J. Mol. Biol. (1971) 56, 209-221

Fate of Transforming DNA following uptake by Competent Bacillus subtilis

I. Formation and Properties of the Donor-Recipient Complex

DAVID DUBNAUAND ROSADAVIDOFF-ABELSON

Department of Microbiobgy, The Public Health Research Institute of the City of New York, Inc., N. Y. 10016, U.S.A.

(Received 4 August 1970, and in revised form 1 December 1970)

Following uptake by competent Bacillm &t&s, transforming DNA is con- verted to two distinct slowly sedimenting molecular forms which possess little transforming activity (eclipse). A few minutes after uptake is initiated, a physical complex of donor and recipient DNA begins to form. The recovery of donor transforming activity following eclipse, and the appearance of recombinant act,ivity, previously reported by Venema, Pritchard & Venema-SchrGder (1965), is shown to be due to changes occurring in the donor-recipient complex. This complex exists transiently in a form with low recombinant-type transforming activity. This transient form may be one in which the donor and recipient com- ponents are joined non-covalently. The donor-recipient complex is shown to be a heteroduplex structure in which the donor moiety has an approximate molecular weight of 750,000.

1. Introduction The integration of genetic material is an important biological process which is still poorly understood in molecular terms. Bacterial transformation presents certain advantages for study, since it involves the use of purified donor DNA which can be characterized and modified in various ways, and since the properties of DNA extracted from recipient cells during transformation can be readily investigated using a variety of physical, chemical and biological tools (Ayad & Barker, 1969; Bodmer, 1966; Bodmer & Ganesan, 1964; Bodmer & Laird, 1968; Fox, 1966; Fox & Hotchkiss, 1960; Harris & Barr, 1969; Lacks, Greenberg & Carlson, 1967; Notani & Goodgal, 1966; Pene & Romig, 1964; Venema, Pritchard & Venema-Schrijder, 1965). Bacterial transformation involves several processes preceding the integration step, such as the uptake and conversion of donor DNA to a form suitable for interaction with recipient DNA. These steps must be understood before a full description of genetic integra- tion is attained, since the molecular properties of the donor moiety are obviously of crucial importance. This paper is the 6rst in a series which will examine the fate of transforming DNA following uptake by competent cells of Bacillus subtilis; both wild type and mutationally altered with respect to transformation. The present paper describes the properties of the donor-recipient complex formed during transformation. This complex appears to be a heteroduplex structure, in which a recipient strand pairs with a donor strand. The molecular weight of the donor moiety appears to be about 750,000. Evidence is presented which indicatea that a transient form of the

14 209

210 D. DUBNAU AND R. DAVIDOFF-ABELSON

donor-recipient complex exists which possesses low transforming activity for the donor and recombinant marker configurations. It is proposed that in this transient form the donor and recipient moieties are associated via non-covalent bonds.

2. Materials and Methods (a) Bacillus subtilis &rains

All B. subtiZ& strains used were 168 derivatives. Strain BD170 (thr-5 &JLZ) was used as the recipient strain for transformation. Strain BD204 @i&Z thy) was the donor strain. The hkB2 and trp-2 markers are 55 to 60% linked by transformation. Strain BD55 (hisB2 &p-2), known also as SB25, was used as a transformation recipient to assay the biological activity of DNA fractions isolated from transformed cells. The ancestry of these markers is described by Dubnau, Goldthwaite, Smith t Marmur (1967).

(b) Media Spizizen salts were used in most media (Anagnostopoulos & Spizizen, 1961). SPI medium

contained Spizizen salts, O*O2o/o Casamino acids, 0.1% Difco yeast extract, 0.5% glucose and 60 &nl. of each required ammo acid. SPII medium contained in addition 5 x 10m4 M-CaCl,, and 2.5 x 10e3 M-MgCI, in addition to that contained in the Spizizen salts.

