 37 C, trypsin ...... 42 147 sodium succinate (pH 6.0), 0.005 M CaC12, 0.005 (f) 100,000 X g supernatant M MnCl2, and 0.02M NaCl. No enzyme was fraction of (e) ...... 31 148 eluted from the column by either washing, but (g) Trypsin t . .<0.01 a large amount of material absorbing at 280 m,u was removed. This was followed by a 400-ml * Assays were carried out at pH 9.2, with de- linear NaCl gradient, running from 0.02 to 0.1 M, natured DNA as substrate, as described in Ma- in 0.05 M sodium succinate (pH 6.0), 0.005 M terials and Methods. CaCl2, and 0.005 M MnCl2. The flow rate was 40 t The nuclease activity of trypsin was assayed ml/hr at 5 C. Forty 10-ml fractions were col- under the same conditions as above; the final con- The centration in the assay was 100 times greater than lected. enzyme was eluted in a fairly sharp in (b), (e), or (f). peak at the middle of the gradient, with very little material absorbing at 280 m,u. The peak tubes was TABLE 4. Purification of Bacillus were pooled, and the solution transferred to subtilis nuclease a dialysis bag and concentrated by contact with dry Carbowax at 4 C for about 8 hr. The con- Units of centrated nuclease degraded native DNA at 2% Fraction prt~Total nuclease aivtSpecific Franprotein (X 10-3) activity of the rate at which denatured DNA was degraded, as measured under the same conditions mg units/mg ofpH and salt. In other experiments, the nuclease Protoplast super- of the protoplast supernatant fluid degraded natant fraction.... 120 56 467 native DNA at 10 to 15% of the rate observed Protoplast lysate.... 535 40 75 with denatured DNA; after purification as de- Ammonium sulfate scribed above, the corresponding rate was re- (A.S.) precipi- duced to 2 to 5 %. The enzyme preparation having tate...... 23.2 17 732 Trypsin-treated 2% activity on native DNA was used for studies A.S. precipitate... 29.2t 66 2,260 of its properties. 100,000 X g...... 25.2 53 2,100 Omission of the trypsin treatment of the am- Concentrated monium sulfate precipitate affected markedly the DEAE cellulose chromatographic behavior of the enzyme. It was fractions ...... 0.5t 23 46,000 eluted from DEAE-cellulose at pH 8.0, at about 0.2M NaCl, as compared with the elution of * Assayed at pH 9.2, with denatured DNA as trypsin-prepared enzyme at pH 6.0, with 0.05 M substrate, as described in Materials and Methods. NaCl. t Includes the added trypsin. Divalent cation requirement of the partially t Concentrated DEAE cellulose fractions were contaminated with traces of Carbowax, used in purified enzyme. The effect of varying concentra- the concentration step. Carbowax interfered with tions of CaCl2 on enzyme rate is shown in Fig. 1. the assay of protein by the method of Lowry et al. When assayed at pH 9.2, maximal stimulation (5), so the protein shown was that assayed before was observed with a Ca+ concentration above the concentration step. 0.001 M. MgCl2 and CuSO4 were tried at concen- trations from 0.002 to 0.01 M, and were found to was incubated at 37 C with gentle shaking. When support activity at less than 10% the rate of Ca+. protoplast formation was nearly complete (45 Optimal pH of the partially purified enzyme. min, as judged microscopically), the suspension In 0.05 M glycylglycinate-0.05M glycinate buff- was chilled on ice and centrifuged at 29,000 x g ers adjusted to the appropriate pH with 1 N VOL. 91, 1966 NUCLEASE OF BACILLUS SUBTILIS 1009

