Transformation in Streptococcus pneumoniae

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1 JOURNAL OF BACTERIOLOGY, Nov. 1983, p /83/ $02.00/0 Copyright , American Society for Microbiology Vol. 156, No. 2 Fate of DNA in Eclipse Complex During Genetic Transformation in Streptococcus pneumoniae MOSES N. VIJAYAKUMARt AND DONALD A. MORRISON* Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois Received 6 June 1983/Accepted 23 August 1983 Uptake of DNA and genetic recombination proceeded normally in competent Streptococcus pneumoniae despite inhibition of DNA replication by 6-(p-hydroxyphenylazo)-uracil. Immediately after a brief uptake period, 68% of donor DNA label was in eclipse complex form, and 22% was in low-molecular-weight products; by the completion of integration at 10 min, 23% was integrated into the chromosome, and the rest was lost from the cell. Throughout the process, less than 1% was found as free single strands. The DNA in eclipse complex is therefore an intermediate in the integration process. In genetic transformation in pneumococcus (Streptococcus pneumoniae), donor DNA is converted to single strands (9) by a membranebound nuclease (8) during uptake by competent cells. This causes an eclipse of the biological activity of the donor strands after uptake (4), as the transforming activity of single strands is much lower than that of native DNA. Further processing results in the integration of donor strands into the chromosome and in restoration of donor marker transforming activity (recovery from eclipse) (5-7, 17). After uptake, part of the transforming DNA is not in single strands of high molecular weight but is in small fragments that enter metabolic pools and are incorporated into the chromosome during replication (9). We have demonstrated that donor strands in eclipse associate with cellular components to form a structure we termed the eclipse complex (10, 11), in which they are protected from nucleases (16). The complex, which was first detected because it has a lower affinity for hydroxylapatite than does single-stranded DNA (10), contains at least one protein component (11, 14). The major protein component of this complex (molecular weight, 19,500) is synthesized specifically at competence (12, 13) and is thought to have roles in transport, protection against nucleases, or promotion of recombination. However, an understanding of the function of the eclipse complex protein should rest first on evidence that the donor DNA in the complex is a precursor of the integration reaction. In previous studies of eclipse complex, the immediate product of a brief period of uptake at t Present address: Biochemistry Department, Duke University, Durham, NC C has been examined. We know that at this temperature integration is slow and exhibits a clear lag phase, whereas at 37 C, it is much faster (17). Thus, it is possible that an important jntermediate forms from eclipse complex only luring integration at 37 C. Furthermore, although the majority of donor label in eclipse appeared to be in the complexed form in earlier experiments (10, 16), there was no direct measure of the actual amount of DNA genetically integrated in those experiments. Thus, it has remained possible that eclipse complex was a dead-end state and that a minority of free single strands represented the only actual precursors to genetic integration. We sought to determine the fate of DNA in eclipse complex by measuring the amount initially present in the cells, the amount of donor material finally integrated into the chromosome, and the amount of noncomplexed single-stranded DNA present before or during integration, if any. Other direct measures of the fraction of donor DNA taken up that is genetically integrated in pneumococcus have ranged from 20 to 40% (5, 7). To determine the amount of donor DNA genetically integrated, we specifically blocked the replicative route of incorporation and measured the remainder. To block DNA synthesis, we used the drug 6-(p-hydroxyphenylazo)-uracil (HPUra), which selectively and reversibly inhibits the action of DNA polymerase III in grampositive bacteria (1). Both in Bacillus subtilis (3) and in pneumococcus strain R6 (18), it has been reported that HPUra is effective in the arrest of DNA synthesis without appreciably altering the yield of transformants. We quantitatively examined the fate of the donor DNA in transforming cells during integra- 644