The deuterium medium used to prepare donor DNA was similar to that of Crespi, Marmur & Katz (1962). It was mod&d by the addition of 0.01 M-Tris adjusted to pD 7.3 by the use of KOD and by the addition of MgSO, to 5 x 10m4 M and MnCls to 10e6 M. The deuterated algal extract was replaced by a deuterated amino acid mixture obtained from Merck (Montreal). (The deuterated sugars used were also obtained from Merck.)

(c) Tramformation

Recipients were made competent by a modification of the method of Anagnostopoulos bi Spizizen (1961). The strains to be made competent were maintained as washed spore suspensions in water. A loop full was used to inoculate a small volume of SPI medium which was then shaken at 30°C overnight. The following morning, fresh SPI medium was inoculated to a Klett calorimeter reading of 10 to 15 (a no. 62 filter was used). The culture was shaken vigorously at 37’C, and readings taken periodically using the Klett calorimeter. When the rate of growth measured in this way began to depart from exponentiality, the cells were diluted tenfold into SPII medium. This culture was shaken slowly at 37°C for 90 mm, at which time competence was close to maximum. The cells were pelleted by centri- fug&ion in the cold, and resuspended in 0.1 vol. of the same culture supernatant, to which 10% glycerol (v/v) had been added. The resuspended culture was distributed in convenient volumes and quickly frozen in a dry ice-ethanol bath. The samples were stored in a Revco freezer at -60°C. To use, the competent cells were thawed rapidly by shaking iu a 45°C water bath. Transformation was carried out at 37°C. When BD55 was used to assay the biological activity of DNA fractions, the cells were incubated with DNA for 30 mm before plating. Transformants were selected by plating on suitable media as described previously (Dubnau et al., 1967).

(d) CaCl density-gradient centri&gation

Neutral CsCl density gradient centrifugation was carried out after adjusting the density of the sample-CsC1 mixture to a value midway between the densities of the DNA species present in the sample. Densities were determined by refractometry, using the relationship : p25”= 10*8601925’- 13.4974 (Vinograd & Hearst, 1962). The CsCl sample mixture con- tamed 10q3 as-Tris, pH 8.2, and 10e3 M-EDTA. Centrifugation was carried out at 28°C in nitrocellulose tubes, in a SW50.1 rotor, at 35,000 rev./mm for 70 hr.

Alkaline CsCl density gradient centrifugation was carried out in a similar manner. The initial pH of the CsCl sample mixture was adjusted to pH 13 with 1 N-NaCH and the den- sity adjusted in the manner described above. An increment of 0.062 g/cm” was allowed between the density of native DNA in neutral CsCl and that of denatured DNA at high pH (Vmogmd, Morris, Davidson & Dove, 1963). Centrifugation was carried out in polyallomer tubes using the conditions described above.

FATE OF TRANSFORMING DNA IN B. SUBTILIS 211

C&l gradients were collected by puncturing the bottom of the tubes and collecting drops. 3aP,nH-labeled B. BubtiZie BDBS DNA and 3aP-labeled light BDSS DNA were included in all centrifugation rims as internal heavy and light density standards. BD66 was used so as not to interfere with the determination of the biological activity of fractions from the gradients. These standards had buoyant densities of l-746 and 1.703 g/cm”, respectively.

(e) Suav86 gradient sedimentation analysis Centrifugation in neutral sucrose gradients was carried out using 6 to 20% linear

gradients containing I.0 M-NaCl. In some cases a cushion of 00% sucrose was placed at the bottom of the gradient to facilitate recovery of high sedimentation value species. Centrifu- gation was carried out in a SW60.1 rotor at 49,000 rev./min and 2O”C, or in a SW27 rotor at 22,000 rev./mm and 2O”C, for 17.6 hr. The times of centrifugation for the experiments performed in the SW60.1 rotor varied with the sedimentation value expected for a given sample. When desired, a sample of [14C]thymidine-labeled T7 coliphage DNA prepared after growth on an Emhwichia coli B thy- strain, wae included as a sedimentation standard. The molecular weights of native DNA were determined using the relationship of Burgi & Hershey (1963).

