JOURNAL OF BACTERIOLOGY, Nov. 1970, p. 684-688 Copyright © 1970 American Society for Microbiology Vol. 104, No. 2 Printed in U.S.A. Uptake of Synthetic Polynucleotides by Competent Cells of Bacillus subtilis ORIO CIFERRI,! SERGIO BARLATI,! ann JOSHUA LEDERBERG Department of Genetics, Stanford University School of Medicine, Stanford, California 94305 Received for publication 21 July 1970 A survey was made of the capacity of competent cells of Bacillus subtilis to take up synthetic polynucleotides. Polydeoxyribonucleotides but not polyribonucleotides are taken up by the cells. Both types of polynucleotide failed to compete with trans- forming deoxyribonucleic acid in the transformation assay whereas both stimulated amino acid incorporation. The latter phenomenon appears to be nonspecific as there is no correlation between the codon composition of the polynucleotides em- ployed and the amino acids whose incorporation was stimulated. Hurwitz et al. (6) and Evans (2) showed that deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybrid complexes are capable of con- ferring the sulfonamide resistance phenotype upon sensitive recipient cells of Diplococcus pneumoniae. More recently Kirtikar and Duerksen (7) reported the induction of penicillinase by treating cultures of Bacillus cereus and Staphylo- coccus aureus with RNA prepared from a peni- cillinase constitutive strain of B. cereus. Amos et al. (1) presented evidence favoring the conten- tion that in ‘“‘soniplasts,” prepared from Esche- richia coli, polyuridilic acid (poly-rU) specifically stimulates the incorporation of phenylalanine but not of leucine. This paper reports the negative results of dili- gent efforts to demonstrate the uptake of ‘‘syn- thetic messenger” polynucleotides by competent cells of B. subtilis. Rather than discard the present results without mention, we present them to illustrate some potential artifacts and save time for others who may have more ingenious approaches to the problem. MATERIALS AND METHODS B. subtilis strain SB893, an arginine and xanthine derivative of the indole-requiring strain 168M, was used. The cells were made competent and were stored and utilized as reported by Stewart (11). DNA was extracted by the Marmur’s procedure (9) from the wild-type B. subtilis SB850. Uptake of labeled poly- nucleotides was measured on suspensions of com- petent cells in Spizizen minimal medium (10) con- taining 0.5% glucose, at a concentration of 2 x 107 to 4 X 107 cells/ml. After the addition of the labeled polynucleotide, 0.2- to 0.5-ml samples of cell sus- 1 Present address: Istituto di Genetica, Universita di Pavia, 27100 Pavia, Italy. pension were withdrawn at different time intervals filtered on membrane filters (Millipore Corp., Bed- ford, Mass.), washed with cold Spizizen medium, dried, and counted. Incorporation of Jabeled amino acids into cold acid-insoluble material was determined on cell sus- pension identical to those reported for the experi- ments on the uptake of polynucleotides except that samples of the cell suspension were pipetted into 0.2 to 0.5 ml of 10% trichloroacetic acid. The samples were left for at least 30 min at OC, filtered through membrane filters, dried, and counted. All experi- ments were performed on cultures incubated at 37 C on a rotary shaker. Chemicals. Unlabeled polyadenilic acid (poly-rA), poly-rU, polycytidylic acid (poly-rC), and polyinosinic acid (poly-rI), as well as tritiated, randomly labeled polyadenilic acid, and polyuridilic acid tritiated in the uracil-5-position (specific activity, 64.4 and 29.9 mc/mmole, respectively) were from Miles Labora- tories, Inc. Tritiated polydeoxyadenilic-thymidilic (poly-dAdT) and polydeoxycytidylic-polydeoxygua- nilic acid (poly-dCdG) (specific activity, 25 mCi per mmole in each case) were from Biopolymers Inc. The molecular weight of both polynucleotides was estimated by sucrose density gradient