2 VOL. 156, 1983 tion in the absence of DNA replication. We present evidence that eclipse complex is an intermediate stage in the pathway leading to recombination. MATERIALS AND METHODS Bacterial strains and media. The bacterial strains used for preparation of competent cells (CP1015, Strr Erys) and isolation of donor DNA (CP1014, Strs Eryr Thy-), the culture media, and the materials used were as described previously (10). HPUra was a gift from Imperial Chemical Industries, Ltd., London. A 20 mm stock solution prepared by dissolving HPUra in 0.05 M NaOH was stored at 4 C. Before use, it was diluted to appropriate concentrations with the growth medium. DNA preparation. For the isolation of donor [3H]DNA, CP1014 was grown in broth with [3H]thymidine (20,uCi/ml) to about 4 x 108 CFU/ml, washed, and lysed in buffer containing 200,ug/ml of RNase, 0.2% Triton X-100, and 0.2% Sarkosyl (10 min, 37'C). After pronase digestion, phenol extraction, and dialysis overnight against 0.05 M Tris (ph 6.8)-0.05 M NaCI M EDTA buffer at 0'C, the DNA preparation was directly used for transformation experiments. The specific activity of the DNA preparation was 5 x 10' cpm/,ug after precipitation on filter paper disks. DNA was sheared in 2-ml volumes at 0 C with the microtip of a sonifier (model W185D of heat systems; Ultrasonics, Inc., Plainview, N.Y.), for a total of 75 s. Determination of the effect of HPUra on transformation and cell viability. Competent cells were exposed to donor [3H]DNA (0.1,ug/ml) in the presence of HPUra at 25'C for 5 min. After treatment with pancreatic DNase I (100,ug/ml, 30 s), the culture was chilled, washed three times with broth, suspended in fresh broth, and divided in half, one-half receiving HPUra at 40,uM. During further incubation at 37 C, samples were withdrawn at the indicated times. Part of each was scored for CFU and transformants; the rest was washed three times in SSC (0.15 M NaCI, M sodium citrate, ph 7) and lysed as described below. The radioactivity in the supernatants and the lysates was determined. Transformation. Competent cells were prepared, concentrated, and stored at -80 C as described previously (10). For experimental use, frozen tubes were thawed at 0 C, incubated at 37 C until 1.5 min before the previously determined time of competence, and then cooled to 25'C. After 1 min, [3H]DNA and HPUra were added simultaneously. After 5 min, uptake was terminated by pancreatic DNase I (100 p.g/ml, 30 s at 25'C); then the culture was quickly chilled and washed three times with broth by centrifugation in the cold. The washed cells were resuspended in broth containing 10% glycerol-40 pm HPUra, frozen, and stored at -80 C. For studies of integration, cultures were thawed at 0 C and incubated at 37 C. Samples were chilled at various times as indicated. A portion of each was scored for transformants and viable cells; the rest was washed twice in SSC, resuspended in a cold lysis buffer (0.03 M EDTA, 0.05 M Tris, [ph 8.0], 0.5% Sarkosyl, 0.01 M NaCI, 0.1% Triton X-100, and RNase [100 p.g/ml]), and incubated at 370C for 5 min. The lysate was frozen with 10%o glycerol and stored at -80 C. For chromatographic analysis, the lysate was thawed at 0 C. INTEGRATION OF ECLIPSE COMPLEX DNA 645 Competence and erythromycin-resistant transformants were assayed as described earlier (10). Column chromatography. Columns of 2% agarose gel beads (Bio-Gel A-50m, (1-cm diameter, 24-ml bed volume; Bio-Rad Laboratories, Richmond, Calif.) were equilibrated with the running buffer (0.1 M NaCI, 0.01 M Tris-hydrochloride [ph 8.0], M EDTA, and 0.05% Sarkosyl) and calibrated for excluded and included volumes with DNA and HPUra, respectively. Samples were applied in 0.2- to 0.4-ml volumes made 5% in glucose. Fractions of 1 ml were collected at a flow rate of 0.30 ml/min. One-fifth or one-half of each fraction was taken for the determination of radioactivity Ḣydroxylapatite chromatography and determination of radioactivity were done as described previously (10, 15), except for addition of 0.05% Sarkosyl to all column buffers. RESULTS The effect of HPUra on DNA synthesis, cell viability, and genetic transformation. The effect of HPUra on the incorporation of [3H]thymidine was tested in the Rx strain of pneumococcus under the conditions employed in transformation experiments (data not shown). Above 80,uM HPUra, DNA synthesis was