JOURNAL OF BACTERIOLOGY, May 1975, p. 610-622 Copyright 0 1975 American Society for Microbiology

Vol. 122, No. 2 Printed in U.SA.

Fate of Heterologous Deoxyribonucleic Acid in Bacillus subtilis MIRO04AWA PIECHOWSKA, ANNA SOZTYK,* AND DAVID SHUGAR Institute of Biochemistry and Biophysics, Academy of Sciences, 02-532 Warszawa, Poland Received for publication 19 February 1975

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thytrp-. Shortly after uptake, most of the radioactive E. coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with E. coli 2H and 3Hlabeled DNA. Association of radioactivity with recipient DNA decreased to onefourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the E. coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication. In some earlier studies it was reported that the fate of heterologous deoxyribonucleic acid (DNA) taken up by competent bacterial cells, and examined by means of fractionation in a CsCl gradient, was somewhat similar to that of homologous DNA. This presumed resemblance was based on the identity of the secondary structures of heterologous and homologous molecules after entry into the recipient cells, and on the fact that, in some instances, the label of heterologous nontransforming DNA was incorporated into recipient DNA like that of homologous transforming DNA. However, the experimental data did not distinguish between incorporation of heterologous DNA label as mononucleotides resulting from intracellular enzymatic degradation or as large fragments via recombination, as in the case of transforming DNA (3, 13, 19, 25). Although generally accepted that recombination requires similarities in base composition and sequence of the reacting DNA molecules (12), the minimal degree of homology necessary remains to be established. Conse-

quently, the absence of transformation by foreign DNA does not, a priori, exclude the possibility of (i) recombination occurring without subsequent expression of the markers introduced, perhaps because of the specificity of the cellular DNA-directed RNA polymerases (15), or (ii) integration of fragments much smaller than those involved in transformation, i.e., so small that, for a limited number of recombination events, the probability of introducing some selected marker is simply that for spontaneous mutation frequencies. The foregoing considerations are of interest in relation to two reported biological effects resulting from the uptake of heterologous nontransforming and nontransfecting DNA by competent bacteria, viz., the lethal effect on streptococci (21) and the mutagenic effect on Bacillus subtilis (17). In neither instance has the molecular mechanism involved been established. When to this is added the increasing number of reports on the biological effects of heterologous DNA on plant and animal cells (5, 11, 14, 18), it

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becomes of interest to obtain more detailed information on the fate of heterologous DNA in a system as simple as competent B. subtilis.