to be about 2 X 105 to 3 X& 10 daltons. '4C-phenylalanine (393 mCi/mmole), “C-lysine (247 mCi/mmole), and 4C- arginine (173 mCi/mmole) were from Calbiochem, Los Angeles, Calif. Polyuridilic-polyadenilic acid (poly-rUrA) was prepared by mixing equimolecular amounts (~500 ug/ml of each) of the polyribonucleotides in 0.1 M NaCl for 15 min at 70 C and cooling slowly to room temperature. Hybrid formation was ascertained by determining the melting temperature that in 0.1 m phosphate buffer (pH 7.8) was estimated to be 52 C. The label was either in the poly-rU or poly-rA moiety. Poly+U:DNA was prepared as described by Kubinski et al. (8). Formation of the hybrid was assessed by the decrease in transforming activity as 684 VoL. 104, 1970 compared to that of DNA renaturated in the absence of poly-rU. The label was in the poly-rU. Polyadenilic- polydeoxythymidilic acid (poly-rAdT) was prepared by mixing 0.1 ml of #H-poly-rA (1 wCi) in 0.4 ml of 0.2 M phosphate buffer (pH 7.8) with 0.5 ml of poly- dT at 10 xg/ml in 10-? Mo tris(hydroxymethyl)amino- methane-hydrochloride buffer (pH 7) at 28 C. Label was in the poly-rA. Poly-dT was generously supplied by R. Lehman. RESULTS The label from the polyribonucleotides tested (poly-rA, poly-rU, poly-rUrA, poly-rAdT, poly- rU:DNA) was not taken up to a significant extent by the cells under any of our conditions. However, a certain amount of radioactivity was recovered with the cells, but the reaction did not show any time dependency so that it could be ascribed to binding of the polymers to the cell surface or to the uptake of contaminating oligomers, or both. Indeed, in the case of poly-rU, treatment of the cells with ribonuclease after exposure to radioactive poly-rU resulted in al- most complete loss of the radioactivity which was bound to the cells. Similar results were obtained in the case of hybrids between one ribopolynu- cleotide and one deoxyribopolynucleotide such as poly-rAdT or poly-rU:DNA. On the contrary, poly-dAdT and poly-dCdG appear to be taken up significantly with kinetics which are time depend- ent (Fig. 1). Attempts were made to alter cell permeability by pretreating the cells with 5% dimethylsulfoxide for 60 min at 37C or 10-2? M ethylenediamine- tetraacetic acid (EDTA), or by incubating cells with lysozyme (10 ug/ml). No significant uptake was observed under such conditions, nor in experiments in which protein synthesis was in- hibited by pretreating the cells with puromycin or chloramphenicol nor when polysome de- polymerization was favored by depriving cells of magnesium or by treating with sodium fluoride. The same type of experiment employing radio- active poly-rU and poly-rA was performed with equally unsuccessful results on the following bacteria: E. coli, Aerobacter aerogenes, Pseudo- monas aeruginosa, Klebsiella pneumoniae, Staphy- loceccus aureus, Proteus vulgaris, Alcaligenes foecalis, and Corynebacterium xerose. Stimulation of amino acid incorporation. Not- withstanding the negative results reported in the previous section, it could be argued that some of the tested polynucleotides were taken up by the cells toa very small extent. This could be tested by using auxotrophic cells of B. subtilis in which pro- tein synthesis is blocked by deprivation of the required amino acid. Under such conditions, the incorporation of amino acids into acid-insoluble SYNTHETIC POLYNUCLEOTIDES AND 8. SUBTILIS 685 0 i 1 1 9 15 30 45 TIME ( MIN.) Fic. 1. Uptake of synthetic deoxyribonuctleotides by competent cells of B. subtilis. °H-poly-dAdT or*H-poly- aCdG were added at a concentration of 0.1 ug per ml per 8.5 X 10) cells to a cell suspension containing: @, poly- dAT; O, poly-dAdT plus B. subtilis DNA (1:1); O, poly-dAdT plus adenine plus thymine (each base at a con- centration of 20 pg/ml); |, poly-dCdG; }, poly-d€dG plus B. subtilis DNA (1:1). Transforming DNA from wild-type B. subtilis was used at