Alkaline sucrose gradients were carried out in the same way, except that the gradients contained 0.9 ar-NaCl and O-1 aa-NaOH. Single-strand molecular weights were determined using [14C]thymidine labeled coliphage T7 sedimentation standard and the relationship of Burgi & Hershey (1963) with the exponential constant of 0.4 determined by Studier (1965) for alkaline conditions. The double- and single-stranded molecular weights of T7 DNA were taken as 26 x lo6 and 12.6 x lo6 (Thomas, 1966).

(f) Preparation of DNA

BD204 was grown in deuterium medium containiug 60 pg L-histidine/ml., 2.4 pg thyminelml. and 76 PC [3H]thymine/ml. (New England Nuclear; spec. act. 14.4 c/m-mole). Growth was followed until early stationary phase, at which time the cells were harvested.

Donor DNA from BD204 (and from BD55 for use as density standards) was isolated as follows. Cells were centrifuged and washed once in 0.06 M-N&~ + 0.1 M-EDTA, pH 6.9. The cells were resuspended in the same solution to which O-6 mg lysozyme (Pentex)/ml. was added. When lysis was almost complete (10 to 12 min), sodium dodecyl sulfate was added to O-1 %. This resulted in a dramatic clearing of the suspension and an increase in viscosity.

Pronase (Calbiochem) was added to 0.5 mgjml. and the suspension incubated at 48°C for 2 hr. The suspension was then cooled and shaken gently until emulsiiied, with an equal volume of buffer-saturated, redistilled phenol, adjusted to pH 8.5 to 9.0. Following centri- fugation to separate the phases, the aqueous layer was removed. The DNA was then “spooled” on a glass rod after precipitation with 2 vol. ethanol, and redissolved in 0.015 M-NaCl + 0*0016 M-sodium citrate (Marmur, 1961). RNA was removed by the addition of 50 pg pancreatic ribonuclease/ml. + 5 units takadiastase T, (Worthington)/ml., followed by incubation at 37°C for 30 min. The phenol treatment and the ethanol precipitation were repeated. Finally, the DNA was dissolved in 0.015 M-N&~ + 0.0015 M-sodium citrate. If necessary, any residual phenol was removed by dialysis. The DNA was stored over CHC13. DNA concentrations were determined calorimetrically by the method of Burton (1956), as modified by Giles & Myers (1965).

The 3H,2H-labeled BD204 DNA so isolated had a specific activity of 1.30 x lo6 cts/min/ pg. The observed buoyant density of this material, determined by CsCl density-gradient centrifugation with internal 32P-labeled BD55 DNA and 32P,2H-labeled BD55 DNA standards, was 1.742 to 1.743. This 3H,2H-labeled BD204 DNA had a double-strand molecular weight of 9.0 x 10’ and a single-strand molecular weight of 847 x 106, deter- mined by sucrose gradient sedimentation as described above. This DNA preparation therefore had about 4 to 6 nicks per single strand.

(g) Design of experiment and preparation of lyaatea

BD170 competent cells (32 ml.) were preincubated for 3 mm at 37°C and 3H,2H-labeled BD204 DNA was then added to 1 rg/ml. After 6 min of incubation at 37°C an 8 ml. sample was taken and chilled rapidly in an ice slurry. After 9 min, further uptake was stopped by

212 D. DUBNAU AND R. DAVIDOFF-ABELSON

the addition of salmon sperm DNA (Calbiochem) to a final conoentration of 200 pg/ml. The culture was shifted to a 30°C water bath, to slow down the events following uptake, and 8 ml. samples were withdrawn at 10, 20 and 60 mm after the addition of the BD204 DNA. The samples were washed 3 times in ice-cold 0.15 M-NaCl + O-02 M-KsHPOI, pH 7.5. Each sample was resuspended in 2.5 ml. of 0.05 M-NaCl + 0.1 M-EDTA, pH 6.9, and was then treated at 37°C with 0.5 mg lysozyme/ml. After 12 mm, 0.1 y. sodium dodecyl sulfate was added, followed by 0.5 mg pronasejml. The lysates were incubated at 48°C for 2 hr, and were then dialyzed extensively against 0.016 M-NaCl + 0.0015 M-sodium citrate to remove sodium dodecyl sulfate.