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cific activity of this DNA was 4.8 x 104 counts/min per Ag. Tritiated phage T6 DNA was prepared as follows: E. coli BB cells, cultured on an M-9 medium (1) supplemented with 0.2% vitamin-free Casamino MATERIALS AND METHODS Bacterial strains. The recipient and donor strains Acids (Difco) were harvested in the early log phase employed, both from the laboratory of M. S. Fox of and transferred to fresh medium to attain a density of the Massachusetts Institute of Technology, were B. 109 cells/ml. 5-Fluorodeoxyuridine (10 Mg/ml), uridine subtilis 168 thy- trp- and B. subtilis 168 thy- trp+ (100 ug/ml), and a mixture of cold and [C8H]thymirespectively. Heterologous DNA was prepared from dine (from UV VVR, Czechoslovakia) was added to the the thymine-dependent strain Escherichia coli 15T- culture medium to a concentration of 2 Ag/ml and to a and from phage T6, obtained from Irena Pietrzykow- specific activity of 2.5 Ci per mmol of thymidine. After ska of this Institute, cultivated on E. coli BB provided 10 min of incubation at 37 C, tryptophan was added by W. Szybalski of the University of Wisconsin; to a concentration of 2 Mg/ml and the cells were whereas non-glucosylated T6 DNA (T*6 DNA) was infected with phage T6 at a multiplicity of infection of prepared from non-glucosylated phage T6 (phage 2. After phage adsorption, the gently stirred culture T*6) cultivated on the uridine 5'-diphosphate/glucose was incubated for about 6 h at 37 C. The cells were phosphorylase defective mutant E. coli B/40 Luria no. then totally lysed by the addition of chloroform. The 56 (10), obtained from S. E. Luria of the Massachu- phage particles were collected and purified by differsetts Institute of Technology. The phage T*6 titer was ential centrifugation, followed by centrifugation in a determined with the aid of the strain Shigella preformed CsCl gradient. The purified phage was dysenteriae Sh, kindly supplied by N. Symons of the treated with phenol and the deproteinized DNA was University of Sussex, England. Absence of glucosyla- dialyzed versus SSC (0.15 M NaCl plus 0.015 M sotion was confirmed by the 300-fold lower phage titer dium citrate) the entire procedure was identical to when cultivated on E. coli BB. Marker DNA for the that described by Thomas and Abelson (27). The speCsCl gradients was prepared from Micrococcus luteus cific activity of the tritiated T6 DNA thus obtained was 3 x 105 counts/min per,g. ATCC 4698. "4C-labeled T6 DNA, for use as a position marker, Competent cultures of the recipient strain, labeled with [4C ]thymine (from UV VVR, Czechoslovakia), was prepared in an analogous manner by cultivation of the bacteria in a medium containing 30 mCi of were prepared as described by Piechowska and Fox (20), using 20 Mg of thymine/ml containing 0.05 gCi of [I"C ]thymine/mmol of thymine; it exhibited a specific activity of 2.5 x 104 counts/min per jig. [14C]thymine. Tritiated, non-glucosylated T6 DNA (T*6 DNA) Preparation of DNA. Labeled transforming DNA was obtained from phage cultivated on E. coli B/40 at was isolated from B. subtilis 168 thy- cultivated in the presence of [C3H]thymine (from UV VVR, Czech- a multiplicity of infection of 4. Total lysis of the cells oslovakia), with a specific activity of 1.7 or 5.2 Ci/mmol. occurred after 1 h of incubation. Subsequent steps The DNA was purified in a CsCl gradient as previ- were identical with those described in the previous ously described (20); the resulting activity was 6.5 x section for the preparation of T6 DNA. The specific 105 counts/min per Mg or 2 x 106 counts/min per Mg, activity of the T*6 DNA was 8 x 104 counts/min per respectively. In some experiments location of the Mg32P-labeled M. luteus DNA was isolated from DNA in the gradient was based on the use of "4C-labeled DNA, isolated from a donor strain, cul- bacteria grown on a medium containing 0.3% beef tivated in the presence of 60 mCi of [14CH.Jthymine extract (Difco), 1% peptone tryptose (Difco), 0.5% NaCl, 1% glucose, 0.01 M buffer (pH 7.6 of tris(hyper mmol (from UV VVR, Czechoslovakia); the specific activity of this DNA was 4.6 x 104 counts/min droxymethyl)aminomethane, and 1% purified agar (Difco). For preparation of the medium, a solution of per gg. Heavy tritiated DNA was isolated from E. coli 15T- cultivated on Roberts medium (22) in the first three components was brought to pH 2 by 99.99% D20 (Techsnabe, Moscow, USSR), contain- addition of 5 N HCl and then was treated with ing thymine (1 gg/ml) and tritiated thymine, to give a Amberlite IR-45 (acetate form). After removal of the specific activity of 2 or 4 Ci/mmol (as indicated below), resin by filtration, the solution was brought to pH 7.6 and a deuterated sugar mixture (Merck, Sharpe & and the other components were added, including 1 Dohme, Montreal, Canada) in place of glucose. The mCi of carrier-free [32P ]phosphoric acid (Institute of procedure for cultivation of the cells in heavy medium, Nuclear Studies, Warsaw) per ml of medium. After and isolation of the DNA, was similar to that previ- incubation, the cells were collected by centrifugation ously described for isolation of heavy transforming and washed. DNA was then extracted as described DNA (20). The specific activity of the DNA thus ob- above for DNA from E. coli. DNA uptake and preparation of cell lysates. tained was 0.7 or 1.4 x 106 counts/min per jig, depending on the activity added to the medium (and Labeled DNA was added to a competent culture to a taken account of in the description of individual ex- concentration of 1 gg/ml. After incubation for 15 min periments). A position marker for use in CsCl gradi- at 30 C, DNA uptake was terminated by addition of ents were derived from heavy DNA isolated from an 20 ug of deoxyribonuclease I (Worthington, Freehold, E. coli 15T- culture containing 60 mCi of [14C]thy- N.J.) per ml for 0.5 min or longer, as indicated, at mine/mmol of cold thymine in the medium; the spe- 37 C, followed by an equal volume of cold 0.15 M NaCl

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plus 0.1 M disodium Versenate (EDTA). The cells were then collected by centrifugation at 4 C, washed with a solution of 0.15 NaCl plus 0.1 M EDTA (pH 8), and once again with a solution of 0.15 M NaCl plus 0.01 M EDTA, pH 8. The washed cells were suspended in the latter solvent at a density fourfold higher than the competent culture treated with 7 mg of lysozyme (Sigma, St. Louis, Mo.) per ml at 2 C, and incubated for 30 min. The mixture was brought to 37 C several times for intervals of 1 or 2 min, so that the total time at 37 C was 9 min. A lysate of protoplasts was then obtained by addition of Sarcosyl NL (Ciba-Geigy) to a concentration of 1% and by warming to 10 C for 1 min. This followed by the addition of nuclease-free Pronase (Calbiochem), to a concentration of 0.2 mg/ml, and by incubation at 37 C for 2 h. NaCl was then added to a concentration of 4 M, and the lysate was heated at 70 C for 20 min. The lysate was cooled and dialyzed overnight, first against two changes of 0.15 M NaCl plus 0.01 M EDTA (pH 8) and then against 0.15 M NaCl plus 0.001 M EDTA (pH 8). Pronase was again added to a concentration of 0.1 mg/ml and the lysate was incubated for 2 h at 37 C. Gradient density fractionation. This was done as previously described (20) using Varlacoid CsCl. For experiments with T*6 DNA, phosphate buffer (pH 11) was employed for the alkaline CsCl gradient. Fractionation in a Cs,SO, (British Drug Houses, London) gradient made use of 1-ml samples to which was added 1.2 ml of 0.01 M EDTA plus 0.02 tris(hydroxymethyl)aminomethane (pH 7.4) and 2.2 ml of 60% (wt/wt) Cs2SO4; the refractive index of this solution was 1.3700 at 20 C. Samples were run in the SW50 rotor of a Beckman model L2 at 27.5 x 10 rpm for 70 h. Radioactive counting. This was done with a Packard Tricarb scintillation counter. The scintillation fluid consisted of 3 g of 2,5-diphenyloxazole and 0.1 g of 1,4-bis-(5-phenyloxazolyl)benzene (Fluka A.G.) in 1 liter of toluene. All samples were collected on 21mm diameter Whatman 3MM paper disks and, in this form, were precipitated with trichloroacetic acid prior to counting. Specific activities of DNA samples were determined under optimal conditions for each label. For samples isolated from gradients, counting was done under conditions where a maximum of 25% of the '4C counts spilled over into the 3H channel, and 2% of the 3H into the 14C channel; appropriate corrections were made for this. Shearing of DNA. Solutions consisting of 26 ug of DNA in 2 ml of SSC, in a 55-mm deep and 14-mm diameter glass vial cooled to 2 C, were subjected to shearing by sonic oscillation for 20 s in a 100-W MSE sonic oscillator at an amplitude of 6.8 Mtm. Molecular weights of sonically oscillated DNA were determined, as described by Rosenthal and Fox (23), by sedimentation in a sucrose gradient versus a labeled standard DNA. The molecular weight of the standard DNA was determined by analytical centrifugation, using Studier's equation (26). Measurement of radioactivity of DNA uptake. At various time intervals after termination of DNA uptake at 37 C, 1-ml samples of recipient cells were