a concentration of 0.1 ug/ml, A 3.5-ml amount of competent cells was added to the mixtures of polynucleotides. At the shown time intervals, 0.4-ml samples were filtered on membrane filters and extensively washed with cold Spizizen medium. The filters were then dried, and the radioac- tivity was determined, material might be stimulated specifically by the synthetic polynucleotides acting as messenger RNA. In the case of poly-rU, for instance, stimulation of phenylalanine incorporation should be evident, whereas stimulation of lysine incorporation should be brought about by the addition of poly-rA. The incorporation of both amino acids should be stimulated when poly- rUrA is employed. An obvious control is the study of the incorporation of a different amino acid which is not coded for by the synthetic polynucleotides employed, e.g., arginine, whose codons are rich in cytidine and guanosine and poor in uridine and adenosine. Addition of poly-rU resulted in stimulation of 686 the incorporation of phenylalanine in trichloro- acetic acid-insoluble material as compared to the control containing no polynucleotides (Fig. 2). However, the incorporation of lysine was also stimulated. The same results were obtained for the incorporation of phenylalanine and arginine in the experiments employing poly-rArU, whereas the incorporation of either lysine or phenylalanine was not stimulated by poly-rA. Such results indicate that the addition of poly-rU brings about a nonspecific stimulation of amino acid incorpo- ration. This could be due to the presence of small amounts of oligomers or mononucleotides present in the polynucleotide preparations or, alternatively, generated upon incubation with the cells. This is substantiated by experiments not reported here, showing that sheared polynucleo- tides stimulate the incorporation of amino acids much more than intact polynucleotides. [Firshein et al. (3-5) have extensively investigated the stimulation of endogenous synthesis in Pneumo- coccus by DNA and RNA digests as well as by synthetic polynucleotides. ] In the case of the hybrid poly-rAdT, stimula- (o) (b) sof Time (Met) Fic. 2. Stimulation of “C-amino acid incorporation in the presence of synthetic polyribonucleotides. (a) Solid lines, 4C-phenylalanine U5 pCi/ml); dashed fines, !C-lysine (1.5 wCi/ml) were added to competent cells previously washed and suspended in Spizizen mini- mal medium (10) containing 0.5% glucose. The tri- chloroacetic acid-insoluble counts were determined on 0.2-ml samples of cell suspension withdrawn at the time intervals indicated. OQ, No addition; @, plus 100 pg of poly-rA per ml; 1, plus 100 pg of poly-rU per ml. (6) Dashed lines, }*C-phenylalanine (0.25 pCi/ml) as in (a) in the presence (qg) or in the absence (QC) of 100 pCi of poly-rUrA per ml; solid line, *C-arginine (0.25 wCi/ml) in the presence (©) or in the absence (@) of 100 yg of poly-rUrA per ml. CIFERRI, BARLATI, AND LEDERBERG J. BACTERIOL. tion of amino acid incorporation was observed, but again not of the amino acids that could be coded for by either poly-rA or poly-dT. Similar results were also obtained in the case of the hybrid formed between single-stranded DNA and poly-rU. Such results further argue against the hypothe- sis that the tested polynucleotides are taken up to a significant extent by the cells or that, if taken up, they are utilized as specific messenger RNA for protein synthesis. The stimulation of the incorporation of amino acids brought about by the tested polynucleotides should then be ascribed to an aspecific stimulation of protein synthesis caused by some of these polynucleo- tides or by the products of their degradation. In the case of poly-dAdT, a polynucleotide that is taken up by competent cells of B. subrilis, no stimulation was observed in the incorporation of tyrosine (one codon of this amino acid is UAU). In this case, however, it may be postulated