800 700 600p 600

500 a.,:400 a.cJ 400 C) 300 200 200 100 7.0 8.0 9.0 10.0 pH 0 2 3 4 5 6 Co" Concentration (mM) FIG. 2. Curve for pH activity. Partially purified nuclease assayed with denatured DNA as substrate, as FIG. 1. Ca++ requirement for Bacillus subtilis nu- described in Materials and Methods. Buffer used was clease. Partially purified nuclease was assayed as de- 0.05 M glycylglycinate-0.05 m glycine-NaOH over the scribed in Materials and Methods at different con- entire pH range studied. centrations ofCaCl2 ; the pH was 9.2 and the substrate used was denatured DNA. NaOH, maximal activity was observed between pH 9.0 and 9.6 (Fig. 2). Evidence for exonucleolytic mode of attack by the partially purified enzyme. The B. subtilis nuclease was characterized as an exonuclease by E 0 means of gel filtration (Birnboim, in press). (0 When denatured DNA was partially digested by a known exonuclease and then chromatographed w C-) on Sephadex G-100, the undigested fragments of z DNA were eluted from the column as a peak after the void volume, and the mononucleotides cnm formed by the digestion were eluted as a second c')0 distinct peak. When denatured DNA was partially £0 digested by a known endonuclease, the poly- disperse products were eluted from a Sephadex G-100 column as a single, broad peak. When the action of the B. subtilis nuclease was examined VOLUME (ML) (Fig. 3), the presence of a DNA peak and mono- FIG. 3. Chromatography of denatured DNA on nucleotide peak strongly suggested that the en- Sephadex G-100 at stages in its digestion by Bacillus zyme was an exonuclease. subtilis nuclease. The left peak corresponds to the DNA Effect of the partially purified enzyme on trans- and the right peak to the mononucleotides formed by the forming DNA. Figure 4 shows the effect of the exonucleolytic digestion. Curves a, b, and c represent partially purified nuclease (2% residual activity three successive stages in the digestion. on radioactively labeled native DNA as measured by acid solubility) on native B. subtilis trans- tured DNA may be readily resolved by analytical forming DNA. Inactivation of transforming ultracentrifugation in CsCl, the effect of the nu- DNA is a sensitive test for endonuclease activity. clease on a mixture of native and denatured B. For comparison, the conversion of radioactive, subtilis DNA was studied. Figure 5 shows micro- denatured DNA to an acid-soluble form was densitometer tracings of ultraviolet-absorption measured under the same conditions. It may be photographs taken in the Spinco model E analyt- seen in Fig. 4 that at 10 min 70% of the denatured ical ultracentrifuge. The upper tracing shows DNA was converted to an acid-soluble form, at native B. subtilis DNA (left peak), denatured a time that the transforming activity of native B. subtilis DNA (center peak), and reference DNA was decreased about 30%. density standard, phage 2C (p = 1.742 g/cm3, Specificity of B. subtilis nuclease with density based on E. coli DNA, taken as 1.710 g/cm3). gradient centrifugation. Since native and dena- The lower tracing is the same mnixture of native 1010 BIRNBOIM J. BACTERIOL.

100 )