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mixed with 1 ml of 0.15 M NaCl plus 0.1 M EDTA (pH 8) at 4 C and then were centrifuged at the same temperature for 10 min at 11,000 x g. The cell pellet was washed with, then suspended in and centrifuged from, 1 ml of 0.15 M NaCl + 0.01 M EDTA (pH 8). This was followed by a similar treatment with 0.15 M NaCl plus 0.001 M EDTA (pH 8). The cells were then suspended in 0.5 ml of 0.15 M NaCl plus 0.001 M EDTA (pH 8) plus 0.5 ml of 10% trichloroacetic acid and were kept on ice for 20 min. The resulting precipitate was collected by filtration under reduced pressure on a Whatman GF/C glass filter and washed with 20 ml of 5% trichloroacetic acid, and the filter with precipitate was rinsed with 3 ml of 5% trichloroacetic acid in a small beaker in an ice bath for 15 min. Then the fluid was decanted. This washing procedure was repeated once more, the filter then was rinsed with cold 96% ethanol and ether and dried under an infrared lamp, and the radioactivity was measured. Rinsing of the filters by pouring trichloroacetic acid solution over them in a beaker, which supplemented the normally applied technique of washing the precipitates on a filter under reduced pressure, was an essential condition for obtaining reproducible results when recipient cells were incubated for periods longer than 0.5 min after termination of DNA uptake. It is probably due to the poor solubility of cell-bound products of degradation of DNA taken up.

RESULTS Fractionation of cell lysates in density gradients after uptake of E. coli DNA. With a view to determining the state of the secondary structure of E. coli DNA shortly after uptake, recipient cell lysates were fractionated on CsCl gradients at pH 8 and 11.2. The analysis was based on a determination of the relative positions of the components of a standard DNA mixture in the same gradients; M. luteus 32Plabeled DNA was used as a position marker both in the control and sample solutions. In the control mixture on a pH 8 gradient, heavy native E. coli DNA formed a band at 12% the length of the gradient from recipient DNA. Heavy denatured E. coli DNA was located at 17% the gradient length from recipient DNA and at 8% the gradient length from the M. luteus marker DNA (Fig. 1A); the corresponding separations in a pH 11.2 gradient were 30 and 21%, respectively (Fig. 1B). Figure 1C and D exhibit the results for a B. subtilis recipient lysate, the cells of which had been incubated for 0.5 min at 37 C after termination of uptake of deuterated, tritiated DNA. In both the pH 8 gradient (Fig. 1C) and the pH 11.2 gradient (Fig. 1D), 70% of the radioactivity of the donor DNA was found at the position characteristic for denatured DNA, the remainder occurring with the recipient DNA. The alkaline gradient led to a shift, characteristic for denatured

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fraction number fraction number FIG. 1. State of E. coli DNA shortly after uptake by B. subtilis recipient culture: (A and B) relative densities of DNA standard (A) in a CsCI gradient at pH 8 and (B) in a CsCl gradient at pH 11.2 (DH-Ec, denatured, heavy E. coli DNA; NH-Ec, native, heavy E. coli DNA; ML, M. lutea 32P-labeled DNA; N-Bs, native B. subtilis DNA); and (C and D) density gradient analysis of [14C]thymine-labeled recipient cell lysate previously incubated for 0.5 min after uptake of E. coli deuterated 'H-labeled DNA (specific activity 7 x 10' counts/min per jsg). The lysate (see Materials and Methods) was fractionated (C) in a CsCI gradient at pH 8 and (D) in a CsCI gradient at pH 11.2. Letters and arrows indicate expected positions for various DNAs denoted by symbols as in (A) and (B) above. Radioactivities denoted by: A, 'H; A, 1'4C; 0, "2P.

molecules, of 70% of the radioactivity of E. coli DNA towards a position of higher density (28). In neither the pH 8 nor the pH 11.2 gradient was it possible to localize a band in the position characteristic for native donor DNA. For longer incubation times (7 and 20 min) of the recipient culture after termination of DNA

uptake, the fate of E. coli-labeled DNA is shown in Fig. 2. When the incubation time was short (0.5 min), fractionation in a pH 11.2 CsCl gradient revealed about 77% of the E. coli DNA in the position of denatured molecules and the remainder associated with the recipient band (Fig. 2A). The longer incubation times led to the