that transcription of the synthetic deoxypolynu- cleotide did not take place. Competition between DNA and synthetic poly- nucleotides. A further control on the possible up- take of the synthetic polynucleotides was made by studying the competition between these polymers and transforming DNA. If synthetic polynucleotides are taken up by the same mecha- nism by which DNA is incorporated during transformation, competition should be observed between natural and synthetic polynucleotides. Likewise, competition between synthetic poly- nucleotides and DNA should be observed if the binding of synthetic polynucleotides to cells takes place at the same sites through which DNA enters the cells. Addition of poly-rU, poly-rA, and poly-rUrA at the same time as DNA does not decrease transformation, even when the synthetic polynu- cleotides are employed at a concentration 100- fold higher than that of the transforming DNA (Table 1). Addition of poly-rUrA to the suspen- sion of competent cells before the addition of DNA has little, if any, effect on transformation (Table 2). Indeed, a 1,000-fold excess of hybrid, even when added 10 min before DNA, does not significantly decrease transformation. It may be added that no competition with DNA for trans- formation was observed by employing the follow- ing polynucleotides at concentrations that were, in certain experiments, 1,000-fold higher than that of DNA:poly-rA, poly-rU, poly-rArU, poly-rC, poly-rI, poly-rCrI, poly-rAdT, poly- dCdG, poly-dAdT, soluble RNA, and ribosomal RNA. On the contrary, addition of different concentrations of heterologous DNA (from calf thymus) reduces stoichiometrically the percentage VoL. 104, 1970 TABLE 1. Effect of the addition of synthetic polyribonucleotides on transformation® Transformation (%) Polyribo- nucleotide concn (ug/ml) poly-rU | poly-rA poly-rUrA 0 4.0 4.0 4.0 1 4.2 4.5 3.6 10 4.0 4.1 3.9 100 3.9 4.0 4.9 * Competent cells were prepared. DNA from wild-type B. subtilis was added at a concentration of 1 ug/ml, and the cells incubated at 37 C for 20 min in a rotary shaker. The reaction was stopped by addition of 10 ug of deoxyribonuclease per ml followed by incubation for 10 min at 37 C. Trans- formation was started by adding the cells to the mixture containing the nucleic acids. TABLE 2. Effect of the time of addition of poly-rUrA on transformation* Transformation (%»): poly-rUrA added at poly-rUrA concn (ug/ml) —10 min? 0 min +10 min 0 2.28 1,25 1.12 10 2.50 0.93 0.74 100 2.05 i 1.22) | 0.67 500 1.84 0.94 0.87 1,000 2.15 | 0.92 | 0.92 * Experimental conditions reported in Table 1. > Respect to the time of addition of DNA. of transformants. It remains to be explained why poly-dCdG and poly-dAdT which are taken up by competent cells (Fig. 1) do not compete with DNA for transformation (Fig. 3). The phenome- non may perhaps be explained by the presence of different sites of entrance for the deoxypolyribo- nucleotides and for DNA or by a difference in the uptake of these polynucleotides by competent and noncompetent cells. Against this last hy- pothesis is the finding that the uptake of poly- dAdT and poly-dCdG is greater when the compe- tence is higher, indicating that only the competent cells are responsible for their uptake. These observations suggest that natural DNA competes at a level other than the uptake step that is possi- bly the same for the synthetic deoxyribopolynu- cleotides. The higher molecular weight of the natural DNA (approximately 100 times) may be one of the discriminating factors. DISCUSSION The data reported indicate that synthetic deoxyribopolynucleotides are taken up by compe- SYNTHETIC POLYNUCLEOTIDES AND B. SUBTILIS 687 {a} (b) ma. 6 TRANSFORMANTS / 1 4 1 1 1 1 ° cs zo 30 9 0 zo 30 TIME | ( MiN.) Fic, 3. Effect of the addition of poly-dCdG and poly-dAT on transformation. Experimental conditions as in Table I except that the concentration of transform- ing