(h) Determination of radioactivity Acid-precipitable radioactive material was collected by filtration through nitrocellulose

membranes after incubation with 5% trichloroacetic acid and 50 pg bovine serum albumin carrier at 0°C for 20 min. The filters were washed, dried and counted in Omnifluor (New England Nuclear). Total radioactivity was determined after mixing the sample, in an appropriate vol. of water, with Scintisol (Isolab). Radioactivity was measured in a Beck- man scintillation spectrometer (LS-BOOB).

3. Results

3H,aH-labeled BD204 DNA, at a concentration of 1 pg/ml. was used to transform a BD170 competent culture at 37°C. After five minutes, a sample was removed and rapidly chilled to 0°C. At nine minutes salmon sperm DNA was added to a final con- centration of 200 pg/ml., and the culture was shifted to 30°C. Further samples were removed at 10, 20 and 50 minutes and similarly chilled. The samples were washed, and total DNA extracted as described in Materials and Methods. Figure 1 shows the total acid-precipitable donor radioactivity taken up per milliliter of original culture.

The amount of donor radioactive material in the washed cells increased between five and ten minutes, but following the addition of excess salmon sperm DNA a loss of counts occurred. (The nature of this loss will be described in a subsequent paper in this series.) The DNA samples were fractionated by preparative C&l density-gradient

11 , , , , ,

IO al 33 40 50 Time after addition of DNA (min)

FIQ. 1. Total acid-preoipitable radioaotivity in extracts of washed competent cells at various times following the addition of 3H,“H-labeled transforming DNA. At the time indicated by the arrow, a 200-fold excess of salmon sperm DNA was added and the culture wtxa shifted from 37°C to 30°C.

FATE OF TRANSFORMING DNA IN B. SUBTILIS

Jmin HH IOmin

3000 - HH LL

I L L,, 40 45 !

213

, Fraction no

FIQ. 2. The distribution of acid-precipitable radioactivity after preparative C&l density gradient oentrifugation of DNA extracted from washed B. subtilis cultures following transformation by sH,sH-labeled DNA. The position of 32P-labeled fully deuterated (HH) and light (LL) density standards is indicated by the arrows.

oentrifugation, and the results are displayed in Figure 2. In the gradient from the five-minute sample, more than 90% of the acid-precipitable radioactivity was found in a band, the midpoint of which was at the density of native fully deuterated B. subtilis DNA. In the ten-minute sample protile, about 40% of the radioactivity was found in a band at the density of the native light B. subtilia DNA, and the remainder was at the heavy density. By 20 minutes, almost all of the radioactivity derived from the donor DNA was present in the light band

Additional information was obtained by examining the sedimentation pattern of the labeled DNA in these four preparations, using centrifugation through neutral 5 to 20% sucrose gradients resting on a shelf of 60% sucrose. The results of such an experiment are shown in Figure 3. At the first time-point studied, the acid-precipitable donor radioactivity was found predominantly in two sedimentation bands; the major one with a higher sedimentation coefficient (S-value). With time, counts disappeared from these two forms and appeared in a third species with a higher S-value, which penetrated the 60% sucrose shelf. Comparison of the approximate proportion of total acid-precipitable radioactivity from each sample found in this high S-value fraction (Fig. 3) with the proportion found in the low density bands shown in Figure 2 indicates that the high S-value and low density species are most likely the same. This compari- son can not be made quantitatively, since resolution is poor in the sucrose gradients and the high S-value and middle value bands overlap. The low density of the high S-value material can be seen directly from Figure 7(a) where the high S-value material isolated from the 20-minute sample, was found to have the buoyant density of the light native B. subtilis standard.

214 D. DUBNAU AND R. DAVIDOFF-ABELSON

4000 Smin

0 16 24

IO min

Froctim no

Fm. 3. The distribution of acid-preoipitable radioactivity after sucrose gradient centrifugation of DNA extracted from washed B. subtilia cultures following transformation by 3H,aH-labeled DNA. The oentrifugation through preformed 5 to 20% sucrose gradients was carried out at 20°C for 17.5 hr at 22,000 rev./min in an SW27 rotor.