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FIG. 2. Fate of E. coli DNA after uptake by competent cells, based on density gradient analysis of lysates of cells previously incubated at 37 C for (A) 0.5 min, (B) 7 min or (C) 20 min after termination of uptake of E. coli deuterated 8H-labeled DNA. Preparation of lysates was as in Materials and Methods. Density gradient, CsCI at pH 11.2. Symbols: A, E. coli deuterated 3H-labeled DNA; A, recipient 14C-labeled DNA; 0, M. luteus 32P_labeled DNA used as a position marker. Arrow (and letters) indicate the positions of: DH-Ec, E. coli denatured heavy DNA; NH-Ec, E. coli native heavy DNA; NL-Bs, B. subtilis native light DNA; ML, M. luteus DNA.

eventual disappearance of the denatured DNA; transforming DNA (20); the loss of donor label simultaneously, the radioactivity associated is presumably due to trichloroacetic acid-soluwith the recipient band increased to 44% of the ble fragments derived from the denatured DNA. labeled DNA taken up (Fig. 2B and C). It should Fractionation of cell lysates after uptake of be noted, however, that the increase in radio- T6 DNA. Cs,SO gradients were employed in activity associated with the recipient band ac- these experiments since, under such conditions, counts for the loss of only 20 to 25% of the de- the difference in density between glucosylated natured DNA, a situation somewhat similar to T6 DNA and B. subtilis DNA is greater than in that previously encountered with homologous CsCl. Figure 3A exhibits the relative positions

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of the DNA samples in a Cs2SO, gradient. It pected position in the gradient of this DNA was should be noted that native and denatured T6 determined by comparison with the control and DNA are separated from recipient DNA by 5.5 with the aid of a T6, 14C-labeled DNA marker and 9%, respectively, of the length of the added immediately prior to centrifugation. In gradient. the 0.5-min sample (Fig. 3B), the T6 DNA Figure 3B and C shows the results for frac- radioactivity fixed by the cells was spread tionation of cell lysates that had been incubated throughout the gradient, with maximal radioacfor 0.5 and 20 min, respectively, after termina- tivity at the position characteristic for native T6 tion of T6 3H-labeled DNA uptake. The ex- DNA. The longer incubation period (Fig. 3C)

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FIG. 3. Fate of phage T6 DNA after uptake by recipient cells: (A) relative positions in Cs2SO4 density gradient of D-T6 (denatured T6 DNA) N-T6 (native T6 DNA), N-Bs (native B. subtilis recipient DNA from a cell lysate); and (B and C) analysis of lysates of cells incubated for (B) 0.5 and (C) 20 min after uptake of T6 3H-labeled DNA. Cell lysates prepared as in Materials and Methods. Symbols: A, T6 3H-labeled DNA taken up by cells; A, B. subtilis recipient "4C-labeled DNA or native T6 "4C-labeled DNA added to cell lysates as position marker. Arrows indicate expected positions of denatured T6 DNA.

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led to the virtual disappearance of this band; the resulting spreading of the label throughout the gradient was most likely due to degradation of the fixed DNA. It should be pointed out that the absolute amount of T6 DNA bound by the cells is fivefold less than for E. coli DNA. The specificity of DNA uptake by B. subtilis cells will be described in detail elsewhere. Fractionation of cell lysates after uptake of T*6 DNA. Bearing in mind that the behavior of T6 DNA may not be typical, since it contains glucosylated 5-hydroxymethylcytosine residues in place of cytosine, identical experiments were performed with non-glucosylated phage DNA, i.e., T*6 DNA. In a CsCl gradient this DNA formed a band together with recipient DNA (Fig. 4A). By contrast, cell-fixed T*6 DNA, from a lysate of cells incubated for 0.5 min after termination of DNA uptake, formed a band in a position characteristic for heavier molecules (Fig. 4B) and, when centrifuged in a pH 11.2 gradient, exhibited an increase in density typical of the behavior of denatured DNA (28) (Fig. 4D). Prolonged incubation led to disappearance of free T*6 DNA and a transfer of about 60% of its radioactive label to the recipient DNA band (Fig. 4C). Effect of exogenous thymidine on recipient DNA-bound label of DNA taken up. After uptake of E. coli or B. subtilis 3H-labeled DNA, the recipient cells were incubated at 37 C in a medium containing cold thymine, and at various time intervals, samples were removed to determine the trichloroacetic acid-precipitable donor DNA radioactivity present in the recipient cells, as described above. The results are shown in Fig. 5. It will be seen that recipientbound radioactivity decreased continuously for 40.to 50 min, during incubation at 37 C, after termination of DNA uptake until it reached a value of about 23% for E. coli DNA, and about 45% for B. subtilis DNA, relative to the radioactivity found in the respective 0.5-min samples. From the results described above on the fate of DNA taken up, as determined by density gradient fractionation (Fig. 2), one might anticipate a minimum being reached after about 20 min of incubation, since at that time there is no longer any free donor DNA present, and all the radioactivity of the donor is located in the recipient DNA band. The time required for reaching a minimum in these experiments may be ascribed to the difference in preparation of the samples for measurement. For, prior to density gradient fractionation, the steps involving cell lysis, Pronase digestion, and prolonged