DNA was 0.1 pg/ml of cell suspension. At the shown time intervals, 0.1 ml of cell suspension was Dipetted into 0.4 ml of Spizizen medium containing 107 Mgt and 5 wg of deoxyribonuclease per ml, and incubated for 10 min at 37 C. Tryptophan transformants were scored. (a2) @, DNA at 0.1 ug/ml; O, 0.1 ng of DNA plus 0.1 ug of poly-dAdT per ml; QO, 0.1 ug of DNA plus 1 yg of poly-dAdT per ml. (b) Same as (a) except that poly-dCdG was used in place of poly-dAdT. 10% tent cells of B. subtilis possibly with the same mechanism by which natural DNA is taken up by these cells. This is not the case for synthetic ribopolynucleotides of either single- or double- stranded structure. Such findings indicate the existence of a highly selective uptake system in competent cells of B. subtilis. The mechanism for the uptake of deoxyribonucleotides appears to be so selective that a 1,000-fold excess of poly- ribonucleotides does not interfere with the transformation process. Although the polyribonucleotides are not significantly taken up by the cells, it has been found that most of the polymers tested, especially poly-rU, stimulate amino acid incorporation. However, this is not a specific effect since no cor- relation exists between the codon present in the polyribonucleotide used and the amino acids that are incorporated. This result is in contrast with the data reported by Amos et al. (1) for E. coli soniplasts in which poly-rU appears to stimulate 688 CIFERRI, BARLATI, specifically the incorporation of phenylalanine. However, they did not deal with the uptake of poly-rU nor of any other synthetic or natural polynucleotides such as DNA. ACKNOWLEDGMENTS This work was supported by Public Health Service research grant AI-05160 from the National Institutes of Allergy and Infec- tious Diseases, and by the cooperative U.S.-Italy Science Program (GB 7785 of the National Science Foundation and 115 0308 04633 of the Consiglio Nazionale delle Ricerche). LITERATURE CITED 1. Amos, H., A. Kuhn, and J. Andre-Schwartz. 1967. Protein synthesis in sonically damaged Escherichia coli. J. Bacteriol. 94:232-240. 2. Evans, A. H. 1964. Introduction of specific drug resistance properties by purified RNA-containing fractions from Pneu- mococcus. Proc. Nat. Acad. Sci. U.S.A. 52:1442-1449. 3. Firshein, W. 1961. Effects of deoxyribonucleic acid products on deoxyribonucleic acid synthesis of virulent and avirulent pneumococci. J. Bacteriol. 82:169-180. 4. Firshein, W. 1965. Influence of deoxyribonucleic acid degrada- AND LEDERBERG J. BACTERIOL. tion products and orthophosphate on deoxyribonucleotide kinase activity and deoxyribonucleic acid synthesis in pneu- mococcus type III. J. Bacteriol. 90:327-336, . Firshein, W., R. C. Benson, and M. Sease. 1967. Deoxycytidy- late and deoxyguanylate kinase activity in pneumococci after exposure to known polyribonucleotides. Science 157: 821-822. . Hurwitz, J., A. Evans, C. Babinet, and A. Skalka. 1963. Syn- thesis and structure of macromolecules. Cold Spring Harbor Symp. Quant. Biol. 28:59-65. . Kirtikar, M. W., and J. D. Duerksen. 1968. A penicillinase- specific ribonucleic acid component from Bacillus cereus. 1. Ribonucleic acid extraction and definition of the in vivo test system. Biochemistry 7:1172~1182. . Kubinski, H., Z. Opara-Kubinska, and W. Szybalski. 1966. Patterns of interactions between polyribonucleotides and individuat DNA strands derived from several vertebrates, bacteria and bacteriophages. J. Mol. Biol. 20:313-329. . Marmur, J. 1961. A procedure for the isolation of deoxyribo- nucleic acid from microorganisms. J. Mol. Biol. 3:208-218. . Spizizen, J. 1958. Transformation of biochemically deficient strain of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 44:1072-1078. . Stewart, C. R. 1969. Physical heterogeneity among Bacillus subtilis deoxyribonucleic acid molecules carrying particular genetic markers. J. Bacteriol. 98:1239-1247.