less than 5% of normal. Cell viability depended both on the concentration of the drug and the length of exposure to the drug; the maximum concentration that did not reduce cell viability within one doubling time was 40 pum. At this level of HPUra, thymidine incorporation was reduced by 90%. Furthermore, the direct measure of recycling incorporation from sonicated transforming DNA described below showed a nearly complete inhibition of reincorporation at 40,uM drug concentration. DNA uptake at 25 C was not altered by this level of drug, and incubation of cells in drug at 37 C for 20 min after DNA uptake did not reduce the yield of transformants or the viability of the culture (data not shown). However, HPUratreated cells cohtinuously released donor label into the medium; 52% of the radioactivity was released by 20 min, compared with 8% in the control. The unreduced yields of transformants indicates that the loss of label from the HPUratreated cells was not due to inhibition of transformation or to cell lysis. Clearly, the portion of donor DNA retained by the cells contained all the transforming molecules. The nature of donor products after uptake. To identify the types of donor molecules present immediately after uptake, we followed a protocol developed earlier (15, 17) in which a brief period of uptake at 25 C provides a substantial amount of donor label in eclipse without much progress of the integration reaction. Lysates were analyzed directly by chromatography on columns of 2% agarose and hydroxylapatite. The size fractionation afforded by agarose gel

3 646 VIJAYAKUMAR AND MORRISON exclusion chromatography (Fig. la) produced a broad peak (I) of fully and partially excluded molecules, reflecting the strand nicking expected as a consequence of uptake (15), and a second peak (II, fractions 15 to 23), containing largely acid-soluble products (data not shown). Hydroxylapatite chromatography separates eclipse complex, single-stranded DNA, and double-stranded DNA, which elute at approximately 0.12, 0.18, and 0.24 M phosphate concentrations, respectively (10). Chromatography of the lysate on hydroxylapatite (Fig. lb) produced two major overlapping labeled peaks; one eluted below 0.1 M phosphate, the other eluted at 0.12 M. There was also a small amount of incorporation into double-stranded DNA. Single strands (control column, not shown) eluted at a phosphate concentration of 0.18 M. The trailing side of the peak centered at 0.12 M extended to the 0.19 M phosphate fractions. Rechromatography of the trailing label eluting near 0.18 M showed that at least 90o eluted at 0.12 M phosphate. Thus, hydroxylapatite chromatography showed that immediately after uptake, less than 0.3% of the label of donor DNA origin was present in the form of free single strands. Pooled fractions of agarose peak I were also fractionated on hydroxylapatite (Fig. lb). They contained the species which eluted at 0.12 M, but not those eluting below 0.1 M, implying that the larger molecules were in eclipse complex and that peak II from agarose was composed of molecules which were eluted at 0.07 M on hydroxylapatite. The small segments of eclipse complex produced by nuclease digestion are also known to elute at this position (16). Thus, this fractionation sequence revealed the existence of two principal species of donor molecules immediately after uptake (in addition to some incorporation into the double-stranded host DNA): larger, acid-precipitable DNA in eclipse complex and small, acid-soluble material eluting below 0.1 M phosphate. Transformation with sonicated DNA. A control experiment was done with donor DNA inactivated (-0.1% residual transforming activity) by sonication (data not shown) (2). This DNA proved to be a rich source of the products eluting from hydroxylapatite at 0.07 M phosphate). In the absence of HPUra, this material quantitatively entered the chromosome; in HPUra, most was released from the cell, and none (<0.1%) became double-stranded DNA. Thus, at least for sheared donor DNA, the molecules eluting at 0.07 M do not participate in genetic recombination. This experiment showed not only that small donor DNA fragments are recycled, but also that the HPUra suppression of recycling of donor DNA via replication was considerably B. IL B. UL A I J. BACTERIOL FRACTION NUMBER FIG. 1. Chromatography of donor DNA products immediately after uptake. Cells transformed at 25C in the presence of HPUra were washed and lysed without further incubation. (A) Agarose chromatography. The arrow indicates the elution position of HPUra. Fractions 6 to 15 (peak I) were pooled for rechromatography on hydroxylapatite. (B) Hydroxylapatite chromatography. Symbols: 0, total lysate and *, pooled fractions from agarose peak I. The columns were run in parallel. The bars indicate the centers of the areas categorized in Table 1: ds, native DNA; ss, singlestranded DNA; and ec, eclipse complex. The acidsoluble material is divided into two components, early and late eluting. stronger than its degree of suppression of exogenous thymidine incorporation. The fate of products derived from high-molecular-weight donor DNA. To inquire whether any substantial part of the donor DNA in eclipse complex is converted to a free single-stranded form at some stage before integration, we monitored the state of transforming molecules during incubation at 37 C for the 10 min required for genetic integration of most donor molecules into the resident chromosome (17). Comptent cells were transformed as above with [ H]DNA of high molecular weight in HPUra. Samples were withdrawn after 0, 2, 5, and 10 min at 37 C, lysed, and analyzed on agarose and hydroxylapatite columns (Table 1). Molecular species of donor origin were as described above (Fig. 1) at 0 min. During incubation at 37 C, the larger molecules of the eclipse v.

4 VOL. 156, 1983 Min at 37 C TABLE 1. INTEGRATION OF ECLIPSE COMPLEX DNA 647 DNA processing and integration in HPUraa Culture Agarose Hydroxylapatite fractionation" fractionation fractionationbhrolatiefctoain Extra- Intra- Peak I Peak II <0.07 M M M M M a The 3H present in transformed cells after uptake, DNase treatment, and washing is taken as 100% in this table. Release from cells and distributions among chromatographic fractions of lysates of washed cells were measured after indicated times at 37 C after uptake. b The first peak (fractions 5 to 13) in agarose fractionation contained acid-precipitable material, the second (fractions 14 to 19) contained acid-soluble material. I Hydroxylapatite fractions were summed in accord with the phosphate gradient elution positions of eclipse complex, single-stranded DNA, double-stranded DNA, and other low-molecular-weight material, as indicated in Fig. 1. complex, eluting at 0.12 M (10), decreased in amount, with a simultaneous appearance of label in native DNA. Hydroxylapatite chromatography did not reveal a distinct population of free single strands at any time during the entire process. At the end of 10 min, about 23% of the total donor radioactivity originally taken up was found in the double-stranded host DNA, representing products of recombination. Thus, at zero time, less than 0.3% of the donor intracellular label behaved as free single strands, 68% was in the form of eclipse complex, and 7% was already in the chromosome. By 10 min, an additional 16% of that label had been integrated into the chromosome, without apparent formation of free single strands. DISCUSSION Two major classes of immediate products of uptake of donor DNA were identified by fractionation according to size and to hydroxylapatite affinity: DNA strands in eclipse complex (68%) and small fragments (22%); less than 1% was found as free single-stranded DNA. To determine the amount of DNA that was genetically integrated, we allowed recombination to proceed at 37 C but blocked replicative incorporation by inhibiting DNA synthesis with HPUra. Under conditions of incubation in which incorporation of thymidine was 90% inhibited and replicative incorporation of products from sheared DNA was inhibited by >99%, genetic integration was shown to be unaffected. In these replication-block conditions, small DNA uptake products were lost from the cells, whereas large DNA was integrated in an amount equal to about 25% of that initially present in the form of eclipse complex. We conclude that the eclipse complex pool provided the reservoir of donor DNA from which genetic integration occurred. This yield of genetically integrated DNA, about 20% of the label taken up, is not far from that reported previously for pneumococcus by Fox (5) and by Lacks et al. (7), reinforcing the conclusion that the DNA incorporated in the presence of HPUra is the genetically important part of the donor DNA after uptake. It was shown previously (15) that most of the products of uptake of small DNA were acid soluble. We show here that many of these bind to hydroxylapatite and elute at 0.07 M P04. This material is blocked from incorporation into the chromosome by HPUra and is then lost from the cell. This implies that unless the resolution of hydroxylapatite columns is good, donor DNA label binding to those columns and eluting before single-stranded DNA is partly eclipse complex and partly very small fragments. We reported earlier that much of the donor DNA label in eclipse showed a unique affinity for hydroxylapatite, distinct from that of singlestranded DNA. We now show that this unique form serves as precursor for genetic integration and persists while integration proceeds at 37 C. Furthermore, most of the small trail of donor label eluting from hydroxylapatite at the position of single-stranded DNA is also eclipse complex. Thus, there is not yet a clear demonstration of any free single strands of donor DNA at all during eclipse. Our earlier observations that virtually all DNA in eclipse was protected from nucleases after extraction is consistent with these results (16). Since donor DNA strands are first incorporated into eclipse complex on uptake, and then integrated into the chromosome and since DNA in eclipse complex is protected from pneumococcal nucleases, the apparent absence of free single strands during eclipse immediately suggests the hypothesis that eclipse complex is the form in which donor DNA encounters resident chromosomes. If this is true, the functions of the

5 648 VIJAYAKUMAR AND MORRISON protein component of eclipse complex should be important for describing the details of that recombination reaction. ACKNOWLEDGMENTS This work was supported by grant PCM from the Genetic Biology Program of the National Science Foundation. J. BACTERIOL. LITERATURE CITED 1. Brown, N (p-hydroxyphenylazo)-uracil: a selective inhibitor of host DNA replication in phage-infected Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 67: Cato, A., and W. Guild Transformation and DNA size. I. Activity offragments of a defined size and a fit to a random double crossover model. J. Mol. Biol. 37: Dubnau, D., and C. Cirigliano Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: nonrequirement of deoxyribonucleic acid replication for uptake and integration of transforming deoxyribonucleic acid. J. Bacteriol. 113: Fox, M. S Fate of transforming deoxyribonucleate following fixation by transformable bacteria. Nature (London) 187: Fox, M. S., and M. K. Allen On the mechanism of deoxyribonucleate integration in pneumococcal transformation. Proc. Natl. Acad. Sci. U.S.A. 52: Gurney, T., Jr., and M. S. Fox. i968. Physical and genetic hybrids formed in bacterial transformation. J. Mol. Biol. 32: Lacks, S., B. Greenberg, and K. Carlson Fate of donor DNA in pneumococcal transformation. J. Mol. Biol. 29: Lacks, S., and M. Neuberger Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae. J. Bacteriol. 124: Lacks, S. A Molecular fate of DNA in genetic transformation of pneumococcus. J. Mol. Biol. 5: Morrison, D. A Transformation in pneumococcus: existence and properties of a complex involving donor deoxyribonucleate single strands in eclipse. J. Bacteriol. 132: Morrison, D. A Transformation in pneumococcus: protein content of eclipse complex. J. Bacteriol. 136: Morrison, D. A Competence-specific protein synthesis in Streptococcus pneumoniae, p In M. Polsinelli and G. Mazza (ed.), Transformation Cotswold Press, Oxford. 13. Morrison, D. A., and M. Baker Competence for genetic transformation in pneumococcus depends on the synthesis of a small set of proteins. Nature (London) 282: Morrison, D. A., M. Baker, and B. M. Mannarelli A protein component of the pneumococcal eclipse complex, p In S. W. Glover and L. 0. Butler (ed.), Transformation Cotswold Press, Oxford. 15. Morrison, D. A., and W. R. Guild Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor. J. Bacteriol. 112: Morrison, D. A., and B. Mannarelli Transformation in pneumococcus: nuclease resistance of deoxyribonucleic acid in the eclipse complex. J. Bacteriol. 140: Shoemaker, N. B., and W. R. Guild Kinetics of integration of transforming DNA in pneumococcus. Proc. Natl. Acad. Sci. U.S.A. 69: Tiraby, J. G., and M. S. Fox Marker effects in pneumococcal transformation, p In R. F. Grell (ed.), Mechanisms of recombination. Plenum Publishing Corp., New York.