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dialysis could conceivably eliminate lowmolecular-weight degradation products of DNA taken up, which do not dissolve in trichloroacetic acid during treatment of intact cells, possibly because these are bound to cell proteins precipitated by the trichloroacetic acid. Both in the present experiments, and in those described above, recipient cultures were prepared in the presence of a thymine concentration fourfold greater than the minimum required for normal growth of B. subtilis 168 thytrp-. Under these conditions, addition of thymidine to a molar concentration fivefold higher than that of thymine was found to decrease the degree of retention of DNA taken up measured as the radioactivity bound to the cells in a form precipitable by trichloroacetic acid. With B. subtilis transforming DNA, the residual radioactivity in the presence of thymidine was 33%; with E. coli DNA this amounted to only 6% (Fig. 5). A similar difference between homologous and heterologous DNA was noted in several experiments which were limited to measurements of the initial and final amounts of labeled DNA taken up and bound by the cells in the presence of thymidine. However, the values obtained after 50 min of incubation were about 60% for B. subtilis DNA (four experiments), and 15% for E. coli DNA (seven experiments). Differences in results of different sets of experiments were due to changes in the recipient cultures. Consequently, a strict comparison of the behavior of homologous and heterologous DNA in the presence of thymidine was carried out with the use of a single recipient culture. The decrease in tightly bound, labeled DNA taken up by B. subtilis in the presence of thymidine did not affect the transformation frequency for the tryptophan synthesis marker, e.g., for a culture on a thymine-containing medium, the average number of trp+ transformants was 3.3 x 106/ml whereas, for a culture on a medium containing a 5:1 ratio of thymidinethymine, the number of transformants was 3.5 x 106.

The values for the plateaus of the curves presented in Fig. 5 characterize the radioactivity of each donor DNA present uniquely in association with the recipient DNA band. This was established by analysis in a CsCl gradient (described in Fig. 6A, below). These values could not be further reduced by removal of thymine or its replacement by thymidine alone (result not shown). Nature of residual radioactivity of E. coli DNA associated with recipient DNA. For this

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FIG. 4. Fate of non-glucosylated T*6 DNA after uptake by recipient cells: (A) relative positions in a CsCI gradient at pH 8 of N-T*6 (native Tr6 DNA) and N-Bs (native B. subtilis DNA); and (B, C, and D) analyses of lysates of cells incubated for 0.5 (B and D) and 20 min (C) after uptake of T*6 3H-labeled DNA. Cell lysates were prepared as described in Materials and Methods. (B and C) Fractionation in a CsCl gradient, pH8, and (D) fractionation in.a CsCI gradient, pH 11.0. Symbols: A, r6 3H-labeled DNA; A, B. subtilis recipient 14Clabeled DNA. Arrows indicate expected positions of denatured *6 DNA (D; r6).

analysis the recipient was cultured on a medium containing 0.043 uCi (50 ug) of [ l4C Ithymidine/ml. To such a competent culture was added E. coli deuterated 8H-labeled DNA with a specific activity of 1.4 x 106 counts/min per jsg, and a density identical to that previously employed (see Fig. 1A and B). After termina-

tion of DNA uptake by deoxyribonuclease treatment, the recipient was incubated for 50 min at 37 C, and a cell lysate was prepared as described in Materials and Methods. The lysate was fractionated in a pH 8 CsCl gradient (Fig. 6A), and the recipient DNA band, including the DNA donor radioactivity, was collected,

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dialyzed versus SSC, and then sonically oscillated to reduce the molecular weight to 0.3 x 10g. Centrifugation of the sonically oscillated DNA in a CsCl gradient (Fig. 6B) showed no shift of the labeled donor band toward densities higher than that of recipient DNA. To obtain a better resolution, the heavy portion of the DNA band (to the left of the position expected for the density of hybrid DNA, i.e., the portion of the left of the vertical line in Fig. 6B) was collected, dialyzed versus 0.1 x SSC, and again centrifuged in a CsCl gradient before (Fig. 6C) and after (Fig. 6D) heat denaturation. For the native sample (Fig. 6C), the band containing the donor label again coincided with the recipient DNA band, with no evidence for formation of a band at the expected position for hybrid molecules. Not only is there no observable band in the position corresponding to denatured heavy E. coli DNA, but there is also no shift of the donor DNA label relative to recipient DNA after thermal denaturation (Fig. 6D). A further attempt to characterize the nature of the E. coli DNA fragments associated with recipient DNA involved measurement of trichloroacetic acid-precipitable radioactivity of DNA taken up and retained by cells cultivated on a thymidine-containing medium under nor40 30 10 50 60 20 mal conditions as described above, and in the min. at 37° C presence of a specific inhibitor of DNA replicaFIG. 5. Competing effect of thymidine on radioac- tion in gram-positive organisms, viz., 6-(ptivity of DNA taken up and retained by competent hydroxyphenylazo)-uracil (4). The inhibitor cells. To 4.9 ml of a competent recipient, in a medium was added to a concentration of 300 mM immecontaining 20 Mg of thymine per ml, 80 pl of a solution diately prior to DNA uptake. The extent to of thymidine (25 mg/mI) was added so that its concen- which the label was retained by the cells, tration was 400 Mg/ml, and 60 gl of a solution of 'H-labeled DNA (85 Mg DNA/ml in SSC) was added measured as described above, decreased to one-half that of the control in the case of E. coli so that its concentration in the medium was 1,Mg/ml. Incubation for 15 min at 30 C was followed by DNA, but only by 4% in the case of B. subtilis addition of 0.1 ml of deoxyribonuclease (1 mg/ml) to DNA. The values obtained in the presence of give deoxyribonuclease concentration of 20 Ag/ml, and the inhibitor were, respectively, 5 and 60% of the temperature of the culture then was raised to the E. coli and B. subtilis DNA taken up (Table 37 C. At 0.5, 10, 20, 40, 60, and 60 mmin of incubation, 1). 1-mI s'mples were withdrawn, the cells were collected by cehtrifugation, washed, and precipitated with trichloroacetic acid, and the radioactivity was determined as described in Materials and Methods. Each measurement was corrected for background due to the radioactivity of a control sample to which was added deoxyribonuclease-hydrolyzed 3H-labeled DNA. The resulting curves show percentage of retention of radioactivity of E. coli 3H-labeled DNA in the presence of thymidine (0), and in a control culture to which was added water in place of thymidine (0); and percentage of retention of radioactivity of B. subtilis 3H-DNA in the presence of thymidine (A), and without thymidine (A). The 100% value was the radioactivity of uptaken DNA in a sample incubated at 37 C for 0.5 min after termination of DNA uptake.