~~0 60-~~

~~Z O 40- _ / v- TRANSFORMING DNA 20~~

~~0 1.5 5 10 20 30 LLI TIME ('MIN) z~~

FIG. 4. Effect of Bacillus subtilis nuclease on B. m subtilis transforming DNA. Native transforming DNA m 0 was incubated with partially purified B. subtilis nu- CJ) clease. At the time intervals shown, samples were re- m moved, and the residual transforming activity with respect to a leucine marker was determined. The conI- version of denatured C'4-DNA to acid-soluble nucleotides was measured under identical conditions for comparison. and denatured DNA which was incubated with B. subtilis nuclease. The center (denatured) peak has largely disappeared as a consequence of the DENSITYT- nuclease digestion, but the native DNA peak is FIG. 5. Specificity ofBacillus subtilis ntuclease shown unchanged. by banding ofnative and denatured DNA in CsCl. Equal amounts of native and denatured B. subtilis DNA were DIscussioN combined. One sample of the mixture was incubated at Although B. subtilis has been widely used for 37 C with B. subtilis nuclease and a second sample was incubated without nuclease. The reaction was stopped other studies, its cellular nucleases have not as by dilution into SSC, and solid CsCI was added. The yet characterized. The present report de- been reference density marker was virulentt B. subtilis phage a in B. subtilis which scribes nuclease degrades 2C DNA (p = 1.742 g/cm3). Photographs were taken denatured DNA at a much faster rate than native after 18 hr ofcentrifugation at 44,770 rev/min at 25 C, DNA, and suggests that the nuclease may be lo- and microdensitometer tracings were made of the ultra- cated at or near the cell surface. This suggestion violet-absorption photographs. In the upper tracing, is based upon three observations: (i) the associa- the left peak is native B. subtilis DNA, the center peak tion of the nuclease with a cell wall-membrane is denatured DNA, and the right peak is the standard. fraction when cells were mechanically disrupted; The lower tracing is ofthe same mixture after treatment (ii) the release of the nuclease from cells which with B. subtilis nuclease. Note that the middle (denatured) peak has largely disappeared as a consequence had been converted to protoplasts by treatment of digestion by the nuclease. with lysozyme; (iii) the demonstration that 10 to 40 % of the total nuclease activity (with denatured DNA as substrate) could be detected in washed present at high levels during the competent state. cells that remained intact during the assay. In the This has not as yet been examined. experiment, the only criterion of "intactness" of The cellular location of penicillinase in a related the cell was that, after the period of incubation, microorganism, B. licheniformis, was studied by the enzyme was still sedimentable at low speeds. Kushner and Pollack (3) and by Lampen (4). This could of course be compatible with marked Like the B. subtilis nuclease, a large part of the changes in the cell wall. penicillinase was sedimentable after mechanical The location of the enzyme near the cell surface disruption of the cells. The B. licheniformis peni- may be related to its excretion, since an extra- cillinase was released from membrane fragments cellular nuclease has been described (2, 9) which by the action of trypsin; the B. subtilis nuclease resembles in several respects the enzyme de- was released from what may be a membrane scribed in this report. Since B. subtilis 168-2 is a structure by the same treatment. After exposure to transformable strain, the location of the nuclease trypsin, the penicillinase was shown to migrate near the cell surface might be expected to inhibit differently during gel electrophoresis (4), whereas the uptake of denatured DNA if the enzyme is the nuclease was found to exhibit a different VOL. 91, 1966 NUCLEASE OF BACILLUS SUBTILIS 101 1 chromatographic behavior on DEAE-cellulose. location of cell-bound penicillinase in Bacillus The mechanism of the activation of the B. subtilis subtilis. J. Gen. Microbiol. 26:255-265. nuclease by trypsin is unknown, but the phe- 4. LAMPEN, J. 0. 1965. Secretion of enzymes by nomenon was observed consistently (see Tables micro-organisms, p. 115-133. In M. R. Pollack and M. H. Richmond [ed.], Function and 3 and 4). structure in microorganisms. Cambridge Univ. Some properties of the B. subtilis nuclease Press, London. have been studied, and it would appear that it 5. LowRy, 0. H., N. J. ROSEBROUGH, A. L. FARR, will be a useful tool, as has been discussed by AND R. J. RANDALL. 1951. Protein measurement Kerr et al. (2). It has been shown that denatured with the Folin phenol reagent. J. Biol. Chem. DNA can be selectively degraded in mixtures of 193:265-275. native and denatured DNA, but the degradation 6. MALAMY, M., AND B. L. HORECKER. 1961. The of, for example, incompletely reannealed DNA localization of alkaline phosphatase in E. coli has not yet been Some K12. Biochem. Biophys. Res. Commun. 5:104- investigated. preliminary 108. evidence from this laboratory suggests that the 7. MARMUR, J. 1961. A procedure for the isolation residual transforming activity of heat-denatured of deoxyribonucleic acid from microorganisms. DNA was resistant to inactivation by the enzyme, J. Mol. Biol. 3:208-218. and therefore possibly due to native molecules of 8. MOMOSE, H., H. NISHIKAWA, AND N. KATSUYA. DNA. RNA was also degraded by the enzyme, 1964. Genetic and biochemical studies on 5'- but this was not studied extensively. nucleotide fermentation. J. Gen. Appl. Micro- biol. (Tokyo) 10:343-358. ACKNOWLEDGMENTS 9. NAKAI, M., Z. MINAMI, T. YAMAZAKI, AND A. TSUGITA. 1965. Studies on the nucleases of a This investigation was supported by Public Health strain of Bacillus subtilis. J. Biochem. 57:96-99. Service grant GM1 1946 from the Division of General 10. NEU, H. C., AND L. A. HEPPEL. 1964. The release Medical Science, by grant GB1869 from the National of ribonuclease into the medium when E. coli Science Foundation, and by grant AT 30-1 3311 from cells are converted to spheroplasts. Biochem. the Atomic Energy Commission to J. Marmur. Biophys. Res. Commun. 14:109-112. I thank Dr. Marmur for providing laboratory space 11. NEU, H. C., AND L. A. HEPPEL. 1964. The release and guidance, and F. Kahan and J. 0. Lampen for of ribonuclease into the medium when Escheri- assistance in preparing the manuscript. A fellowship chia coli cells are converted to spheroplasts. from the Medical Research Council of Canada is J. Biol. Chem. 239:3893-3900. gratefully acknowledged. 12. NEU, H. C., AND L. A. HEPPEL. 1964. On the sur- LITERATURE CITED face localization of enzymes in E. coli.. Biochem. Biophys. Res. Commun. 17:215-219. 1. ANAGNOSTOPOULOS, C., AND J. SPIZIZEN. 1961. 13. POLLACK, J. D., S. RAZIN, AND R. C. CLEVERDON. Requirements for transformation in Bacillus 1965. Localization of enzymes in Mycoplasma. subtilis. J. Bacteriol. 81:741-746. J. Bacteriol. 90:617-622. 2. KERR, I. M., E. A. PRATT, AND I. R. LEHMAN. 14. SALTON, M. R. J. 1964. The bacterial cell wall, 1965. Exonucleolytic degradation of high- p. 56. Elsevier Publishing Co., Amsterdam. molecular-weight DNA and RNA to nucleoside 15. SPIZIZEN, J. 1958. Transformation of biochemi- 3'-phosphates by a nuclease from B. subtilis. cally deficient strains of Bacillus subtilis by Biochem. Biophys. Res. Commun. 20:154-162. deoxyribonucleate. Proc. Natl. Acad. Sci. 3. KUSHNER, D. J., AND M. R. POLLACK. 1961. The U.S. 44:1072-1078.