Venema et al. (1965) have reported that following uptake by competent cells, donor DNA is converted to a form with little or no transforming activity. They further showed that with time, transforming activity for both a donor marker, and for the recombinant marker configuration, dramatically increases. This “eclipse” and re- covery phenomenon is illustrated by the data in Figure 4. These authors postulated that this recovery of donor activity and the appearance of recombinant activity reflect the process of genetic “integration”. This question was examined by comparing the recombinant transforming activity of the total extracted samples with that of the low density form isolated from C&l gradients similar to those shown in Figure 2. The results of such a comparison are shown in Table 1. The BD204 donor strain was trp-2+ hisB2, while the BD170 recipient strain was trp-2 hisBZ+. Thus, Trp+ trans- formation represented donor activity, His + transformation represented recipient activity and Trp+His + cotransformation represented recombinant activity. The recombinant activity is normalized to the recipient activity of each sample to correct for differences in such factors as DNA concentration and molecular weight. Table 1 shows that the ratio of recombinant to recipient transforming activity in the total extracted samples, was comparable to that in the low-density fractions purified from the same samples on CsCl gradients.

The increase of recombinant transforming activity in the total extract was thus

FATE OF TRANSFORMING DNA IN B. SUBTILIS 215

TAFSLE~

Analysis of recombinant-type (Trp + His + ) transforming activity in extracts of competent B. subtilis (trp-2 his+) cultures following exposure to 3H,aH-lubeled

B. subtilis (trp+hisBB) DNA

Time after Total extract Low density fraction

addition Trp + His + t

Trp + His + t

Acid precipitable Trp + His + of DNA (min) His+ His + cts/min/ml. (His+) (cts/min/ml.)

6 1.4 x 10-B 2.2x10-6 - - 10 20.4 x 1O-B 29.8 x 1O-B 1120 0.21 x lo-’ 20 101 x10-6 126 x~O-~ 1980 0.64 x 10-T 50 233 xIO-~ 200 x10-6 2280 0.88 x10-7

t The recombinant transforming activity is normalized to the level of recipient (His+) activity in each sample. The activity in the total extracts is compared to that in the low density fraction isolated following C&l density-gradient centrifugation.

quantitatively accounted for by the increase of this activity in the low-density fraction. A similar comparison showed that sll of the His + , and almost all of the Trp+ trans- forming activity of the total lo-, 20- and 50-minute extracts was present in the low density fraction. These observations together with the buoyant density and sedimen- tation data presented in Figures 2 and 3, indicated that this fraction contained a complex between donor and recipient genetic material. When these comparisons were performed using the high S-value fractions pooled from sucrose gradients similar to those shown in Figure 3, it was found that these fractions contained all of the Trp+His+, all of the His+, and almost all of the Trp + transforming activity found in each sample. On the other hand, when the slower sedimenting fractions from the five- minute sample were isolated and pooled following sedimentation in a neutral sucrose gradient, they were found to possess no detectable recipient (His+) or recombinant (Trp+His+ ) transforming activity and a low but detectable level of donor (Trp +) transforming activity, about 2% of that obtained with an equal amount of the original donor DNA. This and the high buoyant density of these fractions in CsCl gradients (unpublished observations; also compare Figs 2 and 3) indicated that they are en- tirely of donor origin.

The last column of Table 1 shows that the specific recombinant transforming ackivity in the purified donor-recipient complex increased about threefold from the lo- to the 50-minute samples. In other words, donor-recipient complex appeared to form faster when measured by the transfer of radioactivity into the low-density fraction than it did when the appearance of recombinant transforming activity was monitored. This can be seen directly from Figure 4. The appearance of recombinant activity and the recovery of donor activity in the total extract lags well behind the appearance of radioactivity in the donor-recipient complex. This implies that the donor-recipient complex exists transiently in a form with low biological activity. An obvious candidate for such an intermediate is one in which the donor and recipient moieties are held together by non-covalent bonds. Such a molecular species would possess single strand interruptions and would be expected to have a low specific transforming activity (Bodmer, 1966; Laipis, Olivera & Ganesan, 1969; Hutchinson & Hales, 1970). Donor-recipient complex, isolated by sucrose gradient centrifugation

216 D. DUBNAU AND R. DAVIDOFF-ABELSON

Ttp’His+ Trp’ -- His+ His+

I.01 1250 -750

-600

-450

-300

-150

Time afteraddition of DNA (min)

FIG. 4. Comparison of the proportion of donor radioactivity in donor-recipient complex with the level of recombinant-type and donor-type transforming activity in extracts of B. aubtilia, at various times after addition of 3H,aH-labeled transforming DNA. The proportion of radioactivity in donor-recipient complex was determined from CsCl density gradient centrifugation patterns similar to the ones shown in Fig. 2. The transforming activity for the donor (Tip+) and the re- combinant (Trp + His + ) marker configuration in each sample is normalized to the level of recipient type (His+) activity. -O-O-, Proportion of total radioactivity in the light buoyant density fraotion; -@-a--, recombinant transforming activity; - x - x -, donor transforming activity.

5omh H L

c -1

16 20 24 28 32 3t 5 20 24 28 32 ?

FIG. 6. The distribution of acid-precipitable radioactivity from purified donor-recipient DNA complex following alkaline (pH 13.0) CsCl density-gradient centrifugation. The positions of fully deuterated (H) and light (L) B. s&&s density standards are indicated by arrows. -O-O-, sH-labeled donor-reoipient oomplex ; - x - x -. 3aP-labeled deuterated (H) and light (L) marker DNA.

FATE OF TRANSFORMING DNA IN B. SUBTILIS 217

from the 20- and 50-minute samples, was subjected to oentrifugation in alkaline (pH 13-O) C&l gradients. At this pH, DNA strands separate and any non-covalently bonded donor DNA would band at the fully heavy density. This experiment is presented in Figure 5. Both the 20- and 50-minute samples contain a minor proportion of radioactive counts which band at the heavy density. In the neutral CsCl gradients, run on the same samples, almost all of the donor radioactivity banded at the position of the light B. subtilis standard (Fig. 7(a)). The proportion of counts which band at the heavy density seems lower in the 50-minute (Fig. 5(b)) than in the 20-minute (Fig. 5(a)) sample. This difference is only suggestive of the model proposed above. Table 1 shows that the specific transforming activity of the 50-minute sample is only about 38% greater than that of the 20-minute sample. These experiments must be investigated further using samples taken at earlier times which would be expected to reveal more dramatic differences if the model is correct.

In the Pneumowccus and Hemophilw transformation systems a single-stranded donor segment pairs with a recipient strand to form a heteroduplex donor-recipient complex (Fox, 1966; Lacks, 1962; Notani & Goodgal, 1966). It has been suggested that this is true of B. szdtilis as well (Bodmer, 1966; Bodmer & Ganesan, 1964). To test this, donor-recipient complex was isolated from the 20-minute sample, by frac- tionation on sucrose gradients as in Figure 3, and was then sheared in a Sorvall Omnimixer at top speed for 30 minutes at 0°C. This resulted in radioactive material with a double-strand molecular weight of 1.8~ 10” and a single-strand molecular weight of 0.94 x IO6 (average of 2 experiments). Typical sucrose gradient sedimenta- tion patterns for this sample are shown in Figure 6. The unsheared 20-minute donor- recipient complex had a double strand molecular weight of 6.5 x 107. (The single strand molecular weight of the unsheared material was not determined.) The sheared and unsheared samples were resolved by centrifugation in neutral and alkaline C&l

-I

-lcm

.f - 5

;: f

- 500

I IIIIIIIlII I 4 8 12 16 20 4 5 12 16 20

(0) Fraciion no. (b)

Fro. 6. The sedimentation patterns following centrifugation in alkaline (a) and neutral (b) 6 to 20% sucrose gradients, of sheared, donor-recipient oomplex isolated from the 20-min sample. -a-@--, 3H-labeled donor-recipient complex; -O--O--, W-labeled phage T7 DNA.

218 D. DUBNAU AND R. DAVIDOFF-ABELSON

15 20 25 3015 20 25 3015 20 25 30 ? (a) (b) Cc)

Fraction no.

d 15

FIQ. 7. The distribution after CsCl density gradient centrifugation of unshearred (a) and sheared (b) and (c) donor-recipient complex isolated from the 20-min sample. The patterns shown in (a) and (b) were obtained from gradients run at pH 8.2, whereas the pattern of (c) was obtained at pH 13.0. -O-O--, 3H-labeled donor-recipient complex; - x - x -, 32P-labeled deuterated (H) and light (L) marker DNA.

density gradients, and the results are presented in Figure 7. The greater separation

shown in Figure 7(c), compared to that in Figure 7(a) and (b) is a consequence of variation in the drop size during collection of the gradients. The results determined for the unsheared sample in an alkaline CsCl gradient may be seen in Figure 5(a). In the neutral gradient of the unsheared material (Fig. 7(a)) the acid-precipitable radio- activity was located at the density of the light recipient standard with slight skewing toward the dense side. The pattern of the sheared sample in neutral CsCl (Fig. 7(b)) revealed a shift to a position 80 to lOOo/o of the way between the light and hybrid positions. This is compatible with two models for the structure of the sheared donor- recipient complex. The complex may be a heteroduplex structure in which one strand is entirely recipient in origin and the other contains 80 to lOOo/o donor material. Alternately, the complex may consist of two strands, each containing 40 to 50% donor and 50 to 60% recipient DNA. The latter model was excluded by bhe results presented in Figure 7(c), obtained by alkaline CsCl centrifugation of the sheared sample. In this case the donor material was found in a broad band which peaked about 80% of the way between the light and fully deuterated B. subtilis standards. Clearly, most of the single strands derived from sheared donor-recipient complex contained more than 50% donor DNA.

4. Discussion The data presented above show that beginning a few minutes after uptake by

competent B. subtilis cells, donor DNA enters a complex with recipient DNA. Before that time the donor material is in at least two distinct and relatively slowly sediment- ing forms which possess little biological activity. The physical and biological pro- perties of the donor DNA immediately following uptake, will be discussed in a subsequent paper in this series. The presence of a donor-recipient DNA complex confirms the results of other investigators using the B. subtilis (Ayad & Barker, 1969;

FATE OF TRANSFORMING DNA IN B. SUBTILIS 219

Bodmer & Ganesan, 1964; Harris & Barr, 1969; P&e & Romig, 1964), Pneumococcus (Fox & Allen, 1964), and H~UWJJ%ZUS (Noteni & Goodgal, 1966) transformrttion systems, and indicates that transformation utilizes a breakage and reunion type of recombination mechanism. The data presented here, however, do not rigorously exclude the transfer of some genetic information by a mechanism other than breakage and reunion, such as copy-choice.

The recovery of donor transforming markers following eclipse, and the increase in recombinant transforming activity are quantitatively accounted for by events occur- ring within the donor-recipient complex. Our results indicate that the recovery of donor markers reflects the integration process. It does not support the argument of Venema et al. (1965) that recovery of donor activity may precede the integration step. The latter suggestion was based on an apparent slight lag between the increase of donor and of recombinant transforming activity. This lag, which the present suthors have also noticed in total extracts of transformed cells and which may be inferred from the data of Figure 4, is not apparent in the isolated donor-recipient complex (unpublished observations). It is probably a consequence of the low level of donor transforming activity and the total absence of recombinant activity in the slowly sedimenting donor material which predominates at early times following the uptake of DNA.

The present data indicate that the bulk of the donor-recipient complex exists as a heteroduplex containing donor material paired with a recipient strand. This is in accord with previous reports in B. subtilis (Bodmer & Ganesan, 1964) and with the situation in Pneunaowccus (Fox & Allen, 1964) and Hemophilus (Notani & Goodgel, 1966). Experiments on the composition of B. subtilis transformant clones also are consistent with the formation of a heteroduplex donor-recipient complex, although they indicate that substantial correction of these heteroduplexes can occur to yield homoduplex donor-type DNA (Bresler, Kreneva & Kushev, 1968). N. Strauss (per- sonal communication) has carried out experiments with heteroduplex trensforming DNA carrying closely linked markers in the bans configuration, formed by annealing DNA strands previously resolved on methylated albumin kieselguhr columns. The results of these experiments indicate that only one strand from a given duplex mole- cule enters the donor-recipient complex, although either strand can do so. Similar experimente have been reported by Bresler, Kreneva, Kushev & Mosevitskii (1964). The data of Rudner, Karkas & Chargaff (1968) which show that either strand of artificially constructed heteroduplex can carry genetic information during trans- formation of B. subtilis, is consistent with these results. The work of Chilton & Hall (1968) and of Tevethia & Mandel (1970), which demonstrate that single-stranded DNA can transform B. subtilis with high efficiency provides evidence in accord with our data.

The density of the donor-recipient complex from the 20-minute sample in a neutral CsCl gradient is essentially that of native light B. subtilis DNA (Fig. 7(a)). The double-strand molecular weight of this donor-recipient complex is 6.5 x 107. The average contribution of the donor must be less than about 10% of this, since it has little effect on the buoyant density of the complex. The sheared sample has a single- strand molecular weight of 9.4 x lo5 and has a buoyant density 0.8 of the way from that of light to that of fully deuterated DNA. As a first approximation this data suggests that the average size of the donor segment is 0.8 x9.4 x 105, or 7.5 x 105. This is somewhat higher than 4.9 x 105, which was the estimate of Bodmer (1966).

220 D. DUBNAU AND R. DAVIDOFF-ABELSON

In a more recent paper, Bodmer & Laird (1968) suggest that this latter value may be low, since the donor DNA sample used showed a low cotransfer index for four closely linked markers. Several sources of error tend to render our value an approximate one. First, there is uncertainty in the single-stranded molecular weight of the sheared donor-recipient complex DNA. A small shift in the position of the 3H peak in the alkaline sucrose gradient shown in Figure 6(a) would cause an appreciable change in the calculated molecular weight. In two runs the values 1.08 x lo6 and 0.80 x lo6 were obtained, yielding an average of 9.4 x 106. The double-strand molecular weight calculated for the same sample from the neutral sucrose gradient shown in Figure 7(b), was I.83 x 106, which provides independent support for the single-stranded molecular weight measurement. Other very important sources of error lie in the polydispersity of the single-strand molecular weight, and in possible biological dis- persity in the size of the integrated donor segment. Although the sedimentation band shown in Figure 7(a) is quite sharp, these factors are difficult to assess quantitatively, so the estimate given for the size of the donor segment must be regarded as provisional pending further analysis.

The evidence for a non-covalently bonded donor-recipient complex is reminiscent of the situation in phage T4 recombination (Anraku t Tomizawa, 1965; Tomizawa t Anraku, 1964). This interpretation is subject to an important qualification. In the absence of data concerning the average single-stranded molecular weight of the re-extracted recipient DNA, the density profile shown in Figure 6 may reflect mole- cular weight differences. This is unlikely since in other experiments, the single-strand molecular weight of DNA isolated by our methods has been found to be close to 10’. This is more than an order of magnitude larger than the size of the integrated donor [fragment. If the presence of a non-covalently bonded intermediate is shown more rigorously by further work, its isolation should permit a physical and enzymological approach to the exact structure of the early complex.

This work was supported by U. S. Public Health Service grant no. GM-14642 awarded to one of us (D. D.). We acknowledge with pleasure the expert technical assistance of Mrs Sheila Loeb and the excellent secretarial help of Mm Annabel Howard. We are also appreciative of many illuminating discussions with Issar Smith and Leonard Mindich.

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