DISCUSSION The one homologous and three heterologous DNA samples examined in this study exhibit some specific differences in behavior, including: (i) quantitative differences in binding to competent recipient cells; (ii) differences in secondary structure after uptake; (iii) the number and type of fragments associated with recipient DNA. The overall results clearly demonstrate considerably poorer uptake of T6 DNA relative to E. coli DNA and non-glucosylated T6 DNA. This specificity in DNA uptake by B. subtilis

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-6~~~0

[ A~~~~~~~~~0"

50 60 70 80 90 100 110 60 70 80 .106 fraction number fraction number FIG. 6. Analysis of E. coli DNA fragments incorporated into recipient DNA. (A) Analysis of a lysate of recipient cells cultured on a medium containing 0.025 MCi of [14C]thymidine per 50 Mg of thymidine/ml and incubated for 50 min after uptake of E. coli deuterated 3H-labeled DNA with a specific activity of 1.4 x 106 counts/min per ug. Cell lysate was prepared as described in Materials and Methods. CsCI gradient at pH 8. Arrows indicate expected positions of: DH-Ec, denatured heavy E. coli DNA; NH-Ec, native heavy E. coli DNA. (B) Recipient DNA band containing donor DNA label (fractions 66-78 from gradient A) sonically disrupted to a molecular weight of -0.3 x 106 and again centrifuged in a CsCI gradient pH 8. Arrows indicate expected positions of: NH;Ec, native heavy E. coli DNA; HL, hybrid molecules consisting of 50% heavy and 50% light DNA; N-Bs, native B. subtilis recipient DNA. (C and D) The heavy fractions from the sonically treated sample [ractions to the left of the vertical line in (B)] were pooled and again centrifuged in a neutral CsCI gradient as such (C) and after thermal denaturation (D). Arrows indicate expected positions of DNA samples as in (A) and (B) above. Note: native M. Luteus 32P-labeled DNA (denoted by ML) was added as a position marker to all samples in these experiments immediately prior to centrifugation. In (D) the denatured M. luteus DNA originating from the previous centrifugation did not appear as an isolated band, but deformed the left shoulder of the marker band since it made up only 15 of the native M. luteus DNA added to the denatured sample immediately prior to centrifugation.

30

40

50

recipients may be of some significance in rela- Haemophilus influenzae (24) suggests that this tion to the mechanism of binding and transport phenomenon may be quite general. Part of the E. coli, and T*6, DNA is found in and is being subjected to quantitative investigation. During the course of this study, a report on recipient cell lysates in the form of free, denathe specificity of DNA uptake by competent tured molecules as previously shown for homol-

620

PIECHOWSKA, SO-hTYK, AND SHUGAR

J. BACTERIOL.

TABLE 1. Trichloroacetic acid-precipitable radioactivity of DNA uptake retained by B. subtilis competent cells cultured on a thymidine-containing medium in the absence and presence of the DNA replication inhibitor 6-(p-hydroxyphenylazo)-uracil (HPU)a Type of DNA

3H-labeled DNA (E. coli)

HPU

_ +

3H-labeled DNA (B. subtilis)

_ +

Incubation time after DNA uptake

Trichloroacetic acid-

(min)

precipitable of donor DNAradioactivity in

0.5 50

9,573; 9,869 1,056; 1,173

0.5 50

6,647; 6,753 298; 381; 283

recipient

cells (counts/min)

(0.5 Mean min/50%min)

12 5

0.5 50

33,814; 34,566 21,530; 21,068

62

0.5 50

24,720; 32,808 14,554; 18,582

58

To 3.8 ml of a recipient culture 90 Il of a 20 mM solution of HPU in 0.05 M NaOH was added to give a final concentration of 300 MiM HPU and a pH of about 7.5. This was followed by addition of 0.1 ml of a 40 ,ug/ml solution of 3H-labeled DNA in SSC to give a final DNA concentration in the medium of 1 Mg/ml. Incubation at 30 C for 15 min was followed by addition of deoxyribonuclease to a concentration of 20 ug/ml, and incubation was then continued at 37 C. After 0.5 and 50 min of incubation, 2-ml samples were withdrawn, cooled, and centrifuged, and the cells were washed, treated with trichloroacetic acid, and filtered. The precipitate on the filter was washed as described in Materials and Methods for measurement of fixed DNA uptake radioactivity. Each measurement was corrected for background constituted by the radioactivity of a sample prepared from a recipient culture to which was added deoxyribonuclease-hydrolyzed 3H-labeled DNA in place of normal 3H-labeled DNA, in the presence or absence of HPU. Measured background counts were, respectively, 103 and 40 counts/min for E. coli 3H-labeled DNA, and 286 counts/min for B. subtilis 3H-labeled DNA. To controls were added 90 Il of 0.05 M NaOH in place of HPU solution. Specific activities of 3H-labeled DNAs were 1.4 x 106 counts/min per Mg for E. coli, and 2 x 106 counts/min per Mg for B. subtilis. a

ogous DNA (20). By contrast, T6 DNA was found in the native form after uptake. In the case of E. coli and T*6 DNA taken up, the remaining radioactivity is associated with recipient DNA as with labeled transforming DNA (20). But with T6 DNA no incorporation of its radioactivity into recipient DNA could be detected. The source of these differences is being further studied, bearing in mind the possibility that uptake of T6 DNA may not be real uptake as observed for other types of DNA. As regards the comportment of homologous, E. coli and T*6 DNA, where and at what time during or after uptake does denaturation occur? In accordance with the proposal of Lacks et al. (13) for the analogous situation in Diplococcus pneumoniae, it appears reasonable to assume that denaturation takes place during binding of DNA in a form resistant to extracellular deoxyribonuclease. An alternative interpretation is that of Arwert & Venema (2), viz., that DNA taken up is not totally denatured, but rather converted to a destabilized form by cellular proteins and selectively denatured during preparation of samples for gradient fractionation. Additional experimental data are clearly neces-

sary to clarify this problem. It should be emphasized, however, that the experimental procedure employed in this study was such as to leave intact the secondary structure of the recipient DNA, so that this may be considered as an internal control for the above experiments. A marked proportion of the integrated radioactivity of E. coli DNA, as well as part of that of B. subtilis DNA, is derived from the degradation of molecules taken up and the use of the resulting labeled monomers for DNA synthesis. This is testified to by the decrease of such integration in the presence of cold thymidine. The observed influence of thymidine, which competes even with an excess of thymine, is consistent with the preferential utilization of the former for DNA replication (9). In the case of homologous transforming DNA, where most of the radiqactivity is retained in the presence of thymidine, this activity probably emanates from large fragments (0.5 to several million) integrated via recombination (12). In the case of E. coli heavy DNA, the sequential application of sonic oscillation, denaturation, and gradient fractionation did not lead to a separation of labeled donor and

VOL. 122, 1975

FATE OF HETEROLOGOUS DNA IN B. SUBTILIS

recipient DNAs, a finding that differs from that obtained in analogous experiments with homologous DNA (21) and is suggestive of covalent binding of heterologous DNA fragments of a molecular weight lower than 1.5 x 105 (i.e., 0.5 x 0.3 x 105, see above). A second fact which characterizes the residual integration of labeled E. coli DNA into recipient DNA is its decrease in the presence of a DNA replication inhibitor, since under the same conditions integration of labeled homologous DNA remains virtually unchanged, in agreement with Dubnau and Cirigliano (8), who found that this same inhibitor did not affect recombination with transforming DNA. The largest difference in behavior between homologous and heterologous DNA is revealed in the presence of both thymidine and the polymerase inhibitor, viz., the residual binding of only 5% of the E. coli DNA radioactivity taken up, as compared to about 60% for B. subtilis DNA. The overall findings indicate that at least 95% of the E. coli DNA radioactivity found in the recipient DNA is integrated as mononucleotides via DNA replication. In line with current concepts, this finding is consistent with the pronounced difference in base composition and sequence between the heterologous and recipient DNAs. Experimentally, the small similarity between these two DNA species is testified to by the fact that only 4% of E. coli DNA (guanine plus cytosine content 50%) forms hybrids with B. subtilis DNA (guanine plus cytosine content 43.9%) (29). The absence of recombination with E. coli DNA, together with the presence of the latter in the denatured form, suggests that in the case of transforming DNA the denatured form represents the precursor, and not the product, of recombination. This is in accord with the conclusion of Davidoff-Abelson and Dubnau (6), based on studies of the sequential events in the transmutation of transforming DNA in B. subtilis. However, our data demonstrating incorporation of E. coli DNA degradation products into competent cell DNA are at variance with those of Dubnau and Cirigliano (7), who found no incorporation of E. coli DNA label under similar conditions. No immediate interpretation of this discrepancy is forthcoming; one possibility is some difference in properties between the bacterial strains employed. ACKNOWLEDGMENTS We are indebted to M. S. Fox for a gift of part of the deuterated sugar employed in this study; to B. Langley for a sample of 6-(p-hydroxyphenylazo)-uracil; to G. Venema for suggesting the experiment on the competing effect of thymi-

621

dine; and to all those mentioned in Materials and Methods who contributed the vrious bacterial strains used. This investigation was carried out as Project 09.3.1 of the Polish Academy of Sciences.

LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virusresistant mutants of Escherichia coli strain. Proc. Natl. Acad. Sci. U. S. A. 32:120-128. 2. Arwert, F., and G. Venema. 1973. Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA. Mol. Gen. Genet. 123:185-198. 3. Bodmer, W. F., and A. T. Ganesan. 1964. Biochemical and genetic studies of integration and recombination in Bacillus subtilis transformation. Genetics 50:717-738. 4. Brown, N. C. 1971. Inhibition of bacterial DNA replication by 6-(p-hydroxyphenylazo)-uracil: differential effects on repair and semiconservative synthesis in Bacillus subtilis. J. Mol. Biol. 59:1-16. 5. Crawford, L., R. Dulbecco, and M. Fried. 1964. Cell transformation by different forms of polyoma virus DNA. Proc. Natl. Acad. Sci. U.S.A. 52:148-152. 6. Davidoff-Abelson, R., and D. Dubnau. 1973. Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis. J. Bacteriol. 116:154-162. 7. Dubnau, D., and C. Cirigliano. 1972. Fate of transforming DNA following uptake by competent Bacillus subtilis. Ill. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA. J. Mol. Biol. 64:9-29. 8. Dubnau, D., and C. Cirigliano. 1973. Fate of transforming DNA after uptake by competent B. subtilis: nonrequirement of DNA replication for uptake and integration of transforming DNA. J. Bacteriol. 113:1512-1514. 9. Harris, W. J. 1973. The occurrence of two types of synthesis of deoxyribonucleic acid during normal growth in Bacillus subtilis. Biochem. J. 135:315-325. 10. Hattman, S., and T. Fukasawa. 1963. Host-induced modification of T-even phages due to defective glucosylation of their DNA. Proc. Natl. Acad. Sci. U.S.A. 50:297-305. 11. Hess, D. 1973. Transformation experiments in higher plants: evidence for the realization of exosome model in Petunia hybrida. Z. Pflanzenphysiol. 68:432-441. 12. Hotchkiss, R. D., and M. Gabor. 1970. Bacterial transformation, with special reference to recombination process. Annu. Rev. Genet. 4:193-224. 13. Lacks, S., B. Greenberg, and K. Carlson. 1967. Fate of donor DNA in pneumococcal transformation. J. Mol. Biol. 29:327-347. 14. Leff, J., and R. E. Beardsley. 1970. Action tumorigene de l'acide nucleique d'un bacteriophage present dans les cultures de tissue tumoral de tournesol (Heliathus annum). C. R. Acad. Sci. Paris 270:2505-2507. 15. Losick, R., R. G. Shorenstein, and A. L. Sonenshein. 1970. Structural alteration of RNA polymerase during sporulation. Nature (London) 227:910-913. 16. McCarthy, C., and E. W. Nester. 1967. Macromolecular synthesis in newly transformed cells of Bacillus subtilis. J. Bacteriol. 94:131-140. 17. Mergeay, M. 1972. Mutagenic effects of Streptomyces coelicolor DNA detected after streptomycin treatment of competent cultures of Bacillus subtilis. Mol. Gen. Genet. 119:89-92. 18. Merril, C. R., and M. R. Geier. 1971. Bacterial virus gene expression in human cells. Nature (London) 233:398-400. 19. Pene, J. J., and W. R. Romig. 1964. On the mechanism of genetic recombination in transforming Bacillus

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subtilis. J. Mol. Biol. 9:236-245. 20. Piechowska, M., and M. S. Fox.1971. Fate of transforming deoxyribonucleate in Bacillus subtilis. J. Bacteriol. 108:680-689. 21. Piechowska, M., and D. Shugar. 1967. Inhibitory and lethal effects of DNA on transformable streptococci. Biochem. Biophys. Res. Commun. 26:290-295. 22. Roberts, R. B., P. H. Abelson, D. B. Cowie, E. T. Bolton, and R. J. Britten. 1955. Studies of biosynthesis in Escherichia coli. Carnegie Institution of Washington, publication 607, Washington, D. C. 23. Rosenthal, P. N., and M. S. Fox. 1970. Effects of disintegration of incorporated 'H and "P on the physical and biological properties of DNA. J. Mol. Biol. 54:441-468. 24. Scocca, J. J., R. L. Poland, and K. C. Zoon. 1974. Specificity in DNA uptake by transformable Haemophilus influenzae J. Bacteriol. 118:369-373.

J. BACTERIOL.

25. Silverstein, E., and B. M. Mehta. 1971. Fate of Bacillus subtilis transforming DNA incorporated into transformable Diplococcus pneumoniae. Biochemistry 10:683-691. 26. Studier, F. W. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390. 27. Thomas, C. A., and J. Abelson. 1966. The isolation and characterization of DNA from bacteriophage, p. 553-561. In G. L. Cantoni and D. R. Davies (ed.), Harper and Row, Publishers, New York. 28. Vinograd, J., J. Morris, N. Davidson, and W. F. Dove, Jr. 1963. The buoyant behaviour of viral and bacterial DNA in alkaline CsCl. Proc. Natl. Acad. Sci. U.S.A. 49:12-17. 29. Young, F. E., and G. A. Wilson. 1972. Genetics of Bacillus subtilis and other gram-positive sporulating bacilli, p. 77-106. In H. 0. Halvorson (ed.), Spores V. American Society for Microbiology, Washington, D. C.

Fate of heterologous deoxyribonucleic acid in Bacillus subtilis.

JOURNAL OF BACTERIOLOGY, May 1975, p. 610-622 Copyright 0 1975 American Society for Microbiology Vol. 122, No. 2 Printed in U.SA. Fate of